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• Published: 08 March 2018

Meta-analysis and the science of research synthesis

• Jessica Gurevitch 1 ,
• Julia Koricheva 2 ,
• Shinichi Nakagawa 3 , 4 &
• Gavin Stewart 5  

Nature volume  555 ,  pages 175–182 ( 2018 ) Cite this article

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Meta-analysis is the quantitative, scientific synthesis of research results. Since the term and modern approaches to research synthesis were first introduced in the 1970s, meta-analysis has had a revolutionary effect in many scientific fields, helping to establish evidence-based practice and to resolve seemingly contradictory research outcomes. At the same time, its implementation has engendered criticism and controversy, in some cases general and others specific to particular disciplines. Here we take the opportunity provided by the recent fortieth anniversary of meta-analysis to reflect on the accomplishments, limitations, recent advances and directions for future developments in the field of research synthesis.

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Acknowledgements

We dedicate this Review to the memory of Ingram Olkin and William Shadish, founding members of the Society for Research Synthesis Methodology who made tremendous contributions to the development of meta-analysis and research synthesis and to the supervision of generations of students. We thank L. Lagisz for help in preparing the figures. We are grateful to the Center for Open Science and the Laura and John Arnold Foundation for hosting and funding a workshop, which was the origination of this article. S.N. is supported by Australian Research Council Future Fellowship (FT130100268). J.G. acknowledges funding from the US National Science Foundation (ABI 1262402).

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Department of Ecology and Evolution, Stony Brook University, Stony Brook, 11794-5245, New York, USA

Jessica Gurevitch

School of Biological Sciences, Royal Holloway University of London, Egham, TW20 0EX, Surrey, UK

Julia Koricheva

Evolution and Ecology Research Centre and School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, 2052, New South Wales, Australia

Shinichi Nakagawa

Diabetes and Metabolism Division, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, 2010, New South Wales, Australia

School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK

Gavin Stewart

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Gurevitch, J., Koricheva, J., Nakagawa, S. et al. Meta-analysis and the science of research synthesis. Nature 555 , 175–182 (2018). https://doi.org/10.1038/nature25753

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• Published: 11 August 2009

Methods for the synthesis of qualitative research: a critical review

• Elaine Barnett-Page 1 &
• James Thomas 1  

BMC Medical Research Methodology volume  9 , Article number:  59 ( 2009 ) Cite this article

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In recent years, a growing number of methods for synthesising qualitative research have emerged, particularly in relation to health-related research. There is a need for both researchers and commissioners to be able to distinguish between these methods and to select which method is the most appropriate to their situation.

A number of methodological and conceptual links between these methods were identified and explored, while contrasting epistemological positions explained differences in approaches to issues such as quality assessment and extent of iteration. Methods broadly fall into 'realist' or 'idealist' epistemologies, which partly accounts for these differences.

Methods for qualitative synthesis vary across a range of dimensions. Commissioners of qualitative syntheses might wish to consider the kind of product they want and select their method – or type of method – accordingly.

Peer Review reports

The range of different methods for synthesising qualitative research has been growing over recent years [ 1 , 2 ], alongside an increasing interest in qualitative synthesis to inform health-related policy and practice [ 3 ]. While the terms 'meta-analysis' (a statistical method to combine the results of primary studies), or sometimes 'narrative synthesis', are frequently used to describe how quantitative research is synthesised, far more terms are used to describe the synthesis of qualitative research. This profusion of terms can mask some of the basic similarities in approach that the different methods share, and also lead to some confusion regarding which method is most appropriate in a given situation. This paper does not argue that the various nomenclatures are unnecessary, but rather seeks to draw together and review the full range of methods of synthesis available to assist future reviewers in selecting a method that is fit for their purpose. It also represents an attempt to guide the reader through some of the varied terminology to spring up around qualitative synthesis. Other helpful reviews of synthesis methods have been undertaken in recent years with slightly different foci to this paper. Two recent studies have focused on describing and critiquing methods for the integration of qualitative research with quantitative [ 4 , 5 ] rather than exclusively examining the detail and rationale of methods for the synthesis of qualitative research. Two other significant pieces of work give practical advice for conducting the synthesis of qualitative research, but do not discuss the full range of methods available [ 6 , 7 ]. We begin our Discussion by outlining each method of synthesis in turn, before comparing and contrasting characteristics of these different methods across a range of dimensions. Readers who are more familiar with the synthesis methods described here may prefer to turn straight to the 'dimensions of difference' analysis in the second part of the Discussion.

Overview of synthesis methods

Meta-ethnography.

In their seminal work of 1988, Noblit and Hare proposed meta-ethnography as an alternative to meta-analysis [ 8 ]. They cited Strike and Posner's [ 9 ] definition of synthesis as an activity in which separate parts are brought together to form a 'whole'; this construction of the whole is essentially characterised by some degree of innovation, so that the result is greater than the sum of its parts. They also borrowed from Turner's theory of social explanation [ 10 ], a key tenet of which was building 'comparative understanding' [[ 8 ], p22] rather than aggregating data.

To Noblit and Hare, synthesis provided an answer to the question of 'how to "put together" written interpretive accounts' [[ 8 ], p7], where mere integration would not be appropriate. Noblit and Hare's early work synthesised research from the field of education.

Three different methods of synthesis are used in meta-ethnography. One involves the 'translation' of concepts from individual studies into one another, thereby evolving overarching concepts or metaphors. Noblit and Hare called this process reciprocal translational analysis (RTA). Refutational synthesis involves exploring and explaining contradictions between individual studies. Lines-of-argument (LOA) synthesis involves building up a picture of the whole (i.e. culture, organisation etc) from studies of its parts. The authors conceptualised this latter approach as a type of grounded theorising.

Britten et al [ 11 ] and Campbell et al [ 12 ] have both conducted evaluations of meta-ethnography and claim to have succeeded, by using this method, in producing theories with greater explanatory power than could be achieved in a narrative literature review. While both these evaluations used small numbers of studies, more recently Pound et al [ 13 ] conducted both an RTA and an LOA synthesis using a much larger number of studies (37) on resisting medicines. These studies demonstrate that meta-ethnography has evolved since Noblit and Hare first introduced it. Campbell et al claim to have applied the method successfully to non-ethnographical studies. Based on their reading of Schutz [ 14 ], Britten et al have developed both second and third order constructs in their synthesis (Noblit and Hare briefly allude to the possibility of a 'second level of synthesis' [[ 8 ], p28] but do not demonstrate or further develop the idea).

In a more recent development, Sandelowski & Barroso [ 15 ] write of adapting RTA by using it to ' integrate findings interpretively, as opposed to comparing them interpretively' (p204). The former would involve looking to see whether the same concept, theory etc exists in different studies; the latter would involve the construction of a bigger picture or theory (i.e. LOA synthesis). They also talk about comparing or integrating imported concepts (e.g. from other disciplines) as well as those evolved 'in vivo'.

Grounded theory

Kearney [ 16 ], Eaves [ 17 ] and Finfgeld [ 18 ] have all adapted grounded theory to formulate a method of synthesis. Key methods and assumptions of grounded theory, as originally formulated and subsequently refined by Glaser and Strauss [ 19 ] and Strauss and Corbin [ 20 , 21 ], include: simultaneous phases of data collection and analysis; an inductive approach to analysis, allowing the theory to emerge from the data; the use of the constant comparison method; the use of theoretical sampling to reach theoretical saturation; and the generation of new theory. Eaves cited grounded theorists Charmaz [ 22 ] and Chesler [ 23 ], as well as Strauss and Corbin [ 20 ], as informing her approach to synthesis.

Glaser and Strauss [ 19 ] foresaw a time when a substantive body of grounded research should be pushed towards a higher, more abstract level. As a piece of methodological work, Eaves undertook her own synthesis of the synthesis methods used by these authors to produce her own clear and explicit guide to synthesis in grounded formal theory. Kearney stated that 'grounded formal theory', as she termed this method of synthesis, 'is suited to study of phenomena involving processes of contextualized understanding and action' [[ 24 ], p180] and, as such, is particularly applicable to nurses' research interests.

As Kearney suggested, the examples examined here were largely dominated by research in nursing. Eaves synthesised studies on care-giving in rural African-American families for elderly stroke survivors; Finfgeld on courage among individuals with long-term health problems; Kearney on women's experiences of domestic violence.

Kearney explicitly chose 'grounded formal theory' because it matches 'like' with 'like': that is, it applies the same methods that have been used to generate the original grounded theories included in the synthesis – produced by constant comparison and theoretical sampling – to generate a higher-level grounded theory. The wish to match 'like' with 'like' is also implicit in Eaves' paper. This distinguishes grounded formal theory from more recent applications of meta-ethnography, which have sought to include qualitative research using diverse methodological approaches [ 12 ].

• Thematic Synthesis

Thomas and Harden [ 25 ] have developed an approach to synthesis which they term 'thematic synthesis'. This combines and adapts approaches from both meta-ethnography and grounded theory. The method was developed out of a need to conduct reviews that addressed questions relating to intervention need, appropriateness and acceptability – as well as those relating to effectiveness – without compromising on key principles developed in systematic reviews. They applied thematic synthesis in a review of the barriers to, and facilitators of, healthy eating amongst children.

Free codes of findings are organised into 'descriptive' themes, which are then further interpreted to yield 'analytical' themes. This approach shares characteristics with later adaptations of meta-ethnography, in that the analytical themes are comparable to 'third order interpretations' and that the development of descriptive and analytical themes using coding invoke reciprocal 'translation'. It also shares much with grounded theory, in that the approach is inductive and themes are developed using a 'constant comparison' method. A novel aspect of their approach is the use of computer software to code the results of included studies line-by-line, thus borrowing another technique from methods usually used to analyse primary research.

Textual Narrative Synthesis

Textual narrative synthesis is an approach which arranges studies into more homogenous groups. Lucas et al [ 26 ] comment that it has proved useful in synthesising evidence of different types (qualitative, quantitative, economic etc). Typically, study characteristics, context, quality and findings are reported on according to a standard format and similarities and differences are compared across studies. Structured summaries may also be developed, elaborating on and putting into context the extracted data [ 27 ].

Lucas et al [ 26 ] compared thematic synthesis with textual narrative synthesis. They found that 'thematic synthesis holds most potential for hypothesis generation' whereas textual narrative synthesis is more likely to make transparent heterogeneity between studies (as does meta-ethnography, with refutational synthesis) and issues of quality appraisal. This is possibly because textual narrative synthesis makes clearer the context and characteristics of each study, while the thematic approach organises data according to themes. However, Lucas et al found that textual narrative synthesis is 'less good at identifying commonality' (p2); the authors do not make explicit why this should be, although it may be that organising according to themes, as the thematic approach does, is comparatively more successful in revealing commonality.

Paterson et al [ 28 ] have evolved a multi-faceted approach to synthesis, which they call 'meta-study'. The sociologist Zhao [ 29 ], drawing on Ritzer's work [ 30 ], outlined three components of analysis, which they proposed should be undertaken prior to synthesis. These are meta-data-analysis (the analysis of findings), meta-method (the analysis of methods) and meta-theory (the analysis of theory). Collectively, these three elements of analysis, culminating in synthesis, make up the practice of 'meta-study'. Paterson et al pointed out that the different components of analysis may be conducted concurrently.

Paterson et al argued that primary research is a construction; secondary research is therefore a construction of a construction. There is need for an approach that recognises this, and that also recognises research to be a product of its social, historical and ideological context. Such an approach would be useful in accounting for differences in research findings. For Paterson et al, there is no such thing as 'absolute truth'.

Meta-study was developed to study the experiences of adults living with a chronic illness. Meta-data-analysis was conceived of by Paterson et al in similar terms to Noblit and Hare's meta-ethnography (see above), in that it is essentially interpretive and seeks to reveal similarities and discrepancies among accounts of a particular phenomenon. Meta-method involves the examination of the methodologies of the individual studies under review. Part of the process of meta-method is to consider different aspects of methodology such as sampling, data collection, research design etc, similar to procedures others have called 'critical appraisal' (CASP [ 31 ]). However, Paterson et al take their critique to a deeper level by establishing the underlying assumptions of the methodologies used and the relationship between research outcomes and methods used. Meta-theory involves scrutiny of the philosophical and theoretical assumptions of the included research papers; this includes looking at the wider context in which new theory is generated. Paterson et al described meta-synthesis as a process which creates a new interpretation which accounts for the results of all three elements of analysis. The process of synthesis is iterative and reflexive and the authors were unwilling to oversimplify the process by 'codifying' procedures for bringing all three components of analysis together.

Meta-narrative

Greenhalgh et al [ 32 ]'s meta-narrative approach to synthesis arose out of the need to synthesise evidence to inform complex policy-making questions and was assisted by the formation of a multi-disciplinary team. Their approach to review was informed by Thomas Kuhn's The Structure of Scientific Revolutions [ 33 ], in which he proposed that knowledge is produced within particular paradigms which have their own assumptions about theory, about what is a legitimate object of study, about what are legitimate research questions and about what constitutes a finding. Paradigms also tend to develop through time according to a particular set of stages, central to which is the stage of 'normal science', in which the particular standards of the paradigm are largely unchallenged and seen to be self-evident. As Greenhalgh et al pointed out, Kuhn saw paradigms as largely incommensurable: 'that is, an empirical discovery made using one set of concepts, theories, methods and instruments cannot be satisfactorily explained through a different paradigmatic lens' [[ 32 ], p419].

Greenhalgh et al synthesised research from a wide range of disciplines; their research question related to the diffusion of innovations in health service delivery and organisation. They thus identified a need to synthesise findings from research which contains many different theories arising from many different disciplines and study designs.

Based on Kuhn's work, Greenhalgh et al proposed that, across different paradigms, there were multiple – and potentially mutually contradictory – ways of understanding the concept at the heart of their review, namely the diffusion of innovation. Bearing this in mind, the reviewers deliberately chose to select key papers from a number of different research 'paradigms' or 'traditions', both within and beyond healthcare, guided by their multidisciplinary research team. They took as their unit of analysis the 'unfolding "storyline" of a research tradition over time' [[ 32 ], p417) and sought to understand diffusion of innovation as it was conceptualised in each of these traditions. Key features of each tradition were mapped: historical roots, scope, theoretical basis; research questions asked and methods/instruments used; main empirical findings; historical development of the body of knowledge (how have earlier findings led to later findings); and strengths and limitations of the tradition. The results of this exercise led to maps of 13 'meta-narratives' in total, from which seven key dimensions, or themes, were identified and distilled for the synthesis phase of the review.

Critical Interpretive Synthesis

Dixon-Woods et al [ 34 ] developed their own approach to synthesising multi-disciplinary and multi-method evidence, termed 'critical interpretive synthesis', while researching access to healthcare by vulnerable groups. Critical interpretive synthesis is an adaptation of meta-ethnography, as well as borrowing techniques from grounded theory. The authors stated that they needed to adapt traditional meta-ethnographic methods for synthesis, since these had never been applied to quantitative as well as qualitative data, nor had they been applied to a substantial body of data (in this case, 119 papers).

Dixon-Woods et al presented critical interpretive synthesis as an approach to the whole process of review, rather than to just the synthesis component. It involves an iterative approach to refining the research question and searching and selecting from the literature (using theoretical sampling) and defining and applying codes and categories. It also has a particular approach to appraising quality, using relevance – i.e. likely contribution to theory development – rather than methodological characteristics as a means of determining the 'quality' of individual papers [ 35 ]. The authors also stress, as a defining characteristic, critical interpretive synthesis's critical approach to the literature in terms of deconstructing research traditions or theoretical assumptions as a means of contextualising findings.

Dixon-Woods et al rejected reciprocal translational analysis (RTA) as this produced 'only a summary in terms that have already been used in the literature' [[ 34 ], p5], which was seen as less helpful when dealing with a large and diverse body of literature. Instead, Dixon-Woods et al adopted a lines-of-argument (LOA) synthesis, in which – rejecting the difference between first, second and third order constructs – they instead developed 'synthetic constructs' which were then linked with constructs arising directly from the literature.

The influence of grounded theory can be seen in particular in critical interpretive synthesis's inductive approach to formulating the review question and to developing categories and concepts, rejecting a 'stage' approach to systematic reviewing, and in selecting papers using theoretical sampling. Dixon-Woods et al also claim that critical interpretive synthesis is distinct in its 'explicit orientation towards theory generation' [[ 34 ], p9].

Ecological Triangulation

Jim Banning is the author of 'ecological triangulation' or 'ecological sentence synthesis', applying this method to the evidence for what works for youth with disabilities. He borrows from Webb et al [ 36 ] and Denzin [ 37 ] the concept of triangulation, in which phenomena are studied from a variety of vantage points. His rationale is that building an 'evidence base' of effectiveness requires the synthesis of cumulative, multi-faceted evidence in order to find out 'what intervention works for what kind of outcomes for what kind of persons under what kind of conditions' [[ 38 ], p1].

Ecological triangulation unpicks the mutually interdependent relationships between behaviour, persons and environments. The method requires that, for data extraction and synthesis, 'ecological sentences' are formulated following the pattern: 'With this intervention, these outcomes occur with these population foci and within these grades (ages), with these genders ... and these ethnicities in these settings' [[ 39 ], p1].

Framework Synthesis

Brunton et al [ 40 ] and Oliver et al [ 41 ] have applied a 'framework synthesis' approach in their reviews. Framework synthesis is based on framework analysis, which was outlined by Pope, Ziebland and Mays [ 42 ], and draws upon the work of Ritchie and Spencer [ 43 ] and Miles and Huberman [ 44 ]. Its rationale is that qualitative research produces large amounts of textual data in the form of transcripts, observational fieldnotes etc. The sheer wealth of information poses a challenge for rigorous analysis. Framework synthesis offers a highly structured approach to organising and analysing data (e.g. indexing using numerical codes, rearranging data into charts etc).

Brunton et al applied the approach to a review of children's, young people's and parents' views of walking and cycling; Oliver et al to an analysis of public involvement in health services research. Framework synthesis is distinct from the other methods outlined here in that it utilises an a priori 'framework' – informed by background material and team discussions – to extract and synthesise findings. As such, it is largely a deductive approach although, in addition to topics identified by the framework, new topics may be developed and incorporated as they emerge from the data. The synthetic product can be expressed in the form of a chart for each key dimension identified, which may be used to map the nature and range of the concept under study and find associations between themes and exceptions to these [ 40 ].

'Fledgling' approaches

There are three other approaches to synthesis which have not yet been widely used. One is an approach using content analysis [ 45 , 46 ] in which text is condensed into fewer content-related categories. Another is 'meta-interpretation' [ 47 ], featuring the following: an ideographic rather than pre-determined approach to the development of exclusion criteria; a focus on meaning in context; interpretations as raw data for synthesis (although this feature doesn't distinguish it from other synthesis methods); an iterative approach to the theoretical sampling of studies for synthesis; and a transparent audit trail demonstrating the trustworthiness of the synthesis.

In addition to the synthesis methods discussed above, Sandelowski and Barroso propose a method they call 'qualitative metasummary' [ 15 ]. It is mentioned here as a new and original approach to handling a collection of qualitative studies but is qualitatively different to the other methods described here since it is aggregative; that is, findings are accumulated and summarised rather than 'transformed'. Metasummary is a way of producing a 'map' of the contents of qualitative studies and – according to Sandelowski and Barroso – 'reflect [s] a quantitative logic' [[ 15 ], p151]. The frequency of each finding is determined and the higher the frequency of a particular finding, the greater its validity. The authors even discuss the calculation of 'effect sizes' for qualitative findings. Qualitative metasummaries can be undertaken as an end in themselves or may serve as a basis for a further synthesis.

Dimensions of difference

Having outlined the range of methods identified, we now turn to an examination of how they compare with one another. It is clear that they have come from many different contexts and have different approaches to understanding knowledge, but what do these differences mean in practice? Our framework for this analysis is shown in Additional file 1 : dimensions of difference [ 48 ]. We have examined the epistemology of each of the methods and found that, to some extent, this explains the need for different methods and their various approaches to synthesis.

Epistemology

The first dimension that we will consider is that of the researchers' epistemological assumptions. Spencer et al [ 49 ] outline a range of epistemological positions, which might be organised into a spectrum as follows:

Subjective idealism : there is no shared reality independent of multiple alternative human constructions

Objective idealism : there is a world of collectively shared understandings

Critical realism : knowledge of reality is mediated by our perceptions and beliefs

Scientific realism : it is possible for knowledge to approximate closely an external reality

Naïve realism : reality exists independently of human constructions and can be known directly [ 49 , 45 , 46 ].

Thus, at one end of the spectrum we have a highly constructivist view of knowledge and, at the other, an unproblematized 'direct window onto the world' view.

Nearly all of positions along this spectrum are represented in the range of methodological approaches to synthesis covered in this paper. The originators of meta-narrative synthesis, critical interpretive synthesis and meta-study all articulate what might be termed a 'subjective idealist' approach to knowledge. Paterson et al [ 28 ] state that meta-study shies away from creating 'grand theories' within the health or social sciences and assume that no single objective reality will be found. Primary studies, they argue, are themselves constructions; meta-synthesis, then, 'deals with constructions of constructions' (p7). Greenhalgh et al [ 32 ] also view knowledge as a product of its disciplinary paradigm and use this to explain conflicting findings: again, the authors neither seek, nor expect to find, one final, non-contestable answer to their research question. Critical interpretive synthesis is similar in seeking to place literature within its context, to question its assumptions and to produce a theoretical model of a phenomenon which – because highly interpretive – may not be reproducible by different research teams at alternative points in time [[ 34 ], p11].

Methods used to synthesise grounded theory studies in order to produce a higher level of grounded theory [ 24 ] appear to be informed by 'objective idealism', as does meta-ethnography. Kearney argues for the near-universal applicability of a 'ready-to-wear' theory across contexts and populations. This approach is clearly distinct from one which recognises multiple realities. The emphasis is on examining commonalities amongst, rather than discrepancies between, accounts. This emphasis is similarly apparent in most meta-ethnographies, which are conducted either according to Noblit and Hare's 'reciprocal translational analysis' technique or to their 'lines-of-argument' technique and which seek to provide a 'whole' which has a greater explanatory power. Although Noblit and Hare also propose 'refutational synthesis', in which contradictory findings might be explored, there are few examples of this having been undertaken in practice, and the aim of the method appears to be to explain and explore differences due to context, rather than multiple realities.

Despite an assumption of a reality which is perhaps less contestable than those of meta-narrative synthesis, critical interpretive synthesis and meta-study, both grounded formal theory and meta-ethnography place a great deal of emphasis on the interpretive nature of their methods. This still supposes a degree of constructivism. Although less explicit about how their methods are informed, it seems that both thematic synthesis and framework synthesis – while also involving some interpretation of data – share an even less problematized view of reality and a greater assumption that their synthetic products are reproducible and correspond to a shared reality. This is also implicit in the fact that such products are designed directly to inform policy and practice, a characteristic shared by ecological triangulation. Notably, ecological triangulation, according to Banning, can be either realist or idealist. Banning argues that the interpretation of triangulation can either be one in which multiple viewpoints converge on a point to produce confirming evidence (i.e. one definitive answer to the research question) or an idealist one, in which the complexity of multiple viewpoints is represented. Thus, although ecological triangulation views reality as complex, the approach assumes that it can be approximately knowable (at least when the realist view of ecological triangulation is adopted) and that interventions can and should be modelled according to the products of its syntheses.

While pigeonholing different methods into specific epistemological positions is a problematic process, we do suggest that the contrasting epistemologies of different researchers is one way of explaining why we have – and need – different methods for synthesis.

Variation in terms of the extent of iteration during the review process is another key dimension. All synthesis methods include some iteration but the degree varies. Meta-ethnography, grounded theory and thematic synthesis all include iteration at the synthesis stage; both framework synthesis and critical interpretive synthesis involve iterative literature searching – in the case of critical interpretive synthesis, it is not clear whether iteration occurs during the rest of the review process. Meta-narrative also involves iteration at every stage. Banning does not mention iteration in outlining ecological triangulation and neither do Lucas or Thomas and Harden for thematic narrative synthesis.

It seems that the more idealist the approach, the greater the extent of iteration. This might be because a large degree of iteration does not sit well with a more 'positivist' ideal of procedural objectivity; in particular, the notion that the robustness of the synthetic product depends in part on the reviewers stating up front in a protocol their searching strategies, inclusion/exclusion criteria etc, and being seen not to alter these at a later stage.

Quality assessment

Another dimension along which we can look at different synthesis methods is that of quality assessment. When the approaches to the assessment of the quality of studies retrieved for review are examined, there is again a wide methodological variation. It might be expected that the further towards the 'realism' end of the epistemological spectrum a method of synthesis falls, the greater the emphasis on quality assessment. In fact, this is only partially the case.

Framework synthesis, thematic narrative synthesis and thematic synthesis – methods which might be classified as sharing a 'critical realist' approach – all have highly specified approaches to quality assessment. The review in which framework synthesis was developed applied ten quality criteria: two on quality and reporting of sampling methods, four to the quality of the description of the sample in the study, two to the reliability and validity of the tools used to collect data and one on whether studies used appropriate methods for helping people to express their views. Studies which did not meet a certain number of quality criteria were excluded from contributing to findings. Similarly, in the example review for thematic synthesis, 12 criteria were applied: five related to reporting aims, context, rationale, methods and findings; four relating to reliability and validity; and three relating to the appropriateness of methods for ensuring that findings were rooted in participants' own perspectives. Studies which were deemed to have significant flaws were excluded and sensitivity analyses were used to assess the possible impact of study quality on the review's findings. Thomas and Harden's use of thematic narrative synthesis similarly applied quality criteria and developed criteria additional to those they found in the literature on quality assessment, relating to the extent to which people's views and perspectives had been privileged by researchers. It is worth noting not only that these methods apply quality criteria but that they are explicit about what they are: assessing quality is a key component in the review process for both of these methods. Likewise, Banning – the originator of ecological triangulation – sees quality assessment as important and adapts the Design and Implementation Assessment Device (DIAD) Version 0.3 (a quality assessment tool for quantitative research) for use when appraising qualitative studies [ 50 ]. Again, Banning writes of excluding studies deemed to be of poor quality.

Greenhalgh et al's meta-narrative review [ 32 ] modified a range of existing quality assessment tools to evaluate studies according to validity and robustness of methods; sample size and power; and validity of conclusions. The authors imply, but are not explicit, that this process formed the basis for the exclusion of some studies. Although not quite so clear about quality assessment methods as framework and thematic synthesis, it might be argued that meta-narrative synthesis shows a greater commitment to the concept that research can and should be assessed for quality than either meta-ethnography or grounded formal theory. The originators of meta-ethnography, Noblit and Hare [ 8 ], originally discussed quality in terms of quality of metaphor, while more recent use of this method has used amended versions of CASP (the Critical Appraisal Skills Programme tool, [ 31 ]), yet has only referred to studies being excluded on the basis of lack of relevance or because they weren't 'qualitative' studies [ 8 ]. In grounded theory, quality assessment is only discussed in terms of a 'personal note' being made on the context, quality and usefulness of each study. However, contrary to expectation, meta-narrative synthesis lies at the extreme end of the idealism/realism spectrum – as a subjective idealist approach – while meta-ethnography and grounded theory are classified as objective idealist approaches.

Finally, meta-study and critical interpretive synthesis – two more subjective idealist approaches – look to the content and utility of findings rather than methodology in order to establish quality. While earlier forms of meta-study included only studies which demonstrated 'epistemological soundness', in its most recent form [ 51 ] this method has sought to include all relevant studies, excluding only those deemed not to be 'qualitative' research. Critical interpretive synthesis also conforms to what we might expect of its approach to quality assessment: quality of research is judged as the extent to which it informs theory. The threshold of inclusion is informed by expertise and instinct rather than being articulated a priori.

In terms of quality assessment, it might be important to consider the academic context in which these various methods of synthesis developed. The reason why thematic synthesis, framework synthesis and ecological triangulation have such highly specified approaches to quality assessment may be that each of these was developed for a particular task, i.e. to conduct a multi-method review in which randomised controlled trials (RCTs) were included. The concept of quality assessment in relation to RCTs is much less contested and there is general agreement on criteria against which quality should be judged.

Problematizing the literature

Critical interpretive synthesis, the meta-narrative approach and the meta-theory element of meta-study all share some common ground in that their review and synthesis processes include examining all aspects of the context in which knowledge is produced. In conducting a review on access to healthcare by vulnerable groups, critical interpretive synthesis sought to question 'the ways in which the literature had constructed the problematics of access, the nature of the assumptions on which it drew, and what has influenced its choice of proposed solutions' [[ 34 ], p6]. Although not claiming to have been directly influenced by Greenhalgh et al's meta-narrative approach, Dixon-Woods et al do cite it as sharing similar characteristics in the sense that it critiques the literature it reviews.

Meta-study uses meta-theory to describe and deconstruct the theories that shape a body of research and to assess its quality. One aspect of this process is to examine the historical evolution of each theory and to put it in its socio-political context, which invites direct comparison with meta-narrative synthesis. Greenhalgh et al put a similar emphasis on placing research findings within their social and historical context, often as a means of seeking to explain heterogeneity of findings. In addition, meta-narrative shares with critical interpretive synthesis an iterative approach to searching and selecting from the literature.

Framework synthesis, thematic synthesis, textual narrative synthesis, meta-ethnography and grounded theory do not share the same approach to problematizing the literature as critical interpretive synthesis, meta-study and meta-narrative. In part, this may be explained by the extent to which studies included in the synthesis represented a broad range of approaches or methodologies. This, in turn, may reflect the broadness of the review question and the extent to which the concepts contained within the question are pre-defined within the literature. In the case of both the critical interpretive synthesis and meta-narrative reviews, terminology was elastic and/or the question formed iteratively. Similarly, both reviews placed great emphasis on employing multi-disciplinary research teams. Approaches which do not critique the literature in the same way tend to have more narrowly-focused questions. They also tend to include a more limited range of studies: grounded theory synthesis includes grounded theory studies, meta-ethnography (in its original form, as applied by Noblit and Hare) ethnographies. The thematic synthesis incorporated studies based on only a narrow range of qualitative methodologies (interviews and focus groups) which were informed by a similarly narrow range of epistemological assumptions. It may be that the authors of such syntheses saw no need for including such a critique in their review process.

Similarities and differences between primary studies

Most methods of synthesis are applicable to heterogeneous data (i.e. studies which use contrasting methodologies) apart from early meta-ethnography and synthesis informed by grounded theory. All methods of synthesis state that, at some level, studies are compared; many are not so explicit about how this is done, though some are. Meta-ethnography is one of the most explicit: it describes the act of 'translation' where terms and concepts which have resonance with one another are subsumed into 'higher order constructs'. Grounded theory, as represented by Eaves [ 17 ], is undertaken according to a long list of steps and sub-steps, includes the production of generalizations about concepts/categories, which comes from classifying these categories. In meta-narrative synthesis, comparable studies are grouped together at the appraisal phase of review.

Perhaps more interesting are the ways in which differences between studies are explored. Those methods with a greater emphasis on critical appraisal may tend (although this is not always made explicit) to use differences in method to explain differences in finding. Meta-ethnography proposes 'refutational synthesis' to explain differences, although there are few examples of this in the literature. Some synthesis methods – for example, thematic synthesis – look at other characteristics of the studies under review, whether types of participants and their context vary, and whether this can explain differences in perspective.

All of these methods, then, look within the studies to explain differences. Other methods look beyond the study itself to the context in which it was produced. Critical interpretive synthesis and meta-study look at differences in theory or in socio-economic context. Critical interpretive synthesis, like meta-narrative, also explores epistemological orientation. Meta-narrative is unique in concerning itself with disciplinary paradigm (i.e. the story of the discipline as it progresses). It is also distinctive in that it treats conflicting findings as 'higher order data' [[ 32 ], p420], so that the main emphasis of the synthesis appears to be on examining and explaining contradictions in the literature.

Going 'beyond' the primary studies

Synthesis is sometimes defined as a process resulting in a product, a 'whole', which is more than the sum of its parts. However, the methods reviewed here vary in the extent to which they attempt to 'go beyond' the primary studies and transform the data. Some methods – textual narrative synthesis, ecological triangulation and framework synthesis – focus on describing and summarising their primary data (often in a highly structured and detailed way) and translating the studies into one another. Others – meta-ethnography, grounded theory, thematic synthesis, meta-study, meta-narrative and critical interpretive synthesis – seek to push beyond the original data to a fresh interpretation of the phenomena under review. A key feature of thematic synthesis is its clear differentiation between these two stages.

Different methods have different mechanisms for going beyond the primary studies, although some are more explicit than others about what these entail. Meta-ethnography proposes a 'Line of Argument' (LOA) synthesis in which an interpretation is constructed to both link and explain a set of parts. Critical interpretive synthesis based its synthesis methods on those of meta-ethnography, developing an LOA using what the authors term 'synthetic constructs' (akin to 'third order constructs' in meta-ethnography) to create a 'synthesising argument'. Dixon-Woods et al claim that this is an advance on Britten et al's methods, in that they reject the difference between first, second and third order constructs.

Meta-narrative, as outlined above, focuses on conflicting findings and constructs theories to explain these in terms of differing paradigms. Meta study derives questions from each of its three components to which it subjects the dataset and inductively generates a number of theoretical claims in relation to it. According to Eaves' model of grounded theory [ 17 ], mini-theories are integrated to produce an explanatory framework. In ecological triangulation, the 'axial' codes – or second level codes evolved from the initial deductive open codes – are used to produce Banning's 'ecological sentence' [ 39 ].

The synthetic product

In overviewing and comparing different qualitative synthesis methods, the ultimate question relates to the utility of the synthetic product: what is it for? It is clear that some methods of synthesis – namely, thematic synthesis, textual narrative synthesis, framework synthesis and ecological triangulation – view themselves as producing an output that is directly applicable to policy makers and designers of interventions. The example of framework synthesis examined here (on children's, young people's and parents' views of walking and cycling) involved policy makers and practitioners in directing the focus of the synthesis and used the themes derived from the synthesis to infer what kind of interventions might be most effective in encouraging walking and cycling. Likewise, the products of the thematic synthesis took the form of practical recommendations for interventions (e.g. 'do not promote fruit and vegetables in the same way in the same intervention'). The extent to which policy makers and practitioners are involved in informing either synthesis or recommendation is less clear from the documents published on ecological triangulation, but the aim certainly is to directly inform practice.

The outputs of synthesis methods which have a more constructivist orientation – meta-study, meta-narrative, meta-ethnography, grounded theory, critical interpretive synthesis – tend to look rather different. They are generally more complex and conceptual, sometimes operating on the symbolic or metaphorical level, and requiring a further process of interpretation by policy makers and practitioners in order for them to inform practice. This is not to say, however, that they are not useful for practice, more that they are doing different work. However, it may be that, in the absence of further interpretation, they are more useful for informing other researchers and theoreticians.

Looking across dimensions

After examining the dimensions of difference of our included methods, what picture ultimately emerges? It seems clear that, while similar in some respects, there are genuine differences in approach to the synthesis of what is essentially textual data. To some extent, these differences can be explained by the epistemological assumptions that underpin each method. Our methods split into two broad camps: the idealist and the realist (see Table 1 for a summary). Idealist approaches generally tend to have a more iterative approach to searching (and the review process), have less a priori quality assessment procedures and are more inclined to problematize the literature. Realist approaches are characterised by a more linear approach to searching and review, have clearer and more well-developed approaches to quality assessment, and do not problematize the literature.

Mapping the relationships between methods

What is interesting is the relationship between these methods of synthesis, the conceptual links between them, and the extent to which the originators cite – or, in some cases, don't cite – one another. Some methods directly build on others – framework synthesis builds on framework analysis, for example, while grounded theory and constant comparative analysis build on grounded theory. Others further develop existing methods – meta-study, critical interpretive synthesis and meta-narrative all adapt aspects of meta-ethnography, while also importing concepts from other theorists (critical interpretive synthesis also adapts grounded theory techniques).

Some methods share a clear conceptual link, without directly citing one another: for example, the analytical themes developed during thematic synthesis are comparable to the third order interpretations of meta-ethnography. The meta-theory aspect of meta-study is echoed in both meta-narrative synthesis and critical interpretive synthesis (see 'Problematizing the literature, above); however, the originators of critical interpretive synthesis only refer to the originators of meta-study in relation to their use of sampling techniques.

While methods for qualitative synthesis have many similarities, there are clear differences in approach between them, many of which can be explained by taking account of a given method's epistemology.

However, within the two broad idealist/realist categories, any differences between methods in terms of outputs appear to be small.

Since many systematic reviews are designed to inform policy and practice, it is important to select a method – or type of method – that will produce the kind of conclusions needed. However, it is acknowledged that this is not always simple or even possible to achieve in practice.

The approaches that result in more easily translatable messages for policy-makers and practitioners may appear to be more attractive than the others; but we do need to take account lessons from the more idealist end of the spectrum, that some perspectives are not universal.

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The authors would like to acknowledge the helpful contributions of the following in commenting on earlier drafts of this paper: David Gough, Sandy Oliver, Angela Harden, Mary Dixon-Woods, Trisha Greenhalgh and Barbara L. Paterson. We would also like to thank the peer reviewers: Helen J Smith, Rosaline Barbour and Mark Rodgers for their helpful reviews. The methodological development was supported by the Department of Health (England) and the ESRC through the Methods for Research Synthesis Node of the National Centre for Research Methods (NCRM). An earlier draft of this paper currently appears as a working paper on the National Centre for Research Methods' website http://www.ncrm.ac.uk/ .

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Meta-Analysis and Meta-Synthesis Methodologies: Rigorously Piecing Together Research

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• Published: 18 June 2018
• Volume 62 , pages 525–534, ( 2018 )

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For a variety of reasons, education research can be difficult to summarize. Varying contexts, designs, levels of quality, measurement challenges, definition of underlying constructs, and treatments as well as the complexity of research subjects themselves can result in variability. Education research is voluminous and draws on multiple methods including quantitative, as well as, qualitative approaches to answer key research questions. With increased numbers of empirical research in Instructional Design and Technology (IDT), using various synthesis methods can provide a means to more deeply understand trends and patterns in research findings across multiple studies. The purpose of this article is to illustrate structured review or meta-synthesis procedures for qualitative research, as well as, novel meta-analysis procedures for the kinds of multiple treatment designs common to IDT settings. Sample analyses are used to discuss key methodological ideas as a way to introduce researchers to these techniques.

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Leary, H., Walker, A. Meta-Analysis and Meta-Synthesis Methodologies: Rigorously Piecing Together Research. TechTrends 62 , 525–534 (2018). https://doi.org/10.1007/s11528-018-0312-7

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Synthesising quantitative and qualitative evidence to inform guidelines on complex interventions: clarifying the purposes, designs and outlining some methods

1 School of Social Sciences, Bangor University, Wales, UK

Andrew Booth

2 School of Health and Related Research (ScHARR), University of Sheffield, Sheffield, UK

Graham Moore

3 School of Social Sciences, Cardiff University, Wales, UK

Kate Flemming

4 Department of Health Sciences, The University of York, York, UK

Özge Tunçalp

5 Department of Reproductive Health and Research including UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP), World Health Organization, Geneva, Switzerland

Elham Shakibazadeh

6 Department of Health Education and Promotion, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Associated Data

bmjgh-2018-000893supp001.pdf

bmjgh-2018-000893supp002.pdf

bmjgh-2018-000893supp003.pdf

bmjgh-2018-000893supp005.pdf

bmjgh-2018-000893supp004.pdf

Guideline developers are increasingly dealing with more difficult decisions concerning whether to recommend complex interventions in complex and highly variable health systems. There is greater recognition that both quantitative and qualitative evidence can be combined in a mixed-method synthesis and that this can be helpful in understanding how complexity impacts on interventions in specific contexts. This paper aims to clarify the different purposes, review designs, questions, synthesis methods and opportunities to combine quantitative and qualitative evidence to explore the complexity of complex interventions and health systems. Three case studies of guidelines developed by WHO, which incorporated quantitative and qualitative evidence, are used to illustrate possible uses of mixed-method reviews and evidence. Additional examples of methods that can be used or may have potential for use in a guideline process are outlined. Consideration is given to the opportunities for potential integration of quantitative and qualitative evidence at different stages of the review and guideline process. Encouragement is given to guideline commissioners and developers and review authors to consider including quantitative and qualitative evidence. Recommendations are made concerning the future development of methods to better address questions in systematic reviews and guidelines that adopt a complexity perspective.

Summary box

• When combined in a mixed-method synthesis, quantitative and qualitative evidence can potentially contribute to understanding how complex interventions work and for whom, and how the complex health systems into which they are implemented respond and adapt.
• The different purposes and designs for combining quantitative and qualitative evidence in a mixed-method synthesis for a guideline process are described.
• Questions relevant to gaining an understanding of the complexity of complex interventions and the wider health systems within which they are implemented that can be addressed by mixed-method syntheses are presented.
• The practical methodological guidance in this paper is intended to help guideline producers and review authors commission and conduct mixed-method syntheses where appropriate.
• If more mixed-method syntheses are conducted, guideline developers will have greater opportunities to access this evidence to inform decision-making.

Introduction

Recognition has grown that while quantitative methods remain vital, they are usually insufficient to address complex health systems related research questions. 1 Quantitative methods rely on an ability to anticipate what must be measured in advance. Introducing change into a complex health system gives rise to emergent reactions, which cannot be fully predicted in advance. Emergent reactions can often only be understood through combining quantitative methods with a more flexible qualitative lens. 2 Adopting a more pluralist position enables a diverse range of research options to the researcher depending on the research question being investigated. 3–5 As a consequence, where a research study sits within the multitude of methods available is driven by the question being asked, rather than any particular methodological or philosophical stance. 6

Publication of guidance on designing complex intervention process evaluations and other works advocating mixed-methods approaches to intervention research have stimulated better quality evidence for synthesis. 1 7–13 Methods for synthesising qualitative 14 and mixed-method evidence have been developed or are in development. Mixed-method research and review definitions are outlined in box 1 .

Defining mixed-method research and reviews

Pluye and Hong 52 define mixed-methods research as “a research approach in which a researcher integrates (a) qualitative and quantitative research questions, (b) qualitative research methods* and quantitative research designs, (c) techniques for collecting and analyzing qualitative and quantitative evidence, and (d) qualitative findings and quantitative results”.A mixed-method synthesis can integrate quantitative, qualitative and mixed-method evidence or data from primary studies.† Mixed-method primary studies are usually disaggregated into quantitative and qualitative evidence and data for the purposes of synthesis. Thomas and Harden further define three ways in which reviews are mixed. 53

• The types of studies included and hence the type of findings to be synthesised (ie, qualitative/textual and quantitative/numerical).
• The types of synthesis method used (eg, statistical meta-analysis and qualitative synthesis).
• The mode of analysis: theory testing AND theory building.

*A qualitative study is one that uses qualitative methods of data collection and analysis to produce a narrative understanding of the phenomena of interest. Qualitative methods of data collection may include, for example, interviews, focus groups, observations and analysis of documents.

†The Cochrane Qualitative and Implementation Methods group coined the term ‘qualitative evidence synthesis’ to mean that the synthesis could also include qualitative data. For example, qualitative data from case studies, grey literature reports and open-ended questions from surveys. ‘Evidence’ and ‘data’ are used interchangeably in this paper.

This paper is one of a series that aims to explore the implications of complexity for systematic reviews and guideline development, commissioned by WHO. This paper is concerned with the methodological implications of including quantitative and qualitative evidence in mixed-method systematic reviews and guideline development for complex interventions. The guidance was developed through a process of bringing together experts in the field, literature searching and consensus building with end users (guideline developers, clinicians and reviewers). We clarify the different purposes, review designs, questions and synthesis methods that may be applicable to combine quantitative and qualitative evidence to explore the complexity of complex interventions and health systems. Three case studies of WHO guidelines that incorporated quantitative and qualitative evidence are used to illustrate possible uses of mixed-method reviews and mechanisms of integration ( table 1 , online supplementary files 1–3 ). Additional examples of methods that can be used or may have potential for use in a guideline process are outlined. Opportunities for potential integration of quantitative and qualitative evidence at different stages of the review and guideline process are presented. Specific considerations when using an evidence to decision framework such as the Developing and Evaluating Communication strategies to support Informed Decisions and practice based on Evidence (DECIDE) framework 15 or the new WHO-INTEGRATE evidence to decision framework 16 at the review design and evidence to decision stage are outlined. See online supplementary file 4 for an example of a health systems DECIDE framework and Rehfuess et al 16 for the new WHO-INTEGRATE framework. Encouragement is given to guideline commissioners and developers and review authors to consider including quantitative and qualitative evidence in guidelines of complex interventions that take a complexity perspective and health systems focus.

Designs and methods and their use or applicability in guidelines and systematic reviews taking a complexity perspective

Supplementary data

Taking a complexity perspective.

The first paper in this series 17 outlines aspects of complexity associated with complex interventions and health systems that can potentially be explored by different types of evidence, including synthesis of quantitative and qualitative evidence. Petticrew et al 17 distinguish between a complex interventions perspective and a complex systems perspective. A complex interventions perspective defines interventions as having “implicit conceptual boundaries, representing a flexible, but common set of practices, often linked by an explicit or implicit theory about how they work”. A complex systems perspective differs in that “ complexity arises from the relationships and interactions between a system’s agents (eg, people, or groups that interact with each other and their environment), and its context. A system perspective conceives the intervention as being part of the system, and emphasises changes and interconnections within the system itself”. Aspects of complexity associated with implementation of complex interventions in health systems that could potentially be addressed with a synthesis of quantitative and qualitative evidence are summarised in table 2 . Another paper in the series outlines criteria used in a new evidence to decision framework for making decisions about complex interventions implemented in complex systems, against which the need for quantitative and qualitative evidence can be mapped. 16 A further paper 18 that explores how context is dealt with in guidelines and reviews taking a complexity perspective also recommends using both quantitative and qualitative evidence to better understand context as a source of complexity. Mixed-method syntheses of quantitative and qualitative evidence can also help with understanding of whether there has been theory failure and or implementation failure. The Cochrane Qualitative and Implementation Methods Group provide additional guidance on exploring implementation and theory failure that can be adapted to address aspects of complexity of complex interventions when implemented in health systems. 19

Health-system complexity-related questions that a synthesis of quantitative and qualitative evidence could address (derived from Petticrew et al 17 )

It may not be apparent which aspects of complexity or which elements of the complex intervention or health system can be explored in a guideline process, or whether combining qualitative and quantitative evidence in a mixed-method synthesis will be useful, until the available evidence is scoped and mapped. 17 20 A more extensive lead in phase is typically required to scope the available evidence, engage with stakeholders and to refine the review parameters and questions that can then be mapped against potential review designs and methods of synthesis. 20 At the scoping stage, it is also common to decide on a theoretical perspective 21 or undertake further work to refine a theoretical perspective. 22 This is also the stage to begin articulating the programme theory of the complex intervention that may be further developed to refine an understanding of complexity and show how the intervention is implemented in and impacts on the wider health system. 17 23 24 In practice, this process can be lengthy, iterative and fluid with multiple revisions to the review scope, often developing and adapting a logic model 17 as the available evidence becomes known and the potential to incorporate different types of review designs and syntheses of quantitative and qualitative evidence becomes better understood. 25 Further questions, propositions or hypotheses may emerge as the reviews progress and therefore the protocols generally need to be developed iteratively over time rather than a priori.

Following a scoping exercise and definition of key questions, the next step in the guideline development process is to identify existing or commission new systematic reviews to locate and summarise the best available evidence in relation to each question. For example, case study 2, ‘Optimising health worker roles for maternal and newborn health through task shifting’, included quantitative reviews that did and did not take an additional complexity perspective, and qualitative evidence syntheses that were able to explain how specific elements of complexity impacted on intervention outcomes within the wider health system. Further understanding of health system complexity was facilitated through the conduct of additional country-level case studies that contributed to an overall understanding of what worked and what happened when lay health worker interventions were implemented. See table 1 online supplementary file 2 .

There are a few existing examples, which we draw on in this paper, but integrating quantitative and qualitative evidence in a mixed-method synthesis is relatively uncommon in a guideline process. Box 2 includes a set of key questions that guideline developers and review authors contemplating combining quantitative and qualitative evidence in mixed-methods design might ask. Subsequent sections provide more information and signposting to further reading to help address these key questions.

Key questions that guideline developers and review authors contemplating combining quantitative and qualitative evidence in a mixed-methods design might ask

Compound questions requiring both quantitative and qualitative evidence?

Questions requiring mixed-methods studies?

Separate quantitative and qualitative questions?

Separate quantitative and qualitative research studies?

Related quantitative and qualitative research studies?

Mixed-methods studies?

Quantitative unpublished data and/or qualitative unpublished data, eg, narrative survey data?

Throughout the review?

Following separate reviews?

At the question point?

At the synthesis point?

At the evidence to recommendations stage?

Or a combination?

Narrative synthesis or summary?

Quantitising approach, eg, frequency analysis?

Qualitising approach, eg, thematic synthesis?

Tabulation?

Logic model?

Conceptual model/framework?

Graphical approach?

• WHICH: Which mixed-method designs, methodologies and methods best fit into a guideline process to inform recommendations?

Complexity-related questions that a synthesis of quantitative and qualitative evidence can potentially address

Petticrew et al 17 define the different aspects of complexity and examples of complexity-related questions that can potentially be explored in guidelines and systematic reviews taking a complexity perspective. Relevant aspects of complexity outlined by Petticrew et al 17 are summarised in table 2 below, together with the corresponding questions that could be addressed in a synthesis combining qualitative and quantitative evidence. Importantly, the aspects of complexity and their associated concepts of interest have however yet to be translated fully in primary health research or systematic reviews. There are few known examples where selected complexity concepts have been used to analyse or reanalyse a primary intervention study. Most notable is Chandler et al 26 who specifically set out to identify and translate a set of relevant complexity theory concepts for application in health systems research. Chandler then reanalysed a trial process evaluation using selected complexity theory concepts to better understand the complex causal pathway in the health system that explains some aspects of complexity in table 2 .

Rehfeuss et al 16 also recommends upfront consideration of the WHO-INTEGRATE evidence to decision criteria when planning a guideline and formulating questions. The criteria reflect WHO norms and values and take account of a complexity perspective. The framework can be used by guideline development groups as a menu to decide which criteria to prioritise, and which study types and synthesis methods can be used to collect evidence for each criterion. Many of the criteria and their related questions can be addressed using a synthesis of quantitative and qualitative evidence: the balance of benefits and harms, human rights and sociocultural acceptability, health equity, societal implications and feasibility (see table 3 ). Similar aspects in the DECIDE framework 15 could also be addressed using synthesis of qualitative and quantitative evidence.

Integrate evidence to decision framework criteria, example questions and types of studies to potentially address these questions (derived from Rehfeuss et al 16 )

GIS, Geographical Information System; RCT, randomised controlled trial.

Questions as anchors or compasses

Questions can serve as an ‘anchor’ by articulating the specific aspects of complexity to be explored (eg, Is successful implementation of the intervention context dependent?). 27 Anchor questions such as “How does intervention x impact on socioeconomic inequalities in health behaviour/outcome x” are the kind of health system question that requires a synthesis of both quantitative and qualitative evidence and hence a mixed-method synthesis. Quantitative evidence can quantify the difference in effect, but does not answer the question of how . The ‘how’ question can be partly answered with quantitative and qualitative evidence. For example, quantitative evidence may reveal where socioeconomic status and inequality emerges in the health system (an emergent property) by exploring questions such as “ Does patterning emerge during uptake because fewer people from certain groups come into contact with an intervention in the first place? ” or “ are people from certain backgrounds more likely to drop out, or to maintain effects beyond an intervention differently? ” Qualitative evidence may help understand the reasons behind all of these mechanisms. Alternatively, questions can act as ‘compasses’ where a question sets out a starting point from which to explore further and to potentially ask further questions or develop propositions or hypotheses to explore through a complexity perspective (eg, What factors enhance or hinder implementation?). 27 Other papers in this series provide further guidance on developing questions for qualitative evidence syntheses and guidance on question formulation. 14 28

For anchor and compass questions, additional application of a theory (eg, complexity theory) can help focus evidence synthesis and presentation to explore and explain complexity issues. 17 21 Development of a review specific logic model(s) can help to further refine an initial understanding of any complexity-related issues of interest associated with a specific intervention, and if appropriate the health system or section of the health system within which to contextualise the review question and analyse data. 17 23–25 Specific tools are available to help clarify context and complex interventions. 17 18

If a complexity perspective, and certain criteria within evidence to decision frameworks, is deemed relevant and desirable by guideline developers, it is only possible to pursue a complexity perspective if the evidence is available. Careful scoping using knowledge maps or scoping reviews will help inform development of questions that are answerable with available evidence. 20 If evidence of effect is not available, then a different approach to develop questions leading to a more general narrative understanding of what happened when complex interventions were implemented in a health system will be required (such as in case study 3—risk communication guideline). This should not mean that the original questions developed for which no evidence was found when scoping the literature were not important. An important function of creating a knowledge map is also to identify gaps to inform a future research agenda.

Table 2 and online supplementary files 1–3 outline examples of questions in the three case studies, which were all ‘COMPASS’ questions for the qualitative evidence syntheses.

Types of integration and synthesis designs in mixed-method reviews

The shift towards integration of qualitative and quantitative evidence in primary research has, in recent years, begun to be mirrored within research synthesis. 29–31 The natural extension to undertaking quantitative or qualitative reviews has been the development of methods for integrating qualitative and quantitative evidence within reviews, and within the guideline process using evidence to decision-frameworks. Advocating the integration of quantitative and qualitative evidence assumes a complementarity between research methodologies, and a need for both types of evidence to inform policy and practice. Below, we briefly outline the current designs for integrating qualitative and quantitative evidence within a mixed-method review or synthesis.

One of the early approaches to integrating qualitative and quantitative evidence detailed by Sandelowski et al 32 advocated three basic review designs: segregated, integrated and contingent designs, which have been further developed by Heyvaert et al 33 ( box 3 ).

Segregated, integrated and contingent designs 32 33

Segregated design.

Conventional separate distinction between quantitative and qualitative approaches based on the assumption they are different entities and should be treated separately; can be distinguished from each other; their findings warrant separate analyses and syntheses. Ultimately, the separate synthesis results can themselves be synthesised.

Integrated design

The methodological differences between qualitative and quantitative studies are minimised as both are viewed as producing findings that can be readily synthesised into one another because they address the same research purposed and questions. Transformation involves either turning qualitative data into quantitative (quantitising) or quantitative findings are turned into qualitative (qualitising) to facilitate their integration.

Contingent design

Takes a cyclical approach to synthesis, with the findings from one synthesis informing the focus of the next synthesis, until all the research objectives have been addressed. Studies are not necessarily grouped and categorised as qualitative or quantitative.

A recent review of more than 400 systematic reviews 34 combining quantitative and qualitative evidence identified two main synthesis designs—convergent and sequential. In a convergent design, qualitative and quantitative evidence is collated and analysed in a parallel or complementary manner, whereas in a sequential synthesis, the collation and analysis of quantitative and qualitative evidence takes place in a sequence with one synthesis informing the other ( box 4 ). 6 These designs can be seen to build on the work of Sandelowski et al , 32 35 particularly in relation to the transformation of data from qualitative to quantitative (and vice versa) and the sequential synthesis design, with a cyclical approach to reviewing that evokes Sandelowski’s contingent design.

Convergent and sequential synthesis designs 34

Convergent synthesis design.

Qualitative and quantitative research is collected and analysed at the same time in a parallel or complementary manner. Integration can occur at three points:

a. Data-based convergent synthesis design

All included studies are analysed using the same methods and results presented together. As only one synthesis method is used, data transformation occurs (qualitised or quantised). Usually addressed one review question.

b. Results-based convergent synthesis design

Qualitative and quantitative data are analysed and presented separately but integrated using a further synthesis method; eg, narratively, tables, matrices or reanalysing evidence. The results of both syntheses are combined in a third synthesis. Usually addresses an overall review question with subquestions.

c. Parallel-results convergent synthesis design

Qualitative and quantitative data are analysed and presented separately with integration occurring in the interpretation of results in the discussion section. Usually addresses two or more complimentary review questions.

Sequential synthesis design

A two-phase approach, data collection and analysis of one type of evidence (eg, qualitative), occurs after and is informed by the collection and analysis of the other type (eg, quantitative). Usually addresses an overall question with subquestions with both syntheses complementing each other.

The three case studies ( table 1 , online supplementary files 1–3 ) illustrate the diverse combination of review designs and synthesis methods that were considered the most appropriate for specific guidelines.

Methods for conducting mixed-method reviews in the context of guidelines for complex interventions

In this section, we draw on examples where specific review designs and methods have been or can be used to explore selected aspects of complexity in guidelines or systematic reviews. We also identify other review methods that could potentially be used to explore aspects of complexity. Of particular note, we could not find any specific examples of systematic methods to synthesise highly diverse research designs as advocated by Petticrew et al 17 and summarised in tables 2 and 3 . For example, we could not find examples of methods to synthesise qualitative studies, case studies, quantitative longitudinal data, possibly historical data, effectiveness studies providing evidence of differential effects across different contexts, and system modelling studies (eg, agent-based modelling) to explore system adaptivity.

There are different ways that quantitative and qualitative evidence can be integrated into a review and then into a guideline development process. In practice, some methods enable integration of different types of evidence in a single synthesis, while in other methods, the single systematic review may include a series of stand-alone reviews or syntheses that are then combined in a cross-study synthesis. Table 1 provides an overview of the characteristics of different review designs and methods and guidance on their applicability for a guideline process. Designs and methods that have already been used in WHO guideline development are described in part A of the table. Part B outlines a design and method that can be used in a guideline process, and part C covers those that have the potential to integrate quantitative, qualitative and mixed-method evidence in a single review design (such as meta-narrative reviews and Bayesian syntheses), but their application in a guideline context has yet to be demonstrated.

Points of integration when integrating quantitative and qualitative evidence in guideline development

Depending on the review design (see boxes 3 and 4 ), integration can potentially take place at a review team and design level, and more commonly at several key points of the review or guideline process. The following sections outline potential points of integration and associated practical considerations when integrating quantitative and qualitative evidence in guideline development.

Review team level

In a guideline process, it is common for syntheses of quantitative and qualitative evidence to be done separately by different teams and then to integrate the evidence. A practical consideration relates to the organisation, composition and expertise of the review teams and ways of working. If the quantitative and qualitative reviews are being conducted separately and then brought together by the same team members, who are equally comfortable operating within both paradigms, then a consistent approach across both paradigms becomes possible. If, however, a team is being split between the quantitative and qualitative reviews, then the strengths of specialisation can be harnessed, for example, in quality assessment or synthesis. Optimally, at least one, if not more, of the team members should be involved in both quantitative and qualitative reviews to offer the possibility of making connexions throughout the review and not simply at re-agreed junctures. This mirrors O’Cathain’s conclusion that mixed-methods primary research tends to work only when there is a principal investigator who values and is able to oversee integration. 9 10 While the above decisions have been articulated in the context of two types of evidence, variously quantitative and qualitative, they equally apply when considering how to handle studies reporting a mixed-method study design, where data are usually disaggregated into quantitative and qualitative for the purposes of synthesis (see case study 3—risk communication in humanitarian disasters).

Question formulation

Clearly specified key question(s), derived from a scoping or consultation exercise, will make it clear if quantitative and qualitative evidence is required in a guideline development process and which aspects will be addressed by which types of evidence. For the remaining stages of the process, as documented below, a review team faces challenges as to whether to handle each type of evidence separately, regardless of whether sequentially or in parallel, with a view to joining the two products on completion or to attempt integration throughout the review process. In each case, the underlying choice is of efficiencies and potential comparability vs sensitivity to the underlying paradigm.

Once key questions are clearly defined, the guideline development group typically needs to consider whether to conduct a single sensitive search to address all potential subtopics (lumping) or whether to conduct specific searches for each subtopic (splitting). 36 A related consideration is whether to search separately for qualitative, quantitative and mixed-method evidence ‘streams’ or whether to conduct a single search and then identify specific study types at the subsequent sifting stage. These two considerations often mean a trade-off between a single search process involving very large numbers of records or a more protracted search process retrieving smaller numbers of records. Both approaches have advantages and choice may depend on the respective availability of resources for searching and sifting.

Screening and selecting studies

Closely related to decisions around searching are considerations relating to screening and selecting studies for inclusion in a systematic review. An important consideration here is whether the review team will screen records for all review types, regardless of their subsequent involvement (‘altruistic sifting’), or specialise in screening for the study type with which they are most familiar. The risk of missing relevant reports might be minimised by whole team screening for empirical reports in the first instance and then coding them for a specific quantitative, qualitative or mixed-methods report at a subsequent stage.

Assessment of methodological limitations in primary studies

Within a guideline process, review teams may be more limited in their choice of instruments to assess methodological limitations of primary studies as there are mandatory requirements to use the Cochrane risk of bias tool 37 to feed into Grading of Recommendations Assessment, Development and Evaluation (GRADE) 38 or to select from a small pool of qualitative appraisal instruments in order to apply GRADE; Confidence in the Evidence from Reviews of Qualitative Research (GRADE-CERQual) 39 to assess the overall certainty or confidence in findings. The Cochrane Qualitative and Implementation Methods Group has recently issued guidance on the selection of appraisal instruments and core assessment criteria. 40 The Mixed-Methods Appraisal Tool, which is currently undergoing further development, offers a single quality assessment instrument for quantitative, qualitative and mixed-methods studies. 41 Other options include using corresponding instruments from within the same ‘stable’, for example, using different Critical Appraisal Skills Programme instruments. 42 While using instruments developed by the same team or organisation may achieve a degree of epistemological consonance, benefits may come more from consistency of approach and reporting rather than from a shared view of quality. Alternatively, a more paradigm-sensitive approach would involve selecting the best instrument for each respective review while deferring challenges from later heterogeneity of reporting.

Data extraction

The way in which data and evidence are extracted from primary research studies for review will be influenced by the type of integrated synthesis being undertaken and the review purpose. Initially, decisions need to be made regarding the nature and type of data and evidence that are to be extracted from the included studies. Method-specific reporting guidelines 43 44 provide a good template as to what quantitative and qualitative data it is potentially possible to extract from different types of method-specific study reports, although in practice reporting quality varies. Online supplementary file 5 provides a hypothetical example of the different types of studies from which quantitative and qualitative evidence could potentially be extracted for synthesis.

The decisions around what data or evidence to extract will be guided by how ‘integrated’ the mixed-method review will be. For those reviews where the quantitative and qualitative findings of studies are synthesised separately and integrated at the point of findings (eg, segregated or contingent approaches or sequential synthesis design), separate data extraction approaches will likely be used.

Where integration occurs during the process of the review (eg, integrated approach or convergent synthesis design), an integrated approach to data extraction may be considered, depending on the purpose of the review. This may involve the use of a data extraction framework, the choice of which needs to be congruent with the approach to synthesis chosen for the review. 40 45 The integrative or theoretical framework may be decided on a priori if a pre-developed theoretical or conceptual framework is available in the literature. 27 The development of a framework may alternatively arise from the reading of the included studies, in relation to the purpose of the review, early in the process. The Cochrane Qualitative and Implementation Methods Group provide further guidance on extraction of qualitative data, including use of software. 40

Synthesis and integration

Relatively few synthesis methods start off being integrated from the beginning, and these methods have generally been subject to less testing and evaluation particularly in a guideline context (see table 1 ). A review design that started off being integrated from the beginning may be suitable for some guideline contexts (such as in case study 3—risk communication in humanitarian disasters—where there was little evidence of effect), but in general if there are sufficient trials then a separate systematic review and meta-analysis will be required for a guideline. Other papers in this series offer guidance on methods for synthesising quantitative 46 and qualitative evidence 14 in reviews that take a complexity perspective. Further guidance on integrating quantitative and qualitative evidence in a systematic review is provided by the Cochrane Qualitative and Implementation Methods Group. 19 27 29 40 47

Types of findings produced by specific methods

It is highly likely (unless there are well-designed process evaluations) that the primary studies may not themselves seek to address the complexity-related questions required for a guideline process. In which case, review authors will need to configure the available evidence and transform the evidence through the synthesis process to produce explanations, propositions and hypotheses (ie, findings) that were not obvious at primary study level. It is important that guideline commissioners, developers and review authors are aware that specific methods are intended to produce a type of finding with a specific purpose (such as developing new theory in the case of meta-ethnography). 48 Case study 1 (antenatal care guideline) provides an example of how a meta-ethnography was used to develop a new theory as an end product, 48 49 as well as framework synthesis which produced descriptive and explanatory findings that were more easily incorporated into the guideline process. 27 The definitions ( box 5 ) may be helpful when defining the different types of findings.

Different levels of findings

Descriptive findings —qualitative evidence-driven translated descriptive themes that do not move beyond the primary studies.

Explanatory findings —may either be at a descriptive or theoretical level. At the descriptive level, qualitative evidence is used to explain phenomena observed in quantitative results, such as why implementation failed in specific circumstances. At the theoretical level, the transformed and interpreted findings that go beyond the primary studies can be used to explain the descriptive findings. The latter description is generally the accepted definition in the wider qualitative community.

Hypothetical or theoretical finding —qualitative evidence-driven transformed themes (or lines of argument) that go beyond the primary studies. Although similar, Thomas and Harden 56 make a distinction in the purposes between two types of theoretical findings: analytical themes and the product of meta-ethnographies, third-order interpretations. 48

Analytical themes are a product of interrogating descriptive themes by placing the synthesis within an external theoretical framework (such as the review question and subquestions) and are considered more appropriate when a specific review question is being addressed (eg, in a guideline or to inform policy). 56

Third-order interpretations come from translating studies into one another while preserving the original context and are more appropriate when a body of literature is being explored in and of itself with broader or emergent review questions. 48

Bringing mixed-method evidence together in evidence to decision (EtD) frameworks

A critical element of guideline development is the formulation of recommendations by the Guideline Development Group, and EtD frameworks help to facilitate this process. 16 The EtD framework can also be used as a mechanism to integrate and display quantitative and qualitative evidence and findings mapped against the EtD framework domains with hyperlinks to more detailed evidence summaries from contributing reviews (see table 1 ). It is commonly the EtD framework that enables the findings of the separate quantitative and qualitative reviews to be brought together in a guideline process. Specific challenges when populating the DECIDE evidence to decision framework 15 were noted in case study 3 (risk communication in humanitarian disasters) as there was an absence of intervention effect data and the interventions to communicate public health risks were context specific and varied. These problems would not, however, have been addressed by substitution of the DECIDE framework with the new INTEGRATE 16 evidence to decision framework. A d ifferent type of EtD framework needs to be developed for reviews that do not include sufficient evidence of intervention effect.

Mixed-method review and synthesis methods are generally the least developed of all systematic review methods. It is acknowledged that methods for combining quantitative and qualitative evidence are generally poorly articulated. 29 50 There are however some fairly well-established methods for using qualitative evidence to explore aspects of complexity (such as contextual, implementation and outcome complexity), which can be combined with evidence of effect (see sections A and B of table 1 ). 14 There are good examples of systematic reviews that use these methods to combine quantitative and qualitative evidence, and examples of guideline recommendations that were informed by evidence from both quantitative and qualitative reviews (eg, case studies 1–3). With the exception of case study 3 (risk communication), the quantitative and qualitative reviews for these specific guidelines have been conducted separately, and the findings subsequently brought together in an EtD framework to inform recommendations.

Other mixed-method review designs have potential to contribute to understanding of complex interventions and to explore aspects of wider health systems complexity but have not been sufficiently developed and tested for this specific purpose, or used in a guideline process (section C of table 1 ). Some methods such as meta-narrative reviews also explore different questions to those usually asked in a guideline process. Methods for processing (eg, quality appraisal) and synthesising the highly diverse evidence suggested in tables 2 and 3 that are required to explore specific aspects of health systems complexity (such as system adaptivity) and to populate some sections of the INTEGRATE EtD framework remain underdeveloped or in need of development.

In addition to the required methodological development mentioned above, there is no GRADE approach 38 for assessing confidence in findings developed from combined quantitative and qualitative evidence. Another paper in this series outlines how to deal with complexity and grading different types of quantitative evidence, 51 and the GRADE CERQual approach for qualitative findings is described elsewhere, 39 but both these approaches are applied to method-specific and not mixed-method findings. An unofficial adaptation of GRADE was used in the risk communication guideline that reported mixed-method findings. Nor is there a reporting guideline for mixed-method reviews, 47 and for now reports will need to conform to the relevant reporting requirements of the respective method-specific guideline. There is a need to further adapt and test DECIDE, 15 WHO-INTEGRATE 16 and other types of evidence to decision frameworks to accommodate evidence from mixed-method syntheses which do not set out to determine the statistical effects of interventions and in circumstances where there are no trials.

When conducting quantitative and qualitative reviews that will subsequently be combined, there are specific considerations for managing and integrating the different types of evidence throughout the review process. We have summarised different options for combining qualitative and quantitative evidence in mixed-method syntheses that guideline developers and systematic reviewers can choose from, as well as outlining the opportunities to integrate evidence at different stages of the review and guideline development process.

Review commissioners, authors and guideline developers generally have less experience of combining qualitative and evidence in mixed-methods reviews. In particular, there is a relatively small group of reviewers who are skilled at undertaking fully integrated mixed-method reviews. Commissioning additional qualitative and mixed-method reviews creates an additional cost. Large complex mixed-method reviews generally take more time to complete. Careful consideration needs to be given as to which guidelines would benefit most from additional qualitative and mixed-method syntheses. More training is required to develop capacity and there is a need to develop processes for preparing the guideline panel to consider and use mixed-method evidence in their decision-making.

This paper has presented how qualitative and quantitative evidence, combined in mixed-method reviews, can help understand aspects of complex interventions and the systems within which they are implemented. There are further opportunities to use these methods, and to further develop the methods, to look more widely at additional aspects of complexity. There is a range of review designs and synthesis methods to choose from depending on the question being asked or the questions that may emerge during the conduct of the synthesis. Additional methods need to be developed (or existing methods further adapted) in order to synthesise the full range of diverse evidence that is desirable to explore the complexity-related questions when complex interventions are implemented into health systems. We encourage review commissioners and authors, and guideline developers to consider using mixed-methods reviews and synthesis in guidelines and to report on their usefulness in the guideline development process.

Handling editor: Soumyadeep Bhaumik

Contributors: JN, AB, GM, KF, ÖT and ES drafted the manuscript. All authors contributed to paper development and writing and agreed the final manuscript. Anayda Portela and Susan Norris from WHO managed the series. Helen Smith was series Editor. We thank all those who provided feedback on various iterations.

Funding: Funding provided by the World Health Organization Department of Maternal, Newborn, Child and Adolescent Health through grants received from the United States Agency for International Development and the Norwegian Agency for Development Cooperation.

Disclaimer: ÖT is a staff member of WHO. The author alone is responsible for the views expressed in this publication and they do not necessarily represent the decisions or policies of WHO.

Competing interests: No financial interests declared. JN, AB and ÖT have an intellectual interest in GRADE CERQual; and JN has an intellectual interest in the iCAT_SR tool.

Patient consent: Not required.

Provenance and peer review: Not commissioned; externally peer reviewed.

Data sharing statement: No additional data are available.

Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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• Synthesizing Sources | Examples & Synthesis Matrix

Synthesizing Sources | Examples & Synthesis Matrix

Published on July 4, 2022 by Eoghan Ryan . Revised on May 31, 2023.

Synthesizing sources involves combining the work of other scholars to provide new insights. It’s a way of integrating sources that helps situate your work in relation to existing research.

Synthesizing sources involves more than just summarizing . You must emphasize how each source contributes to current debates, highlighting points of (dis)agreement and putting the sources in conversation with each other.

You might synthesize sources in your literature review to give an overview of the field or throughout your research paper when you want to position your work in relation to existing research.

Table of contents

Example of synthesizing sources, how to synthesize sources, synthesis matrix, other interesting articles, frequently asked questions about synthesizing sources.

Let’s take a look at an example where sources are not properly synthesized, and then see what can be done to improve it.

This paragraph provides no context for the information and does not explain the relationships between the sources described. It also doesn’t analyze the sources or consider gaps in existing research.

Research on the barriers to second language acquisition has primarily focused on age-related difficulties. Building on Lenneberg’s (1967) theory of a critical period of language acquisition, Johnson and Newport (1988) tested Lenneberg’s idea in the context of second language acquisition. Their research seemed to confirm that young learners acquire a second language more easily than older learners. Recent research has considered other potential barriers to language acquisition. Schepens, van Hout, and van der Slik (2022) have revealed that the difficulties of learning a second language at an older age are compounded by dissimilarity between a learner’s first language and the language they aim to acquire. Further research needs to be carried out to determine whether the difficulty faced by adult monoglot speakers is also faced by adults who acquired a second language during the “critical period.”

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To synthesize sources, group them around a specific theme or point of contention.

As you read sources, ask:

• What questions or ideas recur? Do the sources focus on the same points, or do they look at the issue from different angles?
• How does each source relate to others? Does it confirm or challenge the findings of past research?
• Where do the sources agree or disagree?

Once you have a clear idea of how each source positions itself, put them in conversation with each other. Analyze and interpret their points of agreement and disagreement. This displays the relationships among sources and creates a sense of coherence.

Consider both implicit and explicit (dis)agreements. Whether one source specifically refutes another or just happens to come to different conclusions without specifically engaging with it, you can mention it in your synthesis either way.

Synthesize your sources using:

• Topic sentences to introduce the relationship between the sources
• Signal phrases to attribute ideas to their authors
• Transition words and phrases to link together different ideas

To more easily determine the similarities and dissimilarities among your sources, you can create a visual representation of their main ideas with a synthesis matrix . This is a tool that you can use when researching and writing your paper, not a part of the final text.

In a synthesis matrix, each column represents one source, and each row represents a common theme or idea among the sources. In the relevant rows, fill in a short summary of how the source treats each theme or topic.

This helps you to clearly see the commonalities or points of divergence among your sources. You can then synthesize these sources in your work by explaining their relationship.

If you want to know more about ChatGPT, AI tools , citation , and plagiarism , make sure to check out some of our other articles with explanations and examples.

• ChatGPT vs human editor
• ChatGPT citations
• Is ChatGPT trustworthy?
• Using ChatGPT for your studies
• What is ChatGPT?
• Chicago style
• Paraphrasing

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• Types of plagiarism
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• Academic integrity
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Synthesizing sources means comparing and contrasting the work of other scholars to provide new insights.

It involves analyzing and interpreting the points of agreement and disagreement among sources.

You might synthesize sources in your literature review to give an overview of the field of research or throughout your paper when you want to contribute something new to existing research.

A literature review is a survey of scholarly sources (such as books, journal articles, and theses) related to a specific topic or research question .

It is often written as part of a thesis, dissertation , or research paper , in order to situate your work in relation to existing knowledge.

Topic sentences help keep your writing focused and guide the reader through your argument.

In an essay or paper , each paragraph should focus on a single idea. By stating the main idea in the topic sentence, you clarify what the paragraph is about for both yourself and your reader.

At college level, you must properly cite your sources in all essays , research papers , and other academic texts (except exams and in-class exercises).

Add a citation whenever you quote , paraphrase , or summarize information or ideas from a source. You should also give full source details in a bibliography or reference list at the end of your text.

The exact format of your citations depends on which citation style you are instructed to use. The most common styles are APA , MLA , and Chicago .

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Ryan, E. (2023, May 31). Synthesizing Sources | Examples & Synthesis Matrix. Scribbr. Retrieved August 12, 2024, from https://www.scribbr.com/working-with-sources/synthesizing-sources/

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Wound-healing properties of Stryphnodendron adstringens (barbatimão) in skin and mucosa injuries: a scoping review protocol

Costa Drigo, Rayane Teresa da Silva 1 ; Becker, Adriana Caroci 1,2 ; Riesco, Maria Luiza Gonzalez 1,3 ; Mascarenhas, Victor Hugo Alves 1 ; Nick, Jan M. 4,5

1 School of Nursing, University of São Paulo, São Paulo, Brazil

2 School of Arts, Sciences and Humanities, University of São Paulo, São Paulo, Brazil

3 The Brazilian Centre for Evidence-based Healthcare: A JBI Centre of Excellence, São Paulo, Brazil

4 LLUH Center for Evidence Synthesis: A JBI Affiliated Group, Loma Linda, CA, USA

5 School of Nursing, Loma Linda University, Loma Linda, CA, USA

The authors declare no conflicts of interest.

Correspondence: Rayane Teresa da Silva Costa Drigo, [email protected]

Objective: 

This review will map the literature on the types of research and methods used to investigate the wound-healing properties of Stryphnodendron adstringens ( barbatimão ) in skin and mucosa injuries.

Introduction: 

Barbatimão is a Brazilian native plant and its wound-healing properties have been described in literature since the colonial period. It is one of the 71 plants included in the Brazilian health system’s national list of medicinal plants of interest. However, existing literature reviews on the subject are limited, not comprehensive, lack a search strategy, and lack peer review.

Inclusion criteria: 

This scoping review will include all types of published and unpublished sources that investigate the wound-healing properties of barbatimão to treat any type of skin or mucosa injury in humans, animals, or in vitro, in any context.

Methods: 

A scoping review will be conducted following JBI methodology. The main databases to be searched will include Embase (EBSCOhost), CINAHL (EBSCOhost), Scopus, PubMed (EBSCOhost), ScienceDirect, Lilacs, SciELO, CUIDEN, MOSAICO, Web of Science, Epistemonikos, and Google Scholar. Unpublished studies will also be considered. Two independent reviewers will examine titles and abstracts and select and read full-text sources for possible inclusion. Subsequently, the reviewers will extract and synthesize the data, which will be presented as a map, diagram, or table, according to the review objectives.

Review registration: 

Open Science Framework osf.io/w57m4

Introduction

S tryphnodendron adstringens , commonly known as barbatimão , is a tannin-rich Brazilian native plant found in the Cerrado and Caatinga phytogeographic areas, mainly in the midwest and southwest regions of the country. It is taxonomically classified in the Fabaceae family, genus Stryphnodendron Mart ., and its scientific name is Stryphnodendron adstringens (Mart.) Coville . 1 Its wound-healing properties have been described by several European naturalists since the Brazilian colonial period. In 1803, the Portuguese physician, Bernardino Antonio Gomes, highlighted the wound-healing properties of barbatimão when applied topically. 2 The naturalist Johann Emanuel Pohl and the botanist Frei Velozzo both extolled its virtues as an astringent curative treatment. 2–4 It is important to note that native plants were used for medicinal purposes by Indigenous peoples long before the Portuguese arrived in Brazil, and that this popular knowledge was passed down from generation to generation through unwritten traditions. The arrival of the Portuguese and other European peoples in Brazil only served to document this traditional use in written format. A recent literature review revealed that there have been 81 traditional uses of the plant’s bark, mainly as an astringent agent and in the treatment of injuries and wound-healing, over the last 500 years of Brazilian history. 3

Although its prevalence is not known, it is widely used in folk medicine in Brazil, especially by economically vulnerable groups and minorities, such as puerperal women undergoing perineal repair, adults without access to conventional health care, indigenous communities, and peoples of African descent. 5,6 Barbatimão is mainly used in topical preparations made from the bark of the stem in infusions, macerations, and decoctions. It is also sold in public markets and local fairs in some regions of the country, where it has an established market. In summary, its use is associated with groups that value folk and traditional medicine. 6–9

The importance of the plant in traditional medicine has been recognized not only nationally, but also globally. To provide guidelines for its purpose and use, barbatimão has been included in the Brazilian Pharmacopoeia and the National List of Medicinal Plants of Interest to the Unified Health System (Sistema Único de Saúd) . This list designates plants with potential therapeutic value that are considered important for public health and welfare. It is one of the 71 plant species traditionally used in the country, with high potential to guide scientific research studies and provide raw materials for phytotherapeutic medications. Recognizing the importance of ensuring safety, the Brazilian Ministry of Health and the World Health Organization (WHO) have established policies to ensure safe access and appropriate use of medicinal plants. 10

A preliminary search was conducted, which identified a large body of experimental literature. The studies demonstrate the wound-healing properties of barbatimão in vitro and in vivo, especially in the rodent population. For example, in one study conducted on Wistar rats, the group treated with barbatimão showed complete epithelialization 14 days after the start of treatment, while the control group, treated only with physiological solution, showed incomplete epithelialization over the same 14-day period ( P < 0.001). 11 In another in vivo study carried out on diabetic rats, the animals were divided into 4 groups corresponding to 4, 7, 10, and 14 days of use of barbatimão . The treatment proved successful in wound healing ( P < 0.05), even in the presence of comorbidities associated with the clinical condition. 12 Unfortunately, human studies are limited, with few case reports. Only one clinical study showed promising results in the treatment of decubitus pressure ulcers, with 70% of the injuries healed within 2 months and 100% of the injuries healed at the end of 6 months. 13–16 There is a notable absence of clinical trials. 5,17 Because barbatimão continues to be widely used by economically disadvantaged and minority groups, reflecting its association with folk and traditional medicine, and because few studies on the efficacy of this treatment have been conducted, a scoping review of the wound-healing properties of barbatimão is appropriate to identify research gaps, guide future investigations, and determine key policy points that will advance research agendas and develop safety aspects of barbatimão . 5–9

In order to identify the types of literature reviews produced on the topic, a second, more refined preliminary search was conducted in PROSPERO, MEDLINE, the Cochrane Database of Systematic Reviews, Open Science Framework, Campbell Collaboration, Carpha database, Epistemonikos, and JBI Evidence Synthesis , and no current or in-progress scoping or systematic reviews on the topic were identified. However, there were non-peer-reviewed studies reporting on historical records of traditional uses of the plant. These reviews did not specify a search strategy, nor did they include unpublished studies. 18 This review will differ from those studies because of its specific emphasis on Stryphnodendron adstringens and its relevance to human applications. The main objective is to encompass a broad spectrum of published and unpublished literature, setting it apart from prior reviews characterized by a lack of comprehensive search strategies and peer-review mechanisms. 16

In summary, this scoping review aims to map the entire literature on barbatimão’s wound-healing properties in skin and mucosal injuries, encompassing research conducted on humans, animals, in vitro, and across various contexts. Its justification lies in the need for a comprehensive understanding of the research methodologies employed and the types of evidence available. This review will be useful for patients and health professionals, and will be in line with the WHO and Brazil’s government agencies, such as the Ministry of Health and the Unified Health System.

Review questions

• What types of studies have been conducted on the wound-healing properties of Stryphnodendron adstringens ( barbatimão ) in skin and mucosa injuries in humans, animals, and in vitro?
• What dosage and formulation of Stryphnodendron adstringens ( barbatimão ) were used in the studies?
• What are the characteristics of the participants and contexts?

Inclusion criteria

Participants.

This review will consider studies that include any animal models, in vitro, along with research involving humans with any type of skin or mucosal lesion, with the use of Stryphnodendron adstringens ( barbatimão ). There will be no limits on comorbidities, age, skin color, race, or gender. It was decided to extend the review to include non-human participants due to the scarcity of human participant research on the topic. Broadening the inclusion criteria beyond humans will allow for a comprehensive analysis of the available evidence, identifying the types of studies, methodologies, and potential gaps that will serve as the basis for future research.

This review will consider sources that assess the wound-healing properties of Stryphnodendron adstringens in skin or mucosa injuries, used topically, in any dosage, duration, and formulation modality of the plant (including extracts, dyes, solutions, ointment, cooked bark, or any part of the plant). We will also consider sources that present the geographical locations where approaches using barbatimão have been developed, as well as the characteristics of the service/community that has used the plant for wound healing. We will also consider participant attributes, such as sex, age, gender, race, species, sample size, comorbidities, and the results observed in these groups.

This review will consider studies carried out in any context (hospital, laboratory, or community), culture, and geographical location.

Types of sources

This review will consider published and unpublished studies with experimental and quasi-experimental designs, including randomized controlled studies, non-randomized controlled studies, before-and-after studies, and interrupted time series studies. In addition, in vivo and in vitro studies will be considered. This review will also consider analytical observational studies, including prospective and retrospective cohort studies, case-control studies, and analytical cross-sectional studies. Descriptive observational study designs, such as case series, individual case reports, and cross-sectional descriptive studies, or other relevant study designs will be considered. Systematic reviews, ethnobotanical, qualitative studies, theses, dissertations, and clinical trial reports that meet the inclusion criteria will also be considered.

The review will be carried out according to the JBI methodology for scoping reviews 19 and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR). 20 The review protocol was registered in Open Science Framework ( osf.io/w57m4 ).

Search strategy

The objective of the search strategy will be to locate published and unpublished literature. A limited initial search of PubMed (EBSCOhost), CINAHL (EBSCOhost), and Scopus was carried out to identify articles on the topic. The keywords contained in the titles and abstracts of the relevant articles and the indexing terms used to describe the articles were used to develop a complete search strategy for PubMed (EBSCOhost; see Appendix I). The search strategy, including all the keywords and indexing terms identified, will be adapted for each database and/or information source. The reference lists of all included evidence sources will be searched for additional documents. There will be no date or language limitations on the articles. Regarding translations, some of the reviewers are native speakers of Portuguese and English; sources written in other languages will be translated using DeepL (DeepL, Cologne, Germany).

The databases to be searched will include Embase (EBSCOhost), CINAHL (EBSCOhost), Scopus, PubMed (EBSCOhost), ScienceDirect, LILACS, SciELO, CUIDEN, MOSAICO, Web of Science, Epistemonikos, and Google Scholar. The sources of unpublished literature to be researched will include clinical trial records such as ClinicalTrials.gov, International Clinical Trials Registry Platform (ICTRP), Brazilian Registry of Clinical Trials (Registro Brasileiro de Ensaios Clínicos , REBEC), as well as theses and dissertations (Brazilian Digital Library of Theses and Dissertations and CAPES Catalog of Theses and Dissertations). Congress annals will also be included. If a source cannot be retrieved, we will attempt to contact the authors of the article via email up to 2 times.

Study selection

After the search, all the identified records will be grouped and loaded to Mendeley Reference Manager v2.67.0 (Mendeley Ltd., Elsevier, Netherlands) and duplicates will be removed. Following a pilot test, 2 independent reviewers will assess the titles and abstracts against the inclusion criteria. Potentially relevant sources will be retrieved in full and their citation details imported into the JBI System for the Unified Management, Assessment and Review of Information (JBI SUMARI; JBI, Adelaide, Australia). 21 The full text of selected citations will be assessed in detail against the inclusion criteria by 2 independent reviewers. Reasons for exclusion of full-text papers that do not meet the inclusion criteria will be recorded and reported in the scoping review. Any disagreements that arise between the reviewers at each stage of the selection process will be resolved through discussion or with a third reviewer. The results of the search and inclusion process corresponding to the sources will be reported in full in the final scoping review and presented in a PRISMA flow diagram. 22

Data extraction

Data will be extracted from papers by 2 independent reviewers using a data extraction tool developed by the reviewers (see Appendix II). The extracted data will include specific details about the participants, concept, context, study characteristics, and main findings relevant to the review question, for example, species examined (eg, humans, mice, rabbits); injury site; intervention frequency or duration; dosage; barbatimão formulation method used; comparison groups; and gaps identified by the study. The data extraction tool will be tested by 2 independent reviewers on 3 randomly selected studies. The tool may be modified and revised as necessary during data extraction. Any modifications will be detailed in the final scoping review. Any disagreements between the reviewers will be resolved through discussion or with a third reviewer. If appropriate, the authors of the articles will be contacted twice to request missing or additional data.

Data analysis and presentation

The data will be presented as a map in diagrammatic or tabular format. A narrative summary will accompany the tabulated results and/or graphs. This narrative will serve as a critical component of the analysis, elucidating how the findings and patterns identified in the data align with the overarching review objective and research questions. It will also facilitate a deeper understanding of the context and implications of the data, thus contributing to broader comprehension of the field.

The results will be categorized and stratified by several relevant criteria. These categorizations will include, but not be limited to, study design, year of publication, context, species examined, specific injury site, frequency or duration of intervention, dosage, formulation modalities of the plant, comparison groups, and any additional conceptual categories that may arise during data extraction. This meticulous categorization will not only facilitate a structured and comprehensive synthesis, but will also make it possible to identify trends, gaps, and potential areas for future research.

RTSCD is supported by a grant from Coordination for the Improvement of Higher Education Personnel (CAPES). The funders had no input into the conduct of this review.

Author contributions

RTSCD conceived the project, performed the literature search, wrote the final version of the manuscript, approved the final version to be published, and agreed to be responsible for all aspects of the work. ACB, MLGR, and VHAM contributed to the design of the project, drafted the manuscript, critically reviewed the final version of the manuscript for intellectual content, approved the final version to be published, and agreed that RTSCD would be responsible for all aspects of the work. JMN contributed to the technical review of the reviewers’ suggestions and improvement of the language, especially with regard to the English language.

Appendix I: Search strategy

Pubmed (ebscohost).

Search conducted: September 11, 2023

Appendix II: Draft data extraction tool

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• Published: 14 August 2024

Experiences of intensive treatment for people with eating disorders: a systematic review and thematic synthesis

• Hannah Webb 1 ,
• Maria Griffiths 1 &
• Ulrike Schmidt 2 , 3  

Journal of Eating Disorders volume  12 , Article number:  115 ( 2024 ) Cite this article

Metrics details

Eating disorders are complex difficulties that impact the individual, their supporters and society. Increasing numbers are being admitted to intensive treatment settings (e.g., for inpatient treatment, day-patient treatment or acute medical treatment). The lived experience perspectives of what helps and hinders eating disorder recovery during intensive treatment is an emerging area of interest. This review aims to explore patients’ perspectives of what helps and hinders recovery in these contexts.

A systematic review was conducted to identify studies using qualitative methods to explore patients’ experiences of intensive treatment for an eating disorder. Article quality was assessed using the Critical Appraisal Skill Programme (CASP) checklist and thematic synthesis was used to analyse the primary research and develop overarching analytical themes.

Thirty articles met inclusion criteria and were included in this review. The methodological quality was mostly good. Thematic synthesis generated six main themes; collaborative care supports recovery; a safe and terrifying environment; negotiating identity; supporting mind and body; the need for specialist support; and the value of close others. The included articles focused predominantly on specialist inpatient care and were from eight different countries. One clear limitation was that ethnicity data were not reported in 22 out of the 30 studies. When ethnicity data were reported, participants predominantly identified as white.

Conclusions

This review identifies that a person-centred, biopsychosocial approach is necessary throughout all stages of eating disorder treatment, with support from a sufficiently resourced and adequately trained multidisciplinary team. Improving physical health remains fundamental to eating disorder recovery, though psychological support is also essential to understand what causes and maintains the eating disorder and to facilitate a shift away from an eating disorder dominated identity. Carers and peers who instil hope and offer empathy and validation are valuable additional sources of support. Future research should explore what works best for whom and why, evaluating patient and carer focused psychological interventions and dietetic support during intensive treatment. Future research should also explore the long-term effects of, at times, coercive and distressing treatment practices and determine how to mitigate against potential iatrogenic harm.

Plain English summary

Some people with eating disorders will need intensive treatment (e.g., inpatient treatment, day-patient treatment or acute medical treatment) during the course of their illness. Understanding what helps and hinders eating disorder recovery during intensive treatment is an important part of developing effective interventions. This review summarises research exploring people with eating disorders’ perspectives of intensive treatment, with the aim of identifying what helps and hinders eating disorder recovery. We searched in scientific databases for all published qualitative studies that explored people with eating disorders’ perspectives of intensive treatment. Thirty studies meet the inclusion criteria of this literature review. The results sections of these studies were analysed by extracting relevant findings relating to eating disorder recovery. We found that a person-centred, holistic approach is necessary throughout all stages of eating disorder treatment, with support from healthcare professionals and carers with specialist knowledge of how to support people with eating disorders. Improving physical health is fundamental to eating disorder recovery. However, psychological support is also essential to help people with eating disorders to understand what causes and maintains the eating disorder and support them to move away from an eating disorder dominated identity. Areas for future research are outlined.

Introduction

Eating disorders (EDs) are a group of mental health disorders, such as anorexia nervosa (AN), bulimia nervosa (BN), and binge eating disorder (BED), that are characterised by severe disturbances of attitudes and behaviours related to food, weight, and shape, and that seriously impact mental and physical health [ 1 ]. ED onset is typically during late adolescence and early adulthood [ 2 ]. With the potential to impact every organ system, EDs can be life threatening, reportedly having the highest mortality rate of all mental health disorders [ 3 , 4 , 5 ]. EDs are burdensome to the individual, their supporters and society [ 6 ]. Covid-19 has only exacerbated this burden: increases in incidence rates, ED symptomatology and hospital admissions have been widely reported [ 7 , 8 , 9 ].

Treatment for people with eating disorders (PwEDs) depends on the severity and chronicity of difficulty [ 10 ]. Most PwEDs are first offered outpatient psychological therapy, which can be complemented with pharmacotherapy, medical monitoring, nursing and/or dietetic support [ 11 ]. For those who do not respond to outpatient treatment, or whose ED cannot be managed safely as an outpatient, intensive treatment may be offered. This typically ranges from day-patient treatment or partial hospitalisation to inpatient or residential treatment in an ED or general psychiatric unit. Though varied, these more intensive treatments typically involve greater multidisciplinary input and direct meal supervision [ 11 ]. Alongside specialist intensive treatments, increasing numbers of PwEDs are being admitted to general medical settings to manage the medical complications associated with EDs [ 12 , 13 ]. Care in medical settings is highly variable, with varying levels of specialist input [ 11 , 13 ]. Importantly, whilst the relative merits of each form of intensive treatment continue to be debated, demand appears to be rising internationally [ 14 , 15 , 16 ].

Clinicians supporting PwEDs encounter challenges due to the egosyntonic nature of the illness [ 17 ]. Many people attach positive value to their ED [ 18 ], as it gives a perceived sense of control, and means of obtaining identity and avoiding negative affect [ 19 , 20 ]. Consequently, PwEDs are often ambivalent towards treatment and display low motivation to change [ 21 , 22 ]. Current treatment efficacy is modest [ 23 ]. A recent rapid review suggested between 30% and 41% of PwEDs relapse within two years of receiving treatment and that less than half achieve recovery at long-term follow up [ 24 ]. Furthermore, across all EDs, 62–70% of people who have received inpatient treatment still meet full diagnostic criteria or have remaining ED symptoms at long-term follow-up [ 6 ].

To improve treatment outcomes for PwEDs, it is vital that we better understand the lived experiences of those who use ED services [ 25 , 26 ]. As such, emerging research explores lived experience perspectives of ED treatment. For example, Babb and colleagues [ 27 ] reviewed qualitative studies exploring PwEDs’ general experiences of ED treatment. This review called for more individualised care and psychological support. Whilst valuable, it did not specifically focus on recovery. It also only identified studies exploring inpatient and outpatient experiences. Yet, some studies have explored PwEDs’ perspectives of other treatment settings, such as day-patient or acute medical settings, which may add important insights. The lifespan approach taken in this review may also mean that a review focused on adult populations is warranted as there are differences in ED treatment accessibility and delivery between child, adolescent and adult services. For instance, the duration of untreated ED (DUED) varies strongly between age groups, with younger age groups seeing shorter DUEDs [ 28 ] and in child and adolescent ED treatment, greater emphasis is placed on family involvement [ 29 ].

Other reviews seek to conceptualise ED recovery from lived experience perspectives. These have led to recovery being described as a complex psychological process that requires commitment, responsibility, development of insight into the function and consequences of the ED, acceptance by others and of the self, and development of meaningful relationships [ 30 ]. Recovery has also been said to include remission of ED symptoms alongside psychological well-being and adaptability, and involves hope, reclaiming identity, meaning and purpose, empowerment and self-compassion as key components [ 31 , 32 , 33 ]. Whilst valuable findings, these reviews do not focus specifically on what aspects of treatment help or hinder recovery.

More recently, two qualitative reviews synthesised literature exploring the lived experiences of inpatient treatment for all EDs [ 34 ] and AN only [ 35 ] within ED-specific treatment settings. These reviews highlight the complex and multifaceted nature of inpatient experiences and the importance of person-centred treatment that involves medical and psychological intervention [ 34 , 35 ]. Undeniably, these reviews provide insight into a neglected area of research. However, they include differing all-age studies and exclude studies exploring different intensities and aspects of intensive treatment (such as the experience of involuntary admission). Yet, many PwEDs move through different intensive treatments, some outside ED-specific treatment settings, and all aspects of intensive treatment may relate to recovery.

ED recovery is a process rather than a singular event, which can begin before and continue beyond inpatient treatment. Therefore, this review aims to extend previous reviews exploring the lived experiences of inpatient treatment. With a focus on recovery, it aims to elucidate what helps and hinders recovery for adults with EDs across all types and aspects of intensive treatment and to provide recommendations for research and clinical practice.

Search strategy

This systematic review was conducted in line with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [ 36 ] and was pre-registered on PROSPERO (ID: CRD42023426052).

Systematic literature searches were carried out using electronic databases (EMBASE, MEDLINE, PsychINFO, and Web of Science), searched from conception to 6th June 2023. Search terms and inclusion and exclusion criteria were formed using the ‘Sample, Phenomenon of Interest, Design, Evaluation and Research type’ (SPIDER) tool [ 37 ], outlined in Table  1 . The search strategy employed was informed by preliminary internet searches and previous reviews. It covered four concepts: [ 1 ] EDs, [ 2 ] intensive treatment, [ 3 ] qualitative methodology, and [ 4 ] lived experiences. Various combinations of search terms were trialled before settling on a broad search strategy that explored all free text to maximise search sensitivity.

Study selection and eligibility criteria

The first author completed the literature search, which yielded 2590 articles. Duplicates were removed, and the titles and abstracts of the remaining articles were screened against predetermined inclusion and exclusion criteria, outlined in Table  2 . Qualitative or mixed method studies (if qualitative results were reported separately) that explored adults’ experiences or views of any aspect of intensive treatment directly related to an eating disorder diagnosis were considered for eligibility. Only studies originally published in English and in peer-reviewed journals were accepted. A decision was made not to search the grey literature due to time constraints and wanting to ensure adequate space and consideration was given to the included studies. Further, grey literature studies are not necessarily subject to the same rigorous academic peer-review processes as non-grey literature studies. Nonetheless, some potentially relevant studies may have been missed.

Eligibility screening resulted in 71 articles which were read in full. Full-text screening excluded a further 45 articles, resulting in a total of 26 articles. The first author also screened the reference lists of included manuscripts to identify other studies that may have met the inclusion criteria and conducted additional searches through Google Scholar throughout the review process. This resulted in an additional four articles, meaning that 30 articles were included in this review. Throughout this process, any discrepancies were discussed with the second author (MG) until a consensus was reached. The complete procedure is detailed in the PRISMA diagram (Fig.  1 ).

PRISMA Flow Diagram

Quality assessment

Though what constitutes “validity” or “quality” in qualitative research is debated, quality appraisal remains a crucial part of any qualitative review [ 38 ]. The Critical Appraisal Skill Programme (CASP) checklist, a commonly used research appraisal tool, offers ten questions that facilitate assessment of qualitative studies. The Cochrane Qualitative and Implementation Methods Group recommends to avoid providing numerical scores, as CASP is not recommended as an absolute score of quality [ 39 ]. Instead, studies are considered according to whether criteria are: “yes well addressed”; “can’t tell”; or “no not addressed”. In this review, “can’t tell” was chosen when insufficient information was reported to make a judgement, as quality issues may be due to poor methodology and/or inadequate reporting [ 40 , 41 ]. The first author conducted the quality assessment and any ambiguities were discussed with the review team until a consensus was reached.

Given the large number of studies in this review, whilst absolute scores were avoided, quality appraisal was used to organise the thematic synthesis, as has been recommended previously [e.g., 41 , 42 ]. This meant studies ( n  = 10) for which “yes” was chosen for all ten questions were first reviewed to generate the coding framework. This was used to code the remaining studies. When particularly meaningful, new codes were generated. No studies were deemed to be low quality, as all studies provided valuable contributions to a limited evidence base. If there had been low quality studies, no new codes would have been generated, though these studies would not have been excluded.

Method of synthesis

Thematic synthesis was chosen to integrate findings of multiple qualitative studies to answer a specific review question and extend what is already known [ 43 ]. All text from “results” or “findings” sections, and any findings in abstracts, were extracted and treated as data. Thematic synthesis followed three iterative stages. Stage one involved line-by-line coding of text according to meaning and content. Stage two involved grouping of codes into hierarchical structures, to develop descriptive themes that remained data-driven and close to the primary studies. Stage three involved the generation of analytical themes through inference of descriptive themes, which go beyond the primary studies to generate new interpretive explanations.

Reflexivity

Reflexivity, the conscious, collaborative appraisal and critique of how one’s subjectivity and context influence the research processes, is an essential component of qualitative research [ 44 , 45 ]. We, the three authors, have psychology/psychiatry and academic and clinical backgrounds. The first author is a trainee clinical psychologist with lived experience of an ED as well as academic and clinical experience in EDs/mental health. The second author is a clinical psychologist with academic and clinical experience in mental health, in particular with adults with experiences of psychosis. The third author is a consultant psychiatrist and expert in the field of EDs, with experience of developing national and international initiatives to improve ED policy and practice. One of us was an insider to the experience of ED treatment and we are all insiders to a culture of working in mental health services with often high levels of need and limited resource. We made every attempt to ensure potential biases (e.g., our combined clinical, academic and experiential understanding that intensive treatment can be challenging for many) were kept in awareness and endeavored to pay attention to the full range of findings. Coding extracts and theme developments were discussed with all authors to check for disagreements or uncertainties before being finalised. Additionally, the first and second author met for monthly supervision to discuss the review development and analysis, and to support a continuous process of self-reflection. This collaborative approach supported development of themes that captured important nuances in the lived experiences of ED treatment, for example identifying the tension between physical versus psychological support. Nonetheless, as with all qualitative research, a different group of researchers who sought to answer the same research question may have extracted different themes from the data.

Studies identified

Thirty papers were identified as relevant. These are summarised in Table  3 .

Included studies totalled 495 participants ranging from 17 to 56 years. 96% identified as female, 2% identified as male, 0.4% identified as non-binary and 0.6% were not reported. 65% of participants were diagnosed with AN, 6.3% with BN, 0.6% with BED, 9.1% with EDNOS, 0.4% with OSFED, and 18.6% as missing or not reported. Ethnicity data were not reported in 22 studies. When ethnicity data were reported, 98.9% of participants identified as white (94/95 participants in reporting studies) and 1% identified as Other.

Included studies were predominantly conducted in the United Kingdom ( N  = 17). Other countries included Australia ( N  = 4), Canada ( N  = 3), Sweden ( N  = 2), Denmark ( N  = 1), Israel ( N  = 1), Norway ( N  = 1) and the USA ( N  = 1). Most studies focused on specialist inpatient units only ( N  = 19), with three studies focusing on inpatient and day-patient settings and one study focusing on inpatient and general psychiatric units. Three studies focused on day-patient settings only and two studies focused on medical settings only. One study focused on intensive community treatment and one study did not report the setting (though it focused on experiences in intensive settings). Most (27/30) studies did not report length of stay and those that did reported a wide range of 0.14 to 27 months.

Recruitment was carried out using various methods, inviting both current and past receivers of treatment. A range of data analysis approaches were used, though half of the studies used thematic analysis. Most studies ( N  = 23) used semi-structured interviews. Other data collection methods included open-ended questions in discharge/feedback questionnaires, narrative interviews, focus groups, diary entries and medical documents.

Quality appraisal

Included studies were of variable quality, but none were considered inadequate (see Table  4 ). All studies provided clear statements of the aims and appropriateness of qualitative methodology. The research design was unclear in three studies [ 46 , 47 , 48 ] and one study [ 49 ] did not explain consideration of ethics. Ten studies did not describe their recruitment strategy and thirteen studies did not provide any/adequate consideration of the relationship between the researcher(s) and participants. This contrasted with many studies that provided clear descriptions of their recruitment strategy (e.g., [ 50 , 51 ]) and researcher reflexivity (e.g., [ 52 , 53 ]). In line with their study methodology, some studies provided more descriptive analyses (e.g., [ 54 , 55 ]) and others provided more in-depth analyses (e.g., [ 48 , 49 , 56 ]). Studies that did not provide sufficient qualitative data for the quality of their analysis to be considered and analysed as part of this review were excluded at the point of screening. All studies showed sufficient rigour, providing clear statements of findings and situating these within the wider literature.

Studies varied significantly in the time-point of data collection (e.g., during treatment, immediately after, retrospectively or a combination), with only some reflecting on the chosen time-point(s). Most studies focused on experiences relating to specialist inpatient treatment and only some adequately described the treatment setting. Moreover, several studies did not provide key participant characteristics, samples were not representative and no study focused exclusively on any ED other than AN.

Thematic synthesis

Six themes were generated from the data: Collaborative Care Supports Recovery; A Safe and Terrifying Environment; Negotiating Identity; Supporting Mind and Body; The Need for Specialist Support; and The Value of Close Others. Themes and subthemes are outlined in Table  5 and discussed below.

Theme 1: collaborative care supports recovery

Active involvement in treatment.

Collaborative care supported recovery across intensive settings. “ Working together ” [ 51 ] and supporting PwEDs to “ make their own decisions ” [ 50 ] strengthened participants’ motivation. However, collaboration was “ often felt to be absent ” [ 54 ]. Several studies identified that participants felt “ alienated from the decision-making process ” [ 55 ], especially those admitted involuntarily. Feeling unheard negatively impacted upon self-esteem and anxiety. Lack of transparency between PwEDs and treatment providers affected treatment experiences and subsequent recovery. Lack of clarity about ward rounds led to “power differences… and anxiety ” [ 57 ]. Participants in both studies exploring medical settings voiced not knowing who was chiefly responsible for their care and “ feeling deceived or given a punishment ” [ 55 ] when starting a refeeding protocol or being detained, due to lack of information. This negatively impacted upon treatment engagement. One study identified that providers should make expectations and regimes clearer and repeat them frequently “ to ensure patients have time to process and understand them ” [ 50 ]. In another study, the option to self-admit (to inpatient treatment) strengthened participants’ agency and motivation, and promoted partnership. However, for some, it risked too much decision-making power – “ too much say… it’ll be bad for me ” [ 56 ].

Collaboration was particularly key during transitions of care. Lack of information and “ uncertainty in what was going to happen ” [ 53 ] contributed to fear and feeling overwhelmed, hindering ongoing recovery. Many studies concurred that “ a graded and planned discharge helped… [re] integration ” [ 58 ]. This involved “ a phased , supportive approach ” [ 61 ], “ communication… with clear goals ” [ 54 ] and consideration of potential “ obstacles and challenges ” [ 63 ]. Several studies identified that treatment intensity dropped too quickly, that little or no further support was offered, or that participants were placed on lengthy outpatient waitlists. Continuity of support was essential.

Temporarily handing over responsibility

Whilst collaborative care generally supported recovery, there were instances in which, for short periods of time, participants found it helpful to not be so involved in care decisions. Several inpatient studies identified that, whilst challenging, many participants actually felt “ saved ” [ 58 ] when providers took responsibility (e.g., implementing clear boundaries around dietary change). “ Handing over” [ 59 ] control was sometimes viewed as a necessary step towards recovery. However, for some, sudden loss of control contributed to heightened distress and “ amped up the ED ” [ 50 ]. For those experiencing involuntary treatment in particular (e.g., forced nasogastric feeding) this led to disconnection from one’s care. One study identified that “ hopelessness and resentment ” [ 58 ] developed. As Fox and Diab [ 49 ] outlined, the ED “ gave participants a sense of control and a method of coping …” and “ refeeding… led to an intense feeling of losing control” – supporting participants to understand the reasons behind care decisions and to process the intensive emotions these activated appeared fundamental to recovery.

Theme 2: a safe and terrifying environment

A bubble that was hard to replicate.

For some, the safety and security afforded by intensive treatment supported recovery. Inpatient and day-patient treatment granted “ permission ” [ 53 , 58 ] to focus on recovery. Inpatients was described as a “ respite from overwhelming everyday demands ” [ 56 ]. Participants felt they “ belonged somewhere ” [ 64 ], finding “ comfort in predictable routines ” [ 65 ]. Inpatients also provided relief for carers. Several studies suggested non-negotiable boundaries supported change – “ completing meals was non-negotiable ” [ 66 ]. Two studies recognised when healthcare professionals (HCPs) made alterations to rules, it gave the ED “ leverage to pathologically negotiate ” [ 65 ]. Nonetheless, one participant identified that the existence of certain rules (e.g., prohibiting of water loading) alerted them to new possibilities.

It was recognised that the certainty and boundaries inpatients afforded was “ not easily replicated ” [ 52 ]. Their loss after discharge contributed to difficulties with continuing recovery. Indeed, inpatients was called a “ bubble ” [ 58 , 59 ], “ greenhouse ” [ 60 ] and “lab… [with] very exact and measured conditions ” [ 60 ]. It left participants “ frozen… and dependent on the unit ” [ 59 ]. Various studies identified that intensive treatment (particularly inpatient treatment) put “ life on hold ” [ 61 ]. For some, this contributed to dependence on treatment and the ED. As O’Connell [ 66 ] outlined, the ED became “ the standpoint from which I related to others ”. A few studies highlighted the importance of providers “ showcasing interest and highlighting aspects of patients’ lives outside of their ED ” [ 50 ] to provide relief from institutionalisation and support motivation. As PwEDs transitioned out of intensive treatment, returning to or beginning careers, relationships, leisure and personal development activities supported “ a sense of routine and purpose ” [ 61 ].

A punitive, distressing environment

Words such as “ miserable ”, “ horrific ”, “ hostile ”, “ traumatic ”, “ distressing ”, “ inhumane ”, “ terrifying ” and “ an assault ” were used to describe treatment (in inpatient and medical settings only) [ 48 , 49 , 54 , 60 , 64 ]. For some, feeling dehumanised, restricted or traumatised negatively impacted upon motivation, engagement and subsequent recovery. Several studies suggested participants felt “ under inspection ” [ 58 ] and treatment was described as “ doing time ” [ 67 ]. “ Exposure to… [and experiences of] distressing events ” [ 54 ] were difficult – described as “ something I’ll never forget ” [ 48 ]. Participants sometimes experienced “ corrective measures as punitive or disciplinary ” [ 65 ]. Moreover, across several studies, participants felt certain boundaries were arbitrary, employed without adequate explanation, or “ rigid and unable to be maintained ” [ 58 ], leaving them feeling disempowered.

Theme 3: negotiating identity

Separating the self and the ed.

Across many studies, attachment to the ED hindered recovery. The ED afforded safety, control and confidence in its success and provided “ emotional and physical detachment ” [ 62 ]. Intensive treatment “ created a state of internal coercion ” [ 48 ]. Several studies identified that a mismatch between treatment requirements and participants’ readiness to change could result in treatment refusal or termination, strengthening attachment to the ED. For those who experienced repeated admissions, lengthy stays or passing between services, “ feelings of hopelessness ” [ 49 ] and “ feelings of failure ” [ 56 ] were prevalent. Consequently, participants “ gripped more tightly onto AN ” [ 66 ] (and the ED identity).

Indeed, being “ reduced to a number and a disorder ” [ 55 ] in inpatient and medical settings hindered recovery. Various studies suggested participants disliked feeling defined by their illness and treated as “ a collective ” [ 60 ] or in accordance with “ an assumed group identity ” [ 68 ]. This “one-size-fits-all approach ” [ 67 ] left participants feeling “ misunderstood , invalidated and stereotyped ” [ 66 ]. There was a desire for “ different tracks for people with different needs ” [ 55 ] and a wish for providers to “ humanise the patient ” [ 50 ]. Indeed, personalised, flexible treatment supported recovery across intensive settings. Day-patients was viewed as more flexible than inpatients, though both groups desired a more “tailored approach ” [ 61 ] (e.g., better consideration of differences in sexuality, gender identity and comorbidities). Intensive community treatment was considered individualised, with “ specific and obtainable goals ” [ 62 ]. Moreover, several studies highlighted that, for some participants, being supported to externalise the ED as separate to their sense of self - recognising “ AN as pathology separate to who they were ” [ 65 ] - supported change and recovery.

Beginning to want something different

Indeed, ambivalence towards treatment, particularly initially, was common. Recovery required moving from ambivalence to acceptance and/or determination. Reflecting back, one participant suggested others should “ surrender a little bit … trust in the treatment ” [ 50 ]. For some, this was difficult. Several studies identified that compliance resulted in discharge, but not necessarily recovery. One participant “ humour [ed]” [ 63 ] providers and another aimed to “ eat their way out ” [ 58 ]. It was these participants where relapse was most likely. Self-criticism, shame, worthlessness and hopelessness kept participants stuck.

Conversely, several studies outlined the value of motivation. In their study exploring experiences of recovered versus relapsed PwEDs, participants’ “ own drive ” [ 63 ] was prevalent in the recovered group. One participant described eventually “ wanting something different ” [ 66 ] and another study noted EDs require “ extremely hard work to be fought against ” [ 62 ]. Key to recovery was self-acceptance, hopefulness, and awareness and insight into the ED: “ compassion… and self-care ” [ 58 ] and “ a sense of self ” [ 64 ] were necessary.

Theme 4: supporting mind and body

Weight restoration and dietary change.

Many participants retrospectively saw intensive treatment as “ saving lives ” [ 48 ], specifically regarding medical stabilisation. However, across inpatient and medical settings, participants struggled with discrepancy between “ normal [weight restored] bodies ” and continued “ anorexic thoughts ” [ 63 ], leading to other maladaptive behaviours or relapse. Overfocus on biological markers, for example “ micro-monitoring of the participant’s weight ” [ 67 ], negatively impacted recovery. Across studies, participants wished for a “ slow pace of change with focus on all aspects of their difficulties ” [ 62 ].

Nonetheless, across specialist settings (i.e., not general medical), support in understanding and implementing dietary changes facilitated recovery. Meal support, plans and routines developed “ behavioural patterns that supported recovery ” [ 52 ] and “ staff eating alongside ” [ 46 ] normalised mealtimes. Nutritional education was also valued. Learning about “ daily nutritional requirements” [ 52 ] and “ their bodies’ need for food ” [ 47 ] helped participants make dietary changes. Similarly, opportunities to engage in practical food groups (e.g., grocery shopping, outings to restaurants/cafes and meal preparation activities) were considered important and increased “confidence to attempt repeating the challenges outside” [ 69 ]. Practicing dietary related cognitive skills and coping strategies supported a “ gradual shift to more independent eating ” [ 70 ].

Psychological awareness and understanding

Understanding what caused and maintained the ED arose as integral to recovery, through individual and group therapy and wider psychological support. Individual therapy supported PwEDs to understand the ED and “ challenge… maladaptive thinking styles and behaviours ” [ 71 ]. A “ strong [therapeutic] connection ” [ 70 ] was essential. Similarly, a range of therapeutic groups, including Cognitive Behavioural Therapy, Dialectical Behavioural Therapy and the Maudsley Anorexia Nervosa Treatment for Adults groups, as well as perfectionism, mindfulness, and value-based groups, were appreciated. Many recognised “the importance of sharing experiences and learning from each other” [ 72 ], though for a minority, the perceived intensity of groups was challenging. A holistic therapy, acupuncture, was “ relaxing , both emotionally and physically ” [ 73 ] particularly after meals. Nonetheless, for some, therapy was “ too structured ” [ 74 ]. There was desire “ for more guidance and practice to help with real life application ” [ 71 ] and several studies identified a need for longer therapeutic intervention. One study identified insufficient psychological input in ward rounds, though one participant did not want their formulation shared due to it being “ very personal ” [ 57 ].

Learning to identify, express and manage emotions emerged as beneficial across intensive settings. For example, developing strategies to “ manage… and label emotions ” [ 74 ] and communicate one’s feelings supported recovery during and after treatment. Self-examination skills (e.g., journaling) helped PwEDs “ continue to work on recovery after discharge ” [ 52 ]. Several studies identified that emotional suppression and avoidance of negative affect limited progress.

Theme 5: the need for specialist support

Genuine care, alliance and trust.

Genuine care, trust and therapeutic alliance between PwEDs and HCPs was important for recovery. Participants wished to be treated with dignity and respect. They valued HCPs who were “ approachable and friendly ” [ 51 ], empathic and non-judgemental, and who validated and managed participants’ emotions. For some, feeling cared for involved nurses adopting a “ motherly or sisterly role ” [ 65 ] and HCPs who went “ beyond their roles ” [ 54 , 75 ]. Several studies noted the importance of strong therapeutic alliances with key workers, characterised by honesty, trust and openness. This promoted “ hope and optimism ” [ 75 ] and led participants to feel “ held or supported ” [ 62 ]. Without a good keyworker relationship “ challenges could feel insurmountable ” [ 51 ].

Correspondingly, across several studies, feeling uncared for negatively impacted recovery. Participants sometimes felt dismissed, patronised or ignored. They struggled with HCPs who “ failed to follow through with promises ” [ 58 ], “ overlooked [them] in comparison to newly admitted patients ” [ 59 ], or offered a “lack of a predictable response” [ 68 ]. Distrust between PwEDs and HCPs was “ an important precursor to some difficult interactions ” [ 67 ]. Described in several studies, conflict often led to further rebellion as the participant sought to “ retain their sense of control ” [ 46 ]. Poor connections resulted in increased anxiety and distrust, which impacted participants’ self-esteem, motivation, and desire to remain in treatment.

Skilled and well Resourced Multidisciplinary Care

Several studies outlined the importance of PwEDs being care for by a skilled and well resourced multidisciplinary team, with “ staff from different disciplines… contributing to residents’ recovery ” [ 70 ]. Changing teams, HCP shortages and use of non-permanent staff decreased standards of care and hindered recovery. Whereas, well trained and skilled HCPs displayed empathy, understanding, knowledge and clear boundaries. Indeed, “ trust and belief in practitioner’s expertise were… fundamentally important ” [ 49 ]. Skilled HCPs were able to separate the person from the ED, facilitate honesty and openness, and develop strong therapeutic alliances.

Theme 6: the Value of Close others

Peer support and comparison.

Peer support and comparison affected recovery. Across intensive settings, “ physical and behavioural comparisons ” [ 59 ] and competitiveness negatively affected “group cohesion and personal recovery ” [ 53 ]. Many found it distressing and triggering being admitted alongside others at various stages of recovery and with differing levels of illness severity. Indeed, participants were susceptible to adopting “new [unhelpful] ED practices ” [ 60 ]. Participants in two studies described comparing themselves (not under section) to those under section. This comparison increased participants’ guilt for choosing to eat and negatively impacted recovery. Correspondingly, participants in one study valued spending time with people without EDs who “ value aspects of life other than shape and weight ” [ 52 ].

In contrast, many of the same studies recognised that being alongside other PwEDs also supported recovery. Peers who understood and were non-judgmental were valued and contributed to connectedness, acceptance and belonging. Peer support “ increased knowledge of effective coping skills and hope for recovery ” [ 59 ]. Several studies noted participants made “ close and lasting friendships… through a sense of camaraderie ” [ 60 ]. Relatedly, one participant valued a peer mentor who had “ been there and got through ” [ 53 ].

Carer Support and understanding

Carer support and understanding during, and upon leaving, intensive treatment supported recovery. Across settings, participants desired for carers to “ provide love , a listening ear ” [ 50 ], particularly “ during the transition period ” [ 61 ]. Carer support groups were also valued. Returning home with “ insufficient or unhelpful social support ” [ 69 ], as well as “ continual emphasis on body weight and dieting within the family or social environment ” [ 63 ], hindered recovery.

Moving from loneliness to connection

Isolation hindered recovery. Particularly upon admission, participants described an emptiness, loneliness and difficulty trusting others. Difficulties developing and maintaining relationships contributed to negative attributions of the self and others and pushed participants further into their ED. Admissions sometimes exacerbated these difficulties as participants were removed from friends and family. Fostering “ meaningful connections after treatment ” [ 52 ] and moving from “ loneliness… to interpersonal connection ” [ 62 ] supported PwEDs to move towards recovery.

This review explored what helps and hinders recovery during intensive treatment for PwEDs. Participants acknowledged that intensive treatment was often necessary, particularly with regards to biomedical recovery. As higher discharge BMI predicts more positive outcomes (for AN) [ 76 ], promoting adequate weight restoration remains a priority. Nonetheless, consistent with existing literature [ 30 , 35 ], a biomedical focus often took precedence over addressing underlying psychosocial difficulties. Participants were weight-restored but not recovered and often discharged without a period of consolidation or without adequate step-down support, placing them at higher risk of relapse following discharge [ 31 ]. Providers should be careful to not over-focus on biological markers and should ensure pace of change is acceptable to the individual.

Correspondingly, a therapeutic milieu, comprising individual and group therapy and the wider care environment, was valued and necessary for recovery, though was not always present or sufficient. Consistent with existing literature [ 77 , 78 ], psychological interventions that supported PwEDs to understand the function and maintenance of their ED, as well as to identify, express and process emotions, facilitated recovery. Externalisation also arose as an important therapeutic technique across the wider care environment to foster separation from an illness identity [ 79 , 80 ].

Ambivalence, resistance to change and hopelessness hindered recovery. Commonly identified as barriers to recovery [ 81 , 82 , 83 ], if these factors were not attended to, change was difficult, and relapse was likely. Imposing actions (e.g., through boundaries and routines) may be necessary for an individual’s safety, but carry a risk of driving them further into their ED, increasing resistance and decreasing motivation and compliance [ 84 ]. These findings support research highlighting the role of holding and actively sharing hope [ 33 , 85 ] and of motivational interviewing [ 86 ].

Consistent dietary support should be embedded into intensive treatment. Across intensive settings (except in medical settings, where they were not mentioned), structured mealtimes, meal support, modelling normal eating, meal plans, nutritional education, and food groups supported PwEDs to move towards recovery. Supporting a small body of literature [ 87 , 88 ], dietary-related interventions allowed PwEDs to practice adaptive coping strategies, improve eating behaviours and self-efficacy, and address social challenges associated with eating.

Compassionate and yet boundaried HCPs were essential. Across intensive settings, collaborative, person-centred care strengthened hope and engagement. PwEDs desired active involvement in treatment, though for some, having responsibility removed initially was a necessary part of recovery. As clinicians have highlighted, balancing PwEDs’ desires with beneficence can be challenging [ 85 , 89 ], however the dominant medical paradigm, that positions HCPs as expert authorities, may harmfully limit choice, autonomy and opportunities for treatment participation. When PwEDs feel unheard or that their needs are not being met, premature treatment termination may result [ 90 ]. Whilst those in intensive settings are often at higher risk, where possible, it remains important to offer choice and clear information. Although few in number, studies exploring day-patient and intensive community settings suggested they afforded greater choice and collaboration, though this may be as these settings generally support less severe ED populations [ 91 ].

Experiences of care were highly individual. At times, intensive environments facilitated recovery. They were safe and supportive, due to firm boundaries, clear routines, and, in inpatient settings, escape from life stressors. Yet, consistent with ED clinicians’ concerns [ 85 ], intensive treatment (especially inpatient) also contributed to treatment dependence and estrangement from life outside. Transition out of intensive treatment was highlighted as a particularly vulnerable period. Day-patient and intensive community treatment discharges were experienced as somewhat more graded and skills learnt as more transferable, perhaps leading to a greater likelihood of maintenance. These findings underscore the value of intensive treatment but also the need for a gradual discharge process. Occupational therapists may be particularly well placed to support development of necessary skills for continuing recovery, supporting PwED’s to identify purpose outside of the ED, cope with external triggers and resume educational, vocational and/or family roles [ 87 ].

Intensive environments (in inpatient and medical settings only) were also experienced as restrictive and traumatising, due to experiences of coercion, scrutiny, and being subjected to, or witnessing of, distressing practices. These iatrogenic factors may hinder recovery and have long-lasting effects, contributing to more severe psychopathology and/or trauma-related symptoms. To date, limited work has explored what aspects render the experience of psychiatric hospitalisation distressing, though experiences of coercion, stress and trauma appear common and distressing [ 92 ]. Moreover, whilst compulsory treatment can be necessary to save lives, the long-term effects are largely unknown [ 93 ].

Adding to the growing literature base surrounding the value of carer support for adults with EDs [ 94 , 95 ], carer support was valued when carers were able to understand the ED and challenges of treatment and offer empathy and validation. Given that carers’ distress and ways of coping can inadvertently maintain or reinforce the ED [ 96 ], this finding affirms the necessity for carers to receive their own support [ 95 ]. Currently, a range of carer interventions show positive outcomes for PwEDs undergoing intensive treatment, though implementation is patchy, and research has predominantly focused on young people with AN and the experiences of mothers [ 95 ].

Peer comparison, competition and contagion were common in intensive settings and often reinforced the ED-dominant identity. Nonetheless, peer support and identification were also common, and frequently decreased isolation while motivating individuals towards recovery. One study also highlighted the value of a peer mentor. As a growing area of research and clinical practice, peer mentors may instil hope and increase motivation for treatment [ 97 ]. Treatment alongside other PwEDs being both helpful and hindering for recovery is a widely reported juxtaposition [ 27 , 85 ]. Helpful peer influence appears to depend on dis-identification with the ED-dominant identity and identification with a recovery identity. Indeed, a sense of shared identity with others in ED recovery promoted recovery in an online support group [ 98 ]. Specialist support is necessary and valued by PwEDs and this generally means PwEDs are treated alongside peers. Peer influence should therefore be considered as part of each individual’s formulation, to explore the potential for support and harm and how this may relate to the ED identity.

Clinical and research implications

To enhance likelihood of ED recovery, a multidisciplinary approach is required across intensive settings. Restoring physical health remains fundamental. However, psychological support is also necessary. Whilst several psychological treatments have evidence supporting use in outpatients, minimal evidence guides implementation of evidence-based practices in intensive settings [ 99 , 100 ]. Interventions that enhance motivation to change [ 86 , 101 ], foster separation from an ED-dominant identity [ 102 , 103 ] and support emotion recognition, regulation and expression [ 104 , 105 ] should be prioritised. Research must determine what works best for whom and why, tailoring processes to PwEDs’ unique needs, contexts and goals [ 30 ] and comorbidities [ 106 ].

Specialist dietetic support should also be employed. Dieticians possess unique skills and knowledge, but the extent to which they are involved in intensive treatment is largely unknown [ 88 ] and limited research guides the content of dietetic interventions or explores the effect of including dietetics [ 107 , 108 ]. Further research should explore what constitutes effective dietetic support across intensive settings [ 87 , 108 ].

Time to consolidate recovery gains alongside planned and phased discharges are vital for ED recovery. Research has begun to explore novel ways to support intensive treatment transitions [ 109 ] and intensive stepped-care treatment programs highlight the value of longer-term multidisciplinary care for PwEDs [ 110 , 111 ]. Further research must explore how to support maintenance of recovery, particularly as PwEDs return to daily life stressors.

Clinical practice guidelines recommend carer involvement in adult ED treatment [ 112 , 113 ] and carers and PwEDs recognise the value of carer support [ 96 , 114 ]. Current carer support is inconsistent, interventions vary, and a sufficient evidence base is lacking, particularly for adult ED populations [ 94 , 115 ]. Carer capacity, skill and knowledge vary and interventions need to be tailored accordingly [ 95 , 96 ]. To develop more routine and individualised care, research needs to elucidate which carer interventions works best for whom and why, taking consideration of different carer types, EDs other than AN, and stages of illness [ 94 , 96 ].

Perhaps most notably, this review highlights the complexity of intensive support for PwEDs. Findings highlight several dilemmas that HCPs face: helpful boundaries and containment versus restriction and coercion; peer support versus contagion; and physical versus psychological recovery. There is a clear need for sufficient resource, specialist training and opportunities for HCPs to engage in reflective spaces. Organisational pressures alongside client complexity mean HCPs can find working with PwEDs emotionally draining, leading to negative judgements, frustration, hopelessness and worry [ 99 , 116 ]. Perhaps it is these feelings that lead HCPs to strive for a practice of safe-certainty (e.g., administering standardised protocols) [ 116 ]. Time and space for reflection may support adoption of positions of safe-uncertainty, and consequently more flexible, person-centred approaches based on formulation and evidence-based interventions [ 116 ].

Specialist skills and knowledge, alongside trust and openness, reduce conflict and enhance therapeutic relationships and treatment engagement [ 117 , 118 , 119 ]. Within intensive settings, HCPs must balance firmness and empathy, communicating with clear boundaries to ensure certain behaviours are minimised whilst at the same time recognising and understanding the defensive nature of the ED and its adaptive function [ 22 ]. Future studies should explore what aspects of intensive treatment may be causing harm and any long-term effects. Moreover, there is need for specialist training and research in general medical settings, given the extent of negative experiences in this area.

Strengths and limitations

This review brings together 495 participants’ perspectives across thirty studies. Extending findings of previous reviews [ 34 , 35 ], this study explores what helps and hinders recovery across the spectrum of intensive treatment specifically for adults with EDs. A rigorous methodological process was employed in the selection, evaluation and interpretation of studies. To ensure findings remained contextualised, details of each included article’s aims, sample, setting, methods and methodological quality were included. However, a number of limitations must also be considered. As grey literature was not searched, some potentially relevant studies may have been missed. However, the sample is purposive rather than exhaustive, as this review aims to offer interpretive explanation and not prediction, therefore it may not be necessary to locate every available study [ 43 ]. The majority of included studies explored inpatient treatment experiences. Whilst the number of studies exploring lived experiences in non-inpatient settings is limited, the included studies offer a glimpse into experiences of these settings and highlight an important research gap. Further research is needed into lived experiences of intensive treatment settings other than specialist inpatient treatment for PwEDs (e.g., exploring lived experiences of day-patient treatment/partial hospitalisation, residential care, intensive community treatment, home-based treatments and acute medical admissions). Moreover, many studies also inadequately described the treatment setting. Given the diversity of intensive treatment approaches for PwEDs, authors should endeavour to describe treatment settings adequately to support transferability of findings [ 120 ]. Additionally, included studies omitted several key participant characteristics, and as has been identified previously, samples lacked ethnic, gender and diagnostic diversity. This limits the generalisability of findings to groups other than white women with AN. Researchers must include ethnicity data, as its absence further maintains underrepresentation. Research prioritising the treatment experiences of marginalised groups is urgently required [ 121 ].

This review explores what helps and hinders recovery during intensive treatment for PwEDs. A sufficiently resourced and adequately trained multidisciplinary service, which includes physical, psychological, dietetic and social support, supports ED recovery. Findings emphasised the vital role psychological support and understanding can have in supporting PwEDs to move from an ED-dominant identity to a sense of self outside of the illness and the value of carers and peers who instil hope and offer empathy and validation. Nonetheless, HCPs face several challenges when supporting PwEDs in intensive settings, as what is helpful for one person may be harmful for another. A person-centred, biopsychosocial approach is necessary throughout all stages of treatment. Further research must evaluate patient and carer focused psychological interventions and the role of dietetic support during intensive treatment. It must explore the long-term effects of, at times, coercive and distressing treatment practices and determine how to mitigate against potential iatrogenic harm.

Data availability

Data is provided within the manuscript. Further data is available on request.

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There was no direct funding for this project. The first author completed the systematic review as part of a Doctorate in Clinical Psychology at the Salomons Institute for Applied Psychology whilst employed by Surrey and Borders Partnership NHS Foundation Trust. US receives salary support from the NIHR Biomedical Research Centre for Mental Health, South London and Maudsley NHS Foundation Trust and Institute of Psychiatry, Psychology and Neuroscience, King’s College London.

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HW conducted the review and analysed data, and was a major contributor in writing the manuscript; MG supervised the project, provided qualitative expertise during analysis and reviewed the manuscript; US supervised the project and reviewed the manuscript. All authors approved the final manuscript.

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Webb, H., Griffiths, M. & Schmidt, U. Experiences of intensive treatment for people with eating disorders: a systematic review and thematic synthesis. J Eat Disord 12 , 115 (2024). https://doi.org/10.1186/s40337-024-01061-5

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Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming

• Junqi Shan 1   na1 ,
• Xinyu Li 2   na1 ,
• Runqi Sun 3   na1 ,
• Yao Yao 1 ,
• Yan Sun 4 ,
• Qin Kuang 4 ,
• Xianling Dai 4 &
• Yanlai Sun 1  

Journal of Experimental & Clinical Cancer Research volume  43 , Article number:  227 ( 2024 ) Cite this article

Metrics details

The failure of proper recognition of the intricate nature of pathophysiology in colorectal cancer (CRC) has a substantial effect on the progress of developing novel medications and targeted therapy approaches. Imbalances in the processes of lipid oxidation and biosynthesis of fatty acids are significant risk factors for the development of CRC. Therapeutic intervention that specifically targets the peroxisome proliferator-activated receptor gamma (PPARγ) and its downstream response element, in response to lipid metabolism, has been found to promote the growth of tumors and has shown significant clinical advantages in cancer patients.

Clinical CRC samples and extensive in vitro and in vivo experiments were carried out to determine the role of ZDHHC6 and its downstream targets via a series of biochemical assays, molecular analysis approaches and lipid metabolomics assay, etc.

To study the effect of ZDHHC6 on the progression of CRC and identify whether ZDHHC6 is a palmitoyltransferase that regulates fatty acid synthesis, which directly palmitoylates and stabilizes PPARγ, and this stabilization in turn activates the ACLY transcription-related metabolic pathway. In this study, we demonstrate that PPARγ undergoes palmitoylation in its DNA binding domain (DBD) section. This lipid-related modification enhances the stability of PPARγ protein by preventing its destabilization. As a result, palmitoylated PPARγ inhibits its degradation induced by the lysosome and facilitates its translocation into the nucleus. In addition, we have identified zinc finger-aspartate-histidine-cysteine 6 (ZDHHC6) as a crucial controller of fatty acid biosynthesis. ZDHHC6 directly interacts with and adds palmitoyl groups to stabilize PPARγ at the Cys-313 site within the DBD domain of PPARγ. Consequently, this palmitoylation leads to an increase in the expression of ATP citrate lyase (ACLY). Furthermore, our findings reveals that ZDHHC6 actively stimulates the production of fatty acids and plays a role in the development of colorectal cancer. However, we have observed a significant reduction in the cancer-causing effects when the expression of ZDHHC6 is inhibited in in vivo trials. Significantly, in CRC, there is a strong positive correlation between the high expression of ZDHHC6 and the expression of PPARγ. Moreover, this high expression of ZDHHC6 is connected with the severity of CRC and is indicative of a poor prognosis.

Conclusions

We have discovered a mechanism in which lipid biosynthesis is controlled by ZDHHC6 and includes the signaling of PPARγ-ACLY in the advancement of CRC. This finding provides a justification for targeting lipid synthesis by blocking ZDHHC6 as a potential therapeutic approach.

Introduction

Colorectal cancer ranks as the third most prevalent form of cancer in the population and is well recognized as a significant global public health concern. Colorectal cancer has the potential to metastasize from the colon’s surface to the colon tissue and other organs such as the liver, prostate gland, seminal vesicle gland, and uterus if not detected early [ 1 , 2 ]. While much research has been conducted on the molecular process behind the development of intestinal cancer, additional investigation is required to identify new and efficient therapeutic approaches. Recent epidemiological studies have discovered a connection between metabolic irregularities and numerous colon disorders, such as ulcerative colitis (UC), Crohn’s disease (CD), and colorectal cancer (CRC) [ 3 , 4 ]. The colon, being a crucial organ for metabolizing the three macronutrients, has been identified as playing a significant role in these diseases. It is important to mention that the worldwide occurrence of CRC is experiencing a significant rise, primarily attributed to the widespread problem of being overweight and the consequent emergence and advancement of obesity-related generalized metabolic syndrome [ 5 , 6 ]. The lipid-rich state has a crucial role in promoting chronic inflammatory bowel disease (IBD) and colorectal cancer (CRC) driven by metabolic syndrome [ 7 , 8 ]. Recent reports have extensively characterized the genetic changes in colorectal cancer (CRC), including mutations in TP53, KRAS, and EGFR [ 9 , 10 ]. CRC is a very diverse malignancy with a low response to treatments and a high fatality rate. The etiology of colorectal cancer (CRC) and its related tumor formation is not yet fully comprehended, however disruption of genetic, immunological, and metabolic homeostasis is regarded to be crucial factors.

Metabolic reprogramming is primarily caused by the dysregulation of metabolic enzyme expression or activity. Palmitoylation is an essential posttranslational modification that controls the breakdown, location, and activation of metabolic enzymes in cells [ 11 , 12 ]. It involves attaching fatty acids to substrate proteins. The disruption of palmitoylation is intricately linked to cellular lipid metabolism and facilitates the development and advancement of several types of malignancies, including colorectal cancer (CRC) [ 13 ]. Recently, there has been extensive research on a class of acyltransferases called zinc finger-aspartate-histidine-cysteine (DHHC)-CRD-type palmitoyl acyltransferases (ZDHHCs) due to their probable involvement in stabilizing oncoproteins [ 14 ]. These enzymes have been investigated in several types of cancer. S -palmitoylation is a posttranslational modification that involves the attachment of C16 fatty acid palmitate (PA) to cysteine (Cys) residues of specific proteins [ 14 , 15 ]. This process regulates the functionality of these proteins in different pathological and physiological situations. Significantly, palmitoylation has been found to regulate the specific localization and movement of proteins. This biological process relies on the catalytic activity of a sequence of palmitoyltransferases. ZDHHC2 and ZDHHC3, which are clearly described enzymes, have significant involvement in the process of lipogenesis [ 16 , 17 ]. They directly palmitoylate and stabilize important lipogenesis components such as stearoyl-CoA desaturase (SCD), fatty acid synthase (FASN), and nuclear factor erythroid-2-related factor 2 (Nrf2) during carcinogenesis. ZDHHC6, a significant palmitoyltransferase, has been identified as a cancer signature gene and has high expression in various types of cancer [ 18 , 19 , 20 ]. ZDHHC6 primarily functions as an oncoprotein by increasing AEG-1 palmitoylation, which in turn activates or stabilizes transcription factors involved in cancer progression [ 21 ]. In addition, ZDHHC6 has a role in promoting cell proliferation by controlling the flow of calcium ions through selenoprotein K during the development of tumor growth [ 22 , 23 ]. Nevertheless, the role of ZDHHC6 in lipid metabolism is largely unknown.

Peroxisome proliferator-activated receptor gamma (PPARγ) plays a crucial role in controlling lipid metabolism and energy balance, which are important biological processes involved in the advancement of colorectal cancer (CRC) [ 24 , 25 ]. PPARγ has influence on diverse metabolic pathways and is present in colorectal epithelial cells, where its activation can stimulate cell differentiation and enhance apoptosis, therefore regulating tumor formation [ 26 ]. Moreover, PPARγ has been recognized as a crucial agent in the process of adipocyte differentiation, lipid storage, and glucose metabolism, all of which play a significant role in creating the tumor microenvironment and facilitating the survival of cancer cells [ 27 , 28 ]. The abnormal lipid metabolism in cancer cells is currently acknowledged as a distinguishing feature of cancer and has a substantial impact on facilitating rapid cell growth and spread to other parts of the body [ 29 ]. CRC cells frequently undergo significant modifications in both fatty acid synthesis and degradation, which play a role in membrane biosynthesis, energy storage, and signal transmission. These changes ultimately support the formation and progression of cancer. PPARγ agonists have been shown to inhibit the development of colon cancer, highlighting the receptor’s potential as a target for therapeutic intervention in the treatment of colorectal cancer (CRC) [ 30 , 31 ]. Changes in PPARγ expression and activity can influence the development of colorectal cancer (CRC) by changing the way lipids are processed, causing inflammation, and impacting insulin sensitivity. These factors are crucial for cancer cells to adapt and survive in the intestinal environment. Nevertheless, the precise involvement of PPARγ in cancer biology is not well understood. Recent research have shown that the process of phosphorylation of AKT stimulates the production of PPARγ, which in turn enhances the synthesis of lipids and the development of tumors in colorectal cancer (CRC) [ 32 , 33 , 34 ]. Our work revealed a correlation between the overexpression of ZDHHC6 and a substantial increase in lipid biosynthesis in CRC. We demonstrated that ZDHHC6 promotes the synthesis of fresh fatty acids and the formation of tumors by palmitoylating and stabilizing PPARγ in colon cancer. The results of our study offer a potential approach to specifically inhibit the production of fatty acids, which could have therapeutic advantages for individuals with elevated ZDHHC6 levels in colorectal cancer.

Materials and methods

Research consent and ethical statement.

The experimental procedures involving animals in this study were conducted in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (1996, in Chinese). Additionally, these procedures were approved by the Institutional Animal Use and Care Committee at Shandong Cancer Hospital and Institute, Shandong First Medical University, and Shandong Academy of Medical Sciences in Jinan, China. The study recruited patients with colorectal cancer (CRC) phenotypes from the Department of Gastrointestinal Surgery and Clinical Trial Research Center at Shandong Cancer Hospital and Institute, Shandong First Medical University, and Shandong Academy of Medical Sciences (Jinan, China). The diagnosis of colorectal cancer (CRC) was established after a comprehensive evaluation that included radiologic, clinical, and endoscopic examinations, as well as careful analysis of histology data. The individuals included in this study had been diagnosed with Crohn’s disease (CD), ulcerative colitis (UC), or colorectal cancer (CRC). The determination of ulcerative colitis (UC) or Crohn’s disease (CD) was achieved through the utilization of histological data in conjunction with radiologic, clinical, and endoscopic assessments. The specific attributes are outlined in Supplemental Tables 1 and 2 . Each patient donor granted informed consent, and the research was approved by the Institutional Research Ethics Committee of Shandong First Medical University and Shandong Academy of Medical Sciences (Jinan, China).

Identification of candidates

We screened candidate genes using the following procedures in order to find genes involved in the progression and metabolism of colorectal cancer (CRC): (1) Data Acquisition: The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO) databases provided gene expression data and pertinent clinical information on CRC patients (GEO: GSE254054, GSE231943, GSE252858, GSE234804, GSE236678, GSE231436, GSE197088, and GSE239549). (2) Finding consistently differentially expressed genes (DEGs): In each data set, the function t -test in R was used to see if there was a significant difference in the level of gene expression between CRC tissues and adjacent or normal tissues. Benjamini-Hochberg correction was then applied. For the ensuing analysis, the lists of upregulated or downregulated DEGs in each of the ten datasets were utilized. (3) Candidate gene survival study in colorectal cancer: Clinical status and follow-up duration were taken from the clinical data of CRC patients. After excluding patients for whom there was no overall survival statistics or information, a survival analysis was carried out using the R packages “survival” and “survminer.” (4) Gene set variation analysis (GSVA): The sample-wise biological processes of gene ontology (GO) activity variation were estimated using the GSVA R package. Single-sample GSVA scores were computed using a gene expression matrix, with each GO gene set including a minimum of ten genes. The GSVA scores of biological processes linked to cancer and the fold change in the expression levels of overlapping genes were analyzed using the Pearson correlation coefficient. (5) GSEA, or gene set enrichment analysis: In each dataset, CRC samples were compared to adjacent normal tissues using the GSEA software. The Molecular Signatures Database provided the gene set ‘c5.all.v6.2.symbols.gmt’, which was utilized in this investigation. Following the use of a 0.05 P value criterion and a 0.25 FDR threshold, the genes found were considered significant. Additionally, we ran GSEA on CRC tissues with high and low ZDHHC6 expression levels from the TCGA and ICGC databases.

Cell culture

The human colorectal cancer cell lines SNU-C2A (Cat#: CCL-250.1), SW48 (Cat#: CCL-231), HT-29 (Cat#: HTB-38), LS1034 (Cat#: CRL-2158), HCT116 (Cat#: CCL-247EMT), Caco-2 (Cat#: HTB-37), and the FHC cells (Cat#: CRL-1831) derived from the human colon were acquired from the American Type Culture Collection (ATCC). The mouse-associated CRC cell lines, namely CT26 (Cat#: CRL-2638), MC38 (Cat#: CVCL_B288), CMT93 (Cat#: CVCL_1986), MC26 (Cat#: CVCL_0240), and the normal mouse colon cell line MODE-K (Cat#: CVCL_B4FG), were acquired from Cellosaurus. The entire cell line was discovered and confirmed to be devoid of mycoplasma. CRC cell lines were cultured in RPMI1640 medium (Cat#: LM87077C, LMAI Bio, China) supplemented with 10% premium quality fetal bovine serum (FBS) (Cat#: abs972, Absin, China) and 1×penicillin-streptomycin solution (Cat#: U31-301 C, YOBIBIO, China). The cells were cultured in culture plates at 37 °C in a humidified atmosphere with 5% CO2. The FHC or MODE-K cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), as specified in the product and instruction manuals. Following the transfection process, specific cells were placed in 6-well, 12-well, or 24-well plates with 60% fusion for 24 h prior to cell transfection. Subsequently, the cells were transfected using Convoy™ Transfection Reagent (Cat#: No. 11103, ACTGene Inc.) as instructed in the product description. Concisely, the transfection mixture was formulated by combining 2 µg of vector, 5 µl of Lipo3000 transfection reagent, and 120 µl of Gibco Opti-MEM™.

Antibodies, chemicals and reagents

The corresponding primary antibodies used in this study were purchased from Abcam Inc ® . The antibodies were specific to the following target proteins: ACTIN (Catalog number: ab179467, diluted at a ratio of 1/2500), PPAR gamma (Catalog number: ab272718, diluted at a ratio of 1/1000), PPAR delta (Catalog number: ab23673, diluted at a ratio of 1/1000), PPAR alpha (Catalog number: ab61182, diluted at a ratio of 1/1000), SREBP1 (Catalog number: ab28481, diluted at a ratio of 1/1000), Myc (Catalog number: ab32, diluted at a ratio of 1/1000), RAB11B (Catalog number: ab228954, diluted at a ratio of 1/150-1/1000), LAMP1 (Catalog number: ab24170, diluted at a ratio of 1/150-1/1000), Lamin B1 (Catalog number: ab229025, diluted at a ratio of 1/1000). The antibodies against ZDHHC6 (Catalog number: ABIN7162558, dilution ratio of 1/200–1/1000) and ZDHHC6 (Catalog number: ABIN6989238, dilution ratio of 1/200–1/1000) were acquired from antibodies-online in Limerick, PA, US. The antibodies against HA (Catalog number: 3724, diluted at a ratio of 1/250–1/1000) and Flag (Catalog number: 14793, diluted at a ratio of 1/250–1/1000) were acquired from Cell Signaling Technology™ (CST). The supplementary anti-Na, K-ATPase antibody (Catalog number: H00000483-B01P) and PPARG antibody (Catalog number: NBP2-22106) were acquired from Novus Biologicals, LLC at a dilution ratio of 1/250-1/1000.

The protein concentration of the samples was determined using the BCA Protein Assay Kit (Catalog number: YSD-500T, Yoche-Biotech, China). The western blotting study employed HRP-tagged secondary antibodies (Catalog number: bs-0297 M-HRP, Bioss, China) at a dilution ratio of 1/15,000–1/20,000 for visualization purposes. The MG-132 (Catalog number: T2154) and cycloheximide (CHX) (Catalog number: T29590) were acquired from TargetMol Chemicals Inc., located in Shanghai, China. The compounds Biotin picolyl azide (Catalog number: 900912), alkynyl myristic acid (Alk14) (Catalog number: 1164), alkynyl palmitic acid (Alk16) (Catalog number: 1165), and alkynyl stearic acid (Alk18) (Catalog number: 1166) were acquired from Click Chemistry Tools. We synthesized and manufactured the alkynyl arachidic acid (Alk20) in our laboratory, achieving a purity level of above 98%. The compounds 2-bromopalmitate (2-BP) (Catalog number: 21604) and BSA (Catalog number: V900933) were acquired from Merck KGaA. The compound known as palmostatin M (Palm M) (Catalog number: 565407) was acquired from MedKoo Biosciences, Inc. The TaqMan ® Universal PCR Master Mix (Catalog Number: P/N4304437) and PowerUp™ SYBR™ Green (Catalog Number: A25742) were acquired from Applied Biosystems.

The pan palmitoylation-specific antibody was acquired as a generous gift from the research laboratory of Dr. Xu at the Chongqing University of Education [ 35 ]. In summary, to create an antibody that specifically targets pan palmitoylation, a brief peptide containing just two Cys C-C (pal) bonds (98.5% purity) was produced. One of the cysteine residues in the peptide was palmitoylated. This peptide was then utilized as the hapten, following established techniques and procedures. Subsequently, the acquired peptide C-C (pal) was linked with keyhole limpet hemocyanin (KLH) to serve as an extra antigen for the immunization of New Zealand white rabbits. The anti-serum was collected following the administration of three doses of passive immunization. Prior to utilization, employ ammonium sulfate precipitation for the purification of this anti-serum. In order to enhance the verification of this antibody, we have employed multiple supplementary validation methodologies. Essentially, the hydroxylamine buffer liquid can generate free sulfhydryl groups through the thioester linkages formed between the protein and palmityls. The TS-6B resin was employed to isolate the palmitoylated valosin-bearing protein that had been treated in a hydroxylamine buffer. Subsequently, an immunoblotting experiment was conducted using an anti-valosin-bearing protein antibody. Additionally, the acquired antibody was subsequently employed to examine the palmitoylation of the short c-terminal domain phosphatase 1, as detailed in the prior investigation. In this study, the pre-existing antibody was used to examine the palmitoylated proteins obtained from cells treated with various drugs, including Palm B, Palm M, or 2-BP. The aforementioned techniques collectively enhanced the sensitivity and specificity of the antibody in detecting the protein palmitoylation process.

Knockout cell and knockdown lines preparation

The pre-existing system for generating knockout cell lines with targeted gene deficiencies, which is relevant to the ongoing research, was established as previously described. Essentially, a CRC cell line was created by using the CRISPR-Cas9 method to delete the ZDHHC6 or PPARG genes. The pre-designed short guide RNA (sgRNA) targeting the human ZDHHC6 or PPARG gene was generated and inserted into pLentiCRISPRV2-GFP vectors (#98290, Testobio Co., Ltd., China) to produce the CRISPR-Cas9 sgRNA-packaging lentivirus. The ready-made CRISPR/Cas9 KO products for human ZDHHC6 plasmid (#sc-418298) and PPARγ plasmid (#sc-400030) were acquired from Santa Cruz Biotechnology, Inc. The sgRNA oligonucleotides were inserted into pLentiCRISPRV2 vectors that had been cleaved by the BsmBI restriction enzyme. The clones carrying gene deletions were extracted and identified using western blotting.

Plasmids and vectors preparation

The segments of DNA encoding the sgRNAs were inserted into PX330 (#PVT6301, Nova Lifetech Inc.) in order to introduce the N-terminal S-protein-Flag-Streptavidin binding peptide (SFB) tag at the endogenic ZDHHC6 gene location. The donor vector for ZDHHC6 knock-in was created using Gibson assembly of 50 homologous arms, Puro-P2A-SFB, and 30 homologous arms into the pUC19 plasmid (#N3041L, New England Biolabs). The ZDHHC6 open reading frames of human origin were produced using PCR amplification using complementary DNA derived from HCT116 cells. In addition, full-length Homo sapiens ZDHHC6 cDNA plasmids were created using PCR-based cDNA amplification. These plasmids were then inserted into pcDNA3.4™ TOPO™ plasmids (#A14697, Thermo Fisher Scientific Inc.) that were tagged with either 3×HA or 3×Flag for use in other in vitro investigations. Expression vectors containing shortened fragments of the PPARG gene, such as Myc-PPARG WT, Myc-PPARG-AF1, Myc-PPARG-DBD, and Myc-PPARG-hinge-LBD, were generated as planned. In addition, to further investigate the functional impacts of ZDHHC6 in laboratory conditions, targeted protein expression vectors were created by loading adenoviruses with the use of a pre-made adenovirus packaging kit (Haixing Biosciences, Suzhou, China). The complete Homo sapiens ZDHHC6 cDNA plasmids and their corresponding ready-made shRNA targeting human ZDHHC6 (sh ZDHHC6 ), shRNA targeting human PPARG (sh PPARG ), human PPARG sequences with the C313S mutant, and Homo sapiens full-length PPARG sequences were individually packaged into adenovirus using a commercially available adenovirus packaging kit (Haixing Biosciences, Suzhou, China). The adenovirus particles were purified and quantified to a titer of 6.0 × 10 10 PFU using the Adenovirus Purification Mini Kit (#V1160-01, Shanghai Juncheng Biotechnology, China) following the instructions provided in the operation handbook.

Histological analysis

The histopathologic assay was conducted by fixing the cells or tumor tissue with a 4% formaldehyde solution called Image-iT™ (#R37814, Thermo Fisher Scientific Inc.), and thereafter cutting it into transverse sections. The cell slices were subjected to treatment with the specified primary antibodies at a temperature of 4 °C for a duration of 24 h. The Leica DM IL LED microscope was used to capture histological pictures for examining specimen sections, while a confocal laser microscopy system (Olympus, Japan) was used for investigating immunofluorescence sections after incubating them with the appropriate secondary antibodies. The tumor specimens were immersed in a 4% formaldehyde solution for 24 h to preserve them, and subsequently embedded in an OCT (optimal cutting temperature) compound. The embedded tissues were sliced into sections of 20 micrometers in thickness. Xylene was employed for the purpose of eliminating the paraffin from the tissue sections during the process of IHC staining, followed by subsequent rehydration. Subsequently, they were immersed in a solution containing 5% normal target serum and 3% BSA (Beyotime) for a duration of 1.5 h. The specified primary antibodies were introduced and left to incubate overnight at a temperature of 4 °C. Following the washing step, a 1:300 dilution of HRP-conjugated secondary antibody (Abcam) was applied and incubated for 1 h. The DAB substrate was introduced to analyze the signals. Photographs were taken with a light microscope.

Immunoprecipitation and S -protein pull down assay

Immunoprecipitation and SFB pull-down experiment was performed as described previously [ 42 ]. Briefly, cells were lysed in E1A lysis buffer (250 mM NaCl, 50 mM HEPES [pH 7.5], 0.1% NP-40, 5 mM EDTA, protease inhibitor cocktail [Sigma]). The antibodie to ZDHHC6 was used for immunoprecipitation. HCT116 cells were transfected with SFB-tagged protein and lysed in NETN buffer (200 mM Tris-HCl [pH 8.0], 100 mM NaCl, 0.05% NP-40, 1 mM EDTA, protease inhibitor cocktail [Sigma]) for 20 min at 4 °C. Crude lysates were subjected to centrifugation at 14,000 × g for 15 min at 4 °C. Supernatants were incubated with S-Protein Agarose for 4 h (Millipore, USA). The agaroses were washed three times with NETN buffer. Proteins were eluted by boiling in 1× SDS loading buffer and subjected to SDS-PAGE for immunoblotting.

GST pull-down assay

GST pull-down assay was used to detect the direct interaction between PPARγ and ZDHHC6. Briefly, GST-tagged PPARγ (GST-PPARγ) and 6×His-tagged ZDHHC6 (His-ZDHHC6) or 3×Flag-tagged ZDHHC6 (Flag-ZDHHC6) proteins were expressed in BL21 (DE3) Escherichia coli via transforming pGEX-4T-1-GST-PPARγ and pET24a-His-ZDHHC6 or pET24a-Flag-ZDHHC6 plasmids, respectively. Then, the E. coli were collected, sonicated, and purified with cOmplete His-Tag Purification Resin (Roche) or Purification of Flag kit (Sigma-Aldrich) to obtain purified ZDHHC6-tagged protein. GST-PPARγ protein was expressed and immobilized with BeyoGold™ GST-tag Purification Resin (Beyotime) following the manufacturer’s instructions. The beads-PPARγ complexes were washed with GST pull-down binding buffer (50 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 10 mM MyCl2, pH 8.0) and incubated with purified ZDHHC6-tagged protein at 4 °C for 4 h on a rotating windmill. Finally, the beads were washed and analyzed by immunoblotting assay.

Quantitative PCR (qPCR) assay

The cells or tissue RNA were obtained and isolated using TRNzol universal reagent (#DP405-02, TIANGEN Biotech Co., Ltd., Beijing) following the instructions provided in the operating handbook. The pre-prepared RNA samples were stored in a refrigerator at a temperature of -80 °C for a maximum of 10 days (#TSX40086V, Thermo Fisher Scientific Inc.). The optimal RNA purity was assessed based on the observation of an absorption ratio (values > 2.00) at 260/280 nm. Subsequently, 1.5 µg of pure RNA was subjected to reverse transcription using the PrimeScript™ RT-PCR Kit (#RR014A, Takara) and the Fast PCR Master Mix (#HY-K0532, MedChemExpress, Beijing). The reverse transcription process was conducted at a temperature of 42 °C for a duration of 1 h, and subsequently, the enzyme was deactivated at a temperature of 70 °C for a period of 10 min. The PCR procedure was conducted utilizing SYBR Green qPCR Master Mix (#HY-K0501, MedChemExpress, Beijing) and SYBR qPCR Master Mix (Universal) (#22204, ToloBio, China) on QuantStudio 7 Pro equipment (Applied Biosystems). The pre-designed, precise primer sequences were acquired from OriGene Technologies, Inc.

Western blotting assay

In order to conduct the western blotting assay, the materials were prepared by homogenizing them with a commercially available RIPA lysis buffer solution (#BL504A, Biosharp, Shanghai, China). Subsequently, the ultimate homogenized extraction was condensed using centrifugation at a temperature of 4 °C and a speed of 13,000 rpm for a duration of 30 min. The protein samples obtained were standardized using the BCA Protein Assay Kit (Catalog number: YSD-500T, Yoche-Biotech, China), using lipid-free BSA as the control. The cell lysis samples were analyzed using a 12% ShineGelTM Plus Tris-Glycine PAGE kit (#SP0511, Shinegene, China) and transferred onto a 0.45 µM hydrophobic polyvinylidene fluoride transfer membrane (#IPFL00010, Sigma-Aldrich). Immunoblotting was performed using the specified primary antibodies. Afterwards, the PVDF membranes were subjected to treatment with Western Blocker™ buffer in a working solution of 1×TBS, which included 0.1% Tween-20 (#T104863, Aladdin, China), for a duration of 1 h. Subsequently, the membranes were incubated with the designated primary antibodies at a temperature of 4 °C for a period of 24 h. The PVDF membranes used in Western blotting were subjected to exposure using the ECL Plus kit test (#E266188, Aladdin, China) and subsequently exposed to medical X-ray film (Blue Ocean Imaging Systems, China). The protein contents were quantified as gray value levels using Image J, version 1.8.0, and then normalized to ACTIN as a fold change.

Labelling, click chemistry, and palmitoylation identification

The Click-iT™ Palmitic Acid (PA), Click-iT™ Cell Reaction Buffer Kit (#C10269), Click-iT™ Protein Reaction Buffer Kit (#C10276), and Azide Kit (#C10265) were acquired from Invitrogen for the purpose of conducting click chemistry and identifying palmitoylated PPARG, following the instructions provided with the products. Following the specified transfection of PPARG WT and PPARG C313S mutants, the cell media was treated with 100 μm Click-iT PA, azide, and kept in a 5% CO 2 , 37 °C environment for 5 h. After a treatment duration of 5 h, the cells were collected and subsequently rinsed four times with pre-cooled D-PBS (#abs9340, Absin, China). This was followed by a combination with lysis buffer containing 1×protease and a general phosphatase inhibitor cocktail (#abs9162, Absin, China). The lysates were thereafter kept at a temperature of 4 °C for a duration of 40 min, after which they were transferred to a centrifugal tube with a volume of 2 ml. The ultimate cell lysates were condensed using centrifugation at a temperature of 4 °C and a speed of 13,500 revolutions per minute for a duration of 10 min. The protein concentration of the collected supernatant was verified using the EZQ™ Protein Quantitation Kit (#R33200, Thermo Fisher Scientific) following the instructions provided in the product manuals. The extracted proteins were incubated with biotin-alkyne using the Click-iT™ Protein Reaction Buffer Kit, following the protocols outlined in the production instruction specification. Subsequently, the biotin-alkyne-palmitic acid and azide-protein complexes were subjected to streptavidin-mediated pulldown experiments using streptavidin magnetic beads (#HY-K0208, MedChemExpress, China). The resulting samples were then analyzed by western blotting using the PPARG antibody.

Furthermore, to enhance the identification of the palmitoylation of PPARG, an additional complementary approach was employed in this section. The cells were treated and lysed using a buffer containing 1% Triton X-100, 150 mM NaCl, 0.2% SDS, 50 mM TEA-HCl at pH 7.4. The buffer also contained 1×protease and phosphatase inhibitor cocktail. Additionally, a click reaction with biotin azide was performed. The cell proteins were extracted using 10 volumes of 100% methyl alcohol at a temperature of -80 °C for a duration of 1.5 h. Subsequently, the proteins were collected again by centrifugation at a speed of 13,500 revolutions per minute for a period of 15 min. The precipitates were resuspended in 100 ml of a 1×suspension buffer and subsequently diluted to a 12-fold immunoprecipitation solution. The immunoprecipitation solution consisted of 0.5% Nonidet P 40, 55 mM Tris-HCl, 160 mM NaCl, 6.5 mM EDTA, and had a pH of 7.5. The cell proteins that were marked with labels were concentrated using streptavidin agarose at a temperature of 30 °C, with a gentle swirling motion for a duration of 1.5 to 2.0 h. The protein-bound streptavidin agarose beads underwent four washes using an immunoprecipitation solution. Subsequently, the protein-bound beads were released using an elution buffer consisting of 95% formamide and 10 mM EDTA with a pH of 8.2, heated to 95 °C for a duration of 5 min. The samples were then analyzed using immunoblotting detection.

Fractionation assay

The separation of components was achieved using the Nuclear/Cytosol Fractionation Kit (#K266-100, BioVision, US) or Nuclear/Cytosol Extraction Kit (#ab289882, Abcam). The nuclear component was disrupted using a homogenization solution consisting of 5% SDS, 1×protease inhibitor cocktail, 2.5 mM PMSF, 160 mM NaCl, 60 mM triethanolamine, 1600 units/ml benzonase nuclease, pH 7.5. After removing the solution, a final homogenization of 6.5 mM was achieved by adding EDTA. Prior to the subsequent fractionation assay, the cytoplasmic component was diluted using a homogenization solution that contained 6 mM EDTA.

Cell proliferation detection

The CCK-8 assay was used to measure the proliferation of cancer cells. The cultured cells were placed in a 96-well plate and incubated for different lengths of time, with a density of 2.5 × 10 4 cells per well. After each treatment, the liquid above the culture was removed, and 15 µl of Cell Counting Kit-8 (CCK-8) from ApexBio Technology (#K1018) was added to each well. After being kept at a temperature of 37 °C for 2 h, the fluorescence intensity at a wavelength of 450 nm was quantified using a microplate reader. Colony formation was utilized as a technique to examine cell proliferation. To summarize, the designated cancer cells were inserted in a 24-well plate, with each well containing 200 cells. Following a period of 2 weeks, colonies with a cell count exceeding 50 were visually observed and measured using crystal violet staining.

Transwell assay

Cell migration and invasion were quantified using Transwell assay. The Corning ® Transwell ® (#CLS3422, Corning, USA) was cultured with Matrigel (#356234, BD, USA). The tumor cells that were identified were cultivated again in a medium without serum (#12–725 F, LONZA, USA) and then placed in the upper chamber with a density of 6.0 × 10 4 cells per well. Subsequently, the medium containing 10% fetal bovine serum (FBS) was introduced into the bottom chamber, and then administered for a duration of 48 h. Subsequently, the cells in the top chamber were entirely eliminated by means of a cotton swab. The cells that moved into the lower chamber were washed, treated with a 4% formaldehyde solution to preserve them, and then colored with a 0.65% crystal violet stain. Finally, the cells were visualized using a microscope. The migration calculation experiment was conducted using identical techniques, with the exception that Matrigel was omitted during the pre-coating of the upper chamber.

In vivo xenograft models construction

Pathogen-free male BALB/c nude mice (5 weeks old, 20–22 g, Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) underwent a subcutaneous procedure in the flank region, where 1.6 × 10 6 HCT116 or SNU-C2A cells were injected in a suspension of 80 µl PBS. The mice were split into treatment groups at random once the xenograft tumors reached the expected volume. Tumor dimensions were measured at regular intervals of 2–3 days using a vernier caliper. The tumor’s volume was determined by applying the formula 1/2×D (major axis)×d2 (minor axis). Mice were euthanized without causing pain after the tumor size reached 2 cm 3 or when ulceration was detected. All animal experimental protocols were performed following the Ethical Animal Care and Use Committee of Shandong University (2021sa0541jk).

Triglyceride detection and lipid accumulation in vitro assay

The measurement of TG was conducted using the Triglyceride Quantification Kit (#K622-100, BioVison, US). A total of 6 × 10 5 cells were gathered and subsequently combined with 1 ml of extraction reagent. The samples were subjected to ultrasonic treatment for a duration of 1 min, followed by centrifugation at a speed of 9000×g at a temperature of 4 °C for a duration of 10 min. The liquid portion was collected for analysis in accordance with the guidelines provided by the manufacturer. The Green Fluorometric Lipid Droplet Assay Kit (BODIPY-493/503) was employed to detect lipid deposition in CRC cells. This kit allowed visualization of the production of lipid droplets in the specified in vitro tests.

RNA sequencing

The TRNzol universal reagent was used to extract RNA from HCT116 cells, following the manufacturer’s procedure. The quantification of RNA samples was performed using the BioPhotometer Plus (Eppendorf), while the assessment of RNA integrity was conducted using the Agilent 2100 bioanalyzer system. The Collibri Stranded RNA Library Prep Kit for Illumina, provided by Thermo Fisher Scientific, was utilized for RNA sequencing library preparation, following the instructions provided by the vendor. The sequencing process was performed on the Illumina HiSeq instrument utilizing a 2 × 150 Paired End configuration, with 35 million reads obtained per sample by GENEWIZ, LLC. The construction of sequencing libraries was performed using the Small RNA-Seq Library Prep Kit (#052.08, Lexogen GmbH, Vienna, Austria) with total RNA as the starting material. In summary, the messenger RNA (mRNA) was extracted from the whole RNA using Sera-Mag SpeedBead particles and subsequently subjected to chemical fragmentation. The RNA fragments were subjected to reverse transcription, where they were converted into complementary DNA (cDNA) using random primers that had a tagging sequence attached to their 3′ ends. The cDNA libraries were subsequently amplified using the KAPA high-fidelity DNA polymerase. The validation of the libraries’ quality was conducted using the Agilent 2100 bioanalyzer. Following that, a high-throughput sequencing procedure was carried out utilizing an Illumina NovaSeq 6000. Following the mapping of sequences using the STAR or HISAT2 software against Homo sapiens or Mus musculus, a biological pathway analysis was conducted using Cluster-Profiler. The RNA sequencing and library creation were carried out by the technical personnel at Nanjing Biomed Sciences Research Bio-Lab.

Metabolomics and lipidomics assay

Approximately 30 milligrams of tissues were combined with 550 µl of a 75% methanol buffer to extract metabolites. The liquid portion was collected and subjected to lyophilization. The freeze-dried powder was dissolved in 60 µL of 80% methanol. Following centrifugation, the liquid remaining after sedimentation, known as the supernatant, can be used for analysis with the TSQ Fortis Plus (#TSQ03-10003, Thermo Fisher Scientific)-Orbitrap Astral High Resolution MS (Thermo Fisher Scientific). The process of extracting metabolites from cells involved the following steps: Firstly, cells collected in a 10-cm dish were washed with pre-cooled PBS and immediately frozen using liquid nitrogen. The cells were subsequently disrupted using 1.5 mL of 80% methanol solution containing internal standards, and then removed from the dish by scraping. The liquid portion obtained after spinning the mixture at high speed and removing the solid particles was preserved. This liquid was then subjected to a process of removing moisture by freezing and subsequent drying. The resulting dried material was used for the examination of small molecules involved in metabolism using a technique called liquid chromatography-mass spectrometry (LC-MS). The process of extracting lipids from cells was conducted as follows: briefly, cells that were gathered in a 10-cm dish were washed with PBS and immediately frozen using liquid nitrogen. The cells were subsequently disrupted using 1.5 mL of methanol containing internal standards, combined with 1.5 mL of chloroform, and vigorously mixed for 30 s using a vortex. Afterward, 500 µL of water were added and vortexed for an additional 30 s. The layer that repels water was gathered and subjected to freeze-drying. The dehydrated powder was reconstituted in 40 µl of an organic solvent mixture (Chloroform: Methanol, 2:1) using a vortex for 30 s, and then combined with 70 µl of another organic solvent mixture (Acetonitrile: Isopropanol: Water, 13:6:1). Following centrifugation, the liquid portion can be specifically utilized for TSQ Fortis Plus-Orbitrap Astral MS analysis.

Glucose [U- 13 C] tracer assay

The medium was replaced with RPMI 1640 supplemented with glucose (2 g/L) labelled with [U- 13 C] when the cell density reached around 80%. After 24 h, the cell culture plates were rinsed with PBS, rapidly frozen in liquid nitrogen, and kept at a temperature of -80 °C.

Statistical analysis

The data were expressed as the mean ± SEM (standard error of the mean) of three independent experiments conducted in triplicate. The software GraphPad Prism 8.0, developed by GraphPad Software in San Diego, USA, was utilized for the purposes of creating visual representations and doing statistical analysis. Unpaired Student’s t-tests were employed to compare the two groups. A unidirectional analysis of variance (ANOVA) was conducted, followed by a Bonferroni post-hoc test to compare different groups. A P value less than 0.05 was deemed to be statistically significant. The scientists were unaware of the animal genotype and grouping information.

Identification of potential genes strongly associated with the course and prognosis of colorectal cancer (CRC)

We examined the expression matrix of colorectal cancer and adjacent healthy tissues in public datasets that included high-coverage gene profiling data in the TCGA, ICGC, and NCBI Gene Expression Omnibus (GEO) databases (GEO: GSE254054, GSE231943, GSE252858, GSE234804, GSE236678, GSE231436, GSE197088, and GSE239549) in order to identify intriguing genes that regulate the progression of colorectal cancer (Fig.  1 A). The CRC tissues exhibited a substantial enrichment of events linked to tumors and processes related to metabolism (Fig.  1 B). Across the ten datasets we selected, we identified 41 differentially expressed genes (DEGs) that were conservatively upregulated and 101 DEGs that were downregulated (Fig.  1 C). Notably, based on clinical data from the TCGA and ICGC databases, 6 upregulated and 4 downregulated DEGs were identified because they showed a substantial correlation with survival rates (Fig.  1 D). Additionally connected to molecular processes influencing the development of colorectal cancer and metabolic reprogramming are these 6 upregulated and 4 downregulated DEGs (Fig.  1 E).

Identification of potential genes implicated in colorectal cancer (CRC) and cancer metabolism-associated biological processes. ( A ) A screening procedure to find putative gene candidates. ( B ) Colorectal cancer (CRC) samples were found to differ from adjacent controls in terms of physiopathology and biological processes related to metabolism in a number of databases, including TCGA, ICGC, and the NCBI Gene Expression Omnibus (GEO) datasets (GEO: GSE254054, GSE231943, GSE252858, GSE234804, GSE236678, GSE231436, GSE197088, and GSE239549). ( C ) Following gene differential expression analysis, the total number of differentially expressed genes that crossed over into various databases was counted. ( D ) Six upregulated and four downregulated DEGs were identified based on a survival analysis of differentially expressed genes across six databases.In the databases of TCGA and ICGC, P  < 0.05 was deemed statistically significant. ( E ) Six upregulated and four downregulated DEGs represent the molecular mechanisms impacting the onset of colorectal cancer and metabolic reprogramming. ( F ) Palmitoyltransferase ZDHHC6 expression in the ICGC and TCGA databases. ( G ) Pancarcinoma analysis using TCGA datasets to measure ZDHHC6 expression levels in various malignancies. ( H ) The overall survival (OS) of colorectal cancer patients in the TCGA and ICGC databases according to different ZDHHC6 expression levels. ( I ) After dividing the TCGA and ICGC samples’ ZDHHC6 expression levels into groups of high and low expression levels, the grouped samples underwent GSEA analysis. The data were expressed as the mean ± SEM. A P value less than 0.05 was considered statistically significant. *** P  < 0.001

Of the top four elevated DEGs most closely connected with CRC occurrences, we concentrated on ZDHHC6, the sole gene having the most underappreciated biological significance in CRC (Fig.  1 E). Using the TCGA and ICGC datasets, we further confirmed that CRC had higher levels of ZDHHC6 expression than surrounding tissues (Fig.  1 F, G). The samples in the TCGA and ICGC databases were split into two groups based on the ZDHHC6 expression value: one group was ZDHHC6 high-expression, and the other ZDHHC6 low-expression. Crucially, compared to those with low ZDHHC6 expression levels, those with high ZDHHC6 expression levels had a noticeably worse survival prognosis (Fig.  1 H). There were significantly more biological processes associated with cancer growth and metabolic reprogramming in the group with high ZDHHC6 expression on a regular basis. This demonstrated that ZDHHC6 is potentially associated with biological processes of CRC (Fig.  1 I).

In addition, we examined the potential involvement of ZDHHC6 expression in colitis and the advancement of colorectal cancer (CRC) related with colitis, by analysing the intersection of ZDHHC6 in many datasets. Colon samples obtained from persons diagnosed with Crohn’s disease (CD), ulcerative colitis (UC), and colorectal cancer (CRC), as well as from healthy donors (HC), exhibited markedly elevated levels of ZDHHC6 protein compared to the healthy individuals in the control group. This was particularly accurate for individuals with symptoms of ulcerative colitis (UC) and Crohn’s disease (CD). Individuals exhibiting characteristics of colorectal cancer (CRC) demonstrated elevated levels of ZDHHC6 protein content in comparison to those in the Crohn’s disease (CD), ulcerative colitis (UC), and healthy control (HC) groups (Supplementary Fig.  1 A). The Pearson multiple correlation and multiple linear regression analyses demonstrated a positive correlation between ZDHHC6 protein levels and plasma concentrations of ESR, CRP, and ProCT. These laboratory markers are commonly used in the diagnosis of clinic colitis (Supplementary Fig.  1 A, B). Significantly, we also verified a negative correlation between the levels of albumin (ALB) in the bloodstream, a potential indicator of inflammatory bowel disease (IBD), and the levels of ZDHHC6 in the colon. The results suggest a link between inflammatory bowel disease (IBD) and the concentrations of ZDHHC6 in the tissue of the colon (Supplementary Fig.  1 A, B). A similar observation was documented in mice with colitis caused by dextran sulphate sodium (DSS), a frequently employed chemically-induced experimental model for colitis. Also, a notable elevation in ZDHHC6 protein levels as the disease advanced, as observed using the western blotting experiment (Supplementary Fig.  1 C). Furthermore, considering the various physical characteristics and underlying factors that worsen both sudden and long-lasting inflammation of the colon, the objective of this study was to investigate whether changes in ZDHHC6 expression played a role in the advancement of colorectal cancer (CRC) and the development of chronic colitis caused by DSS. The control mice were given AOM + DSS treatment for a period of ten weeks to promote the onset of colitis-associated carcinogenesis (Supplementary Fig.  1 D). Not surprisingly, there was a noticeable and significant increase in ZDHHC6 expression in colon samples from the mouse model with the CRC phenotype (Supplementary Fig.  1 D). These findings indicated that abnormally upregulated ZDHHC6 levels are correlated with the severity of colitis and the development of CRC.

Disturbed lipid metabolism in human CRC with upregulated ZDHHC6 levels

In addition to the substantial rise in ZDHHC6 observed in the TCGA dataset (Fig.  1 G) and the ICGC database (Supplementary Fig.  2 A), In order to confirm the increased expression of ZDHHC6 in colorectal cancer (CRC), we obtained 73 pairs of CRC samples and their matching adjacent samples. Our analysis revealed a significant increase in the mRNA expression levels of ZDHHC6 in CRC tissues compared to nearby normal tissues (Fig.  2 A). In addition, the analysis of proteins revealed that both human CRC tumour tissues and CRC-related cell lines (SNU-C2A, SW48, HT-29, LS1034, HCT116, and Caco-2) as well as mouse-associated colon cancer cell lines (CT26, MC38, CMT93, and MC26) exhibited elevated levels of ZDHHC6 expression. This finding was further validated through western blotting and immunofluorescence detection (Fig.  2 B-E, Supplementary Fig.  2 B). Hence, we examined potential processes that could contribute to the overexpression of ZDHHC6 in colorectal cancer (CRC). Subsequently, we identified the changes in its expression profile when exposed to 2-bromopalmitate (2-BP), a broad inhibitor of protein palmitoylation. The ZDHHC6 protein expression in human CRC cell lines was significantly decreased in a dose-dependent manner when exposed to a concentration gradient of 2-BP, as shown in Fig.  2 F and G. In addition, the administration of 2-BP leads to the inhibition of ZDHHC6, which is directly associated with the decrease in Ki67-positive colon cancer cells. This correlation is supported by the results of an in vitro immunofluorescence experiment (Fig.  2 H).

Increased ZDHHC6 is positively associated with the development of human colorectal cancer (CRC). ( A ) ZDHHC6 mRNA expression levels in 73 pairs of CRC sample pairs (T) and their corresponding adjacent sample pairs (N). n  = 73 pairs. ( B ) ZDHHC6 protein expression levels in sixteen pairs of similar adjacent tissues and colorectal cancer tissues selected at random. For each group, n  = 3. ( C ) ZDHHC6 mRNA expression levels in relation to a range of CRC-associated cell lines, such as SNU-C2A, SW48, HT-29, LS1034, HCT116, and Caco-2, as well as the matching human normal colonic epithelial cell line (FHC), are displayed in qPCR analysis. For each group, n  = 5. ( D , E ) ZDHHC6 protein expression in SNU-C2A, SW48, HT-29, LS1034, HCT116, Caco-2, and FHC cell line as demonstrated by western blotting ( D ) and immunofluorescence analysis ( E ). 200 μm; each group has n  = 5. ( F , G ) qPCR analysis ( F ) and western blotting experiment ( G ) demonstrate the effect of the gradually increased dosage of 2-bromopalmitate (2-BP) on the relative ZDHHC6 mRNA and protein expression levels in HCT116, SNU-C2A, SW48, and Caco-2 cell lines. For each group, n  = 3. ( H ) An immunofluorescence assay demonstrating the co-expression of ZDHHC6 and Ki67 in response to 40 µM 2-bromopalmitate (2-BP) in HCT116, SNU-C2A, SW48, and Caco-2 cell lines. 200 μm; each group has n  = 3. Data are expressed as mean ± SEM. The relevant experiments presented in this section were performed independently at least three times. P  < 0.05 indicates statistical significance

Considering the significant impact of ZDHHC6 on the advancement of colorectal cancer (CRC) and its probable involvement in the metabolic processes of tumours, we further examined the influence of ZDHHC6 on the molecular metabolism of CRC. To characterise the disrupted metabolic processes in colorectal cancer (CRC), a high-throughput metabolomics analysis was conducted to identify the metabolites that were significantly altered in 10 sets of CRC and adjacent normal tissues (Fig.  3 A, B). In comparison to the adjacent normal tissues, there are 39 metabolites that exhibit significant changes in the cancerous tissues. The elevated metabolites consist primarily of lipids and lipid-like compounds, specifically fatty acids (FAs), phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), sphingomyelin (SM), and lysophosphatidylcholine (LPC) (Fig.  3 C). An additional analysis was conducted to determine the enrichment of specific pathways utilising 39 metabolites that showed differential expression. The results indicated that the production of triacylglycerol, glycerol phosphate shuttle, and palmitoylated protein were enriched (Fig.  3 D, E). The findings validated that the lipidome composition exhibited a substantial increase in CRC tissues. However, the potential explanation for this abnormally vigorous lipid metabolism in CRC tissues remains uncertain. To investigate the potential connection between the abnormally increased lipidome in CRC and the expression of ZDHHCs, we analysed the expression of ZDHHCs in the aforementioned pairings of cancerous and neighbouring normal tissues. Our findings revealed that ZDHHC6 was the ZDHHC member with the highest level of expression, as shown in Fig.  3 F. The modified expression of ZDHHC6 was strongly associated with the synthesis of lipids and lipid-like metabolites in SNU-C2A and HCT116 cells when ZDHHC6 was either suppressed (sh ZDHHC6 ) or enhanced (Ad ZDHHC6 ) (Supplementary Fig.  2 C, D). The Pearson multiple correlation and multiple linear regression analyses revealed a significant association between the levels of ZDHHC6 protein and various tumour markers, including CA125, CA50, CA724, CA199, CEA, CA242, CK-BB, and HCG (Fig.  3 G, H). These findings indicate a potential correlation between elevated levels of ZDHHC6 and an aberrant rise in lipid and lipid-like metabolites in CRC. However, the precise function of ZDHHC6 in lipid metabolism in CRC remains uncertain.

Upregulated ZDHHC6 levels contribute to disturbed lipid metabolism in human colorectal cancer (CRC). ( A ) To identify significantly altered metabolites in ten pairs of colorectal cancer (CRC) samples and adjacent normal samples from CRC patients, a high-throughput LC-MS-based untargeted metabolomic analysis was carried out. Postoperative pathology confirmed that all patients had colorectal cancer; no other cancers or long-term medical problems were present. ( B ) The selected ten pairs of colorectal cancer (CRC) samples and adjacent normal samples from CRC patients were subjected to principal component analysis (PCA). ( C ) Heatmap of tumor samples (T) with significantly altered metabolites compared to corresponding normal adjacent tissue (N). Significant changes have been observed in 39 metabolites in cancerous tissues. Wilcoxon test with paired two-samples, P  < 0.05. The fold change is indicated by -2.0 ~ 2.0 (Fc). ( D ) Based on 39 significantly changed metabolite clusters discovered by pathway analysis ( https://www.metaboanalyst.ca/ ), the pie chart illustrates the improved metabolic signaling pathways. ( E ) A chord diagram shows the direction and distribution of data flow as well as the relationships between different metabolic signaling pathways. ( F ) Heatmap analysis of the ZDHHCs protein expression fold change (T/N) in the normal adjacent samples and the CRC samples. Wilcoxon test with paired two-samples, P  < 0.05. The fold change is indicated by -2.0 ~ 2.0 (Fc). ( G ) The levels of ZDHHC6 protein and several tumor markers, such as CA125, CA50, CA724, CA199, CEA, CA242, CK-BB, and HCG, significantly correlate, according to Pearson multiple correlation analyses ( n  = 73 per parameter). ( H ) Pearson r: multiple linear regression showing the overall association between ZDHHC6 protein levels and other tumor markers, such as CK-BB, HCG, CEA, CA242, CA724, CA199, CA125, and CA50 ( n  = 73 per parameter) Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P  < 0.05 indicates statistical significance

ZDHHC6 enhances lipid deposition and carcinogenesis in CRC cells

Consistent with our previous findings, we conducted additional experiments to specifically determine the function of ZDHHC6 in colorectal cancer (CRC). We investigated the impact of introducing Ad ZDHHC6 or Adsh ZDHHC6 into SNU-C2A and HCT116 cells on their rate of cell division (Supplementary Fig.  3 A, B). Interestingly, SNU-C2A and HCT116 cells that were transfected with AdshZDHHC6 showed a significant decrease in cell viability and EdU staining intensity. Conversely, cells transfected with Ad ZDHHC6 exhibited an increase in cell viability and EdU intensity. These findings suggest that alterations in ZDHHC6 expression in CRC cells may have an impact on proliferation in a laboratory setting (Supplementary Fig.  3 C, D). Furthermore, our current study supports the notion that ZDHHC6 may play a role in the development of colorectal cancer (CRC) pathogenesis. This is consistent with our findings, as demonstrated by the transwell analysis, which showed a significant decrease in the invasion and migration of tumor cells when ZDHHC6 was silenced, compared to the control group (Supplementary Fig.  3 E, F). Consistent with expectations, the levels of epithelial-mesenchymal transition (EMT)-related markers such as vimentin, N-cadherin, fibronectin, TGFβ1, and MMP13 were significantly reduced in SNU-C2A and HCT116 cells. Conversely, the expression of E-cadherin was greatly increased (Supplementary Fig.  3 G, H). Furthermore, in vivo experiments demonstrated that inhibiting ZDHHC6 effectively decreased the rates of tumor growth and the weights of tumors in mice that had been implanted with the SNU-C2A tumor models (Supplementary Fig.  3 I). The findings confirmed that the lack of ZDHHC6 played a role in inhibiting the growth of colon cancer cells and the process of EMT. Afterward, we discovered 36 metabolites that showed substantial changes in ZDHHC6 -deletion CRC cells. Fatty acid metabolites exhibited a considerable drop in HCT116 cells lacking ZDHHC6 . Furthermore, we conducted pathway enrichment analysis on the set of 36 metabolites and observed a significant association between ZDHHC6 and the pathways related to fatty acid production (Fig.  4 A-C). Glucose is a crucial source for the creation of fatty acids through de novo biosynthesis. Subsequently, we employed glucose [U- 13 C] to monitor the process of fatty acid production. Our findings revealed that the cells with ZDHHC6 knockdown exhibited a substantial reduction in the labeling of palmitic acid and stearic acid from the glucose trackers (Fig.  4 D, Supplementary Fig.  4 A). In contrast, the HCT116 cells that had an enhanced expression of ZDHHC6 showed a considerable increase in the labeling of palmitic acid and stearic acid from glucose (Fig.  4 E, Supplementary Fig.  4 A). In addition, ZDHHC6 markedly enhanced the formation of triglycerides in HCT116 cells, as demonstrated by the bodipy staining experiment (Fig.  4 F). Additionally, we conducted analogous tests on SW48, HT-29, and Caco-2 cell lines. We noted a substantial increase in the expression of several fatty acids and triglycerides in the colorectal cancer cells that were overexpressing ZDHHC6 (Supplementary Fig.  4 B, C). Collectively, these findings offer proof that ZDHHC6 is crucial in the buildup of lipid content and may stimulate the production of fatty acids from scratch in CRC cells.

ZDHHC6 facilitates lipid deposition and carcinogenesis in CRC cells. ( A ) A venn diagram shows the variations in metabolites produced by HCT116 cells with ZDHHC6 knockout (KO) and wild-type (WT) phenotypes. ZDHHC6 and fatty acid synthesis pathways have a significant association, according to pathway enrichment analysis of the 36 metabolites. Total peak area was used to correct the LC-MS-based untargeted metabolomic study and its findings. ( B ) Using these 36 differential metabolites, pathway analysis showed enhanced signaling pathways. ( www.metaboanalyst.ca ). ( C ) A heatmap showing how these 36 significantly altered metabolites changed. Student’s t -test, unpaired, two-tailed, P  < 0.05. The fold change is indicated by -2.0 ~ 2.0 (Fc). ( D , E ) The ratios of various isotopic forms of FFA C16:0 (palmitate) in ZDHHC6 (KO) (D) and Ad ZDHHC6 (E) HCT116 cells after a brief exposure to glucose [U- 13 C]. When the cell density was around 85%, the media was changed to RPMI 1640 containing 2 g/L glucose tagged with [U- 13 C]. Following a 24-hour period, the PBS-rinsed cell culture plates were quickly frozen in liquid nitrogen and subjected to an LC-MS assay analysis ( n  = 4 per group). ( F ) Representative immunofluorescence pictures of HCT116 cells with ZDHHC6 (WT) and ZDHHC6 (KO) phenotypic, demonstrating ZDHHC6 expression, lipid accumulation (Bodipy staining), and corresponding intracellular triglyceride (TG) levels ( n  = 4 per group). ( G , H ) ZDHHC6 (WT) and ZDHHC6 (KO) HCT116 cells were injected into the right flanks of nude mice. Every two days, tumor volumes were measured. On day 22 following dissection, tumor pictures (G), growth curves, and weight (H) were recorded ( n  = 4 per group). Scale bars, 1 cm. ( I ) A heatmap utilizing untargeted metabolomic analysis comparing significantly changed metabolites between tumors originating from ZDHHC6 (KO) HCT116 cells and ZDHHC6 (WT) cell lines. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P  < 0.05 indicates statistical significance

Subsequently, we conducted experiments involving both loss-of-function and gain-of-function assays on CRC cell lines to validate the involvement of ZDHHC6 in carcinogenesis. To investigate the role of ZDHHC6 in colorectal cancer (CRC) cells in living organisms, we implanted ZDHHC6 -deficient HCT116 cells and ZDHHC6 -overexpressing HCT116 cells under nude mice. Tumor sizes and weights were significantly reduced in animals implanted with ZDHHC6 -deficient HCT116 cells compared to those implanted with control cells (Fig.  4 G, H). In contrast, the upregulation of ZDHHC6 enhanced the tumorigenic capacity of HCT116 cells (Supplementary Fig.  4 D). Conversely, the tumours originating from ZDHHC6 -deleted HCT116 cells exhibited a notable reduction in several lipid metabolites, such as fatty acids, when compared to tumours formed from control cells (Fig.  4 I). In summary, our data validate that ZDHHC6 significantly contributes to lipid buildup and carcinogenesis in CRC.

ZDHHC6 specifically targets and interacts with lipid metabolism key transcription factor of PPARγ

To explore the impact of ZDHHC6 on lipid accumulation in colorectal cancer (CRC), we conducted high-throughput RNA sequencing analysis to examine the possible effects of ZDHHC6 on key enzymes involved in fatty acid synthesis in CRC cells. Out of the top 36 genes that showed significant differences in expression, previously identified genes involved in the production of free fatty acids (FFA) were shown to be responsive to the depletion of ZDHHC6 (Supplementary Fig.  5 A). The overexpression of ZDHHC6 was found to be linked to the process of lipid biosynthesis, as proven by GO-biological process analysis (Supplementary Fig.  6 A). Subsequent GSEA analysis revealed that the gene sets related to the production of fatty acids were considerably increased in HCT116, Caco-2, SNU-C2A, and SW48 cells that were transduced with ZDHHC6 overexpression, as supported by Supplementary Fig.  6 B. The abnormal increase in fatty acid synthesis is strongly associated with the main metabolic enzymes ACLY, ACC, FASN, and SCD1. To validate the role of ZDHHC6 in regulating fatty acid metabolic genes, we conducted qPCR analysis on CRC cells with ZDHHC6 knockdown and cells with ZDHHC6 overexpression. The mRNA levels of ACC, ACLY, FASN, and SCD1 were reduced in the ZDHHC6-knockdown HCT116 and SNU-C2A cells, but increased in the ZDHHC6-overexpressing cells (Supplementary Fig.  5 B). In addition, we conducted a thorough investigation to determine the potential involvement of ZDHHC6 in the regulation of fatty acid degradation. To achieve this, we analyzed the expression of enzymes related to fatty acid degradation (ACOX-1, CPT1A, LCAD, UCP2, and MCAD) in stable cells engineered with ZDHHC6. We employed qRT-PCR and observed that ZDHHC6 indeed influenced the expression of these enzymes in CRC cells (Supplementary Fig.  6 C-G). To verify the role of ZDHHC6 in promoting lipidome accumulation via ACC and ACLY, we introduced ZDHHC6 into HCT116 and SNU-C2A cells that had been depleted of ACC or ACLY. Our findings indicate that the introduction of ZDHHC6 did not significantly enhance the buildup of intracellular fatty acids and triglycerides in the ACC or ACLY knockdown cells, as observed in cells transfected with ZDHHC6 alone (Supplementary Fig.  5 C, D). Collectively, these findings suggest that ZDHHC6 enhances the accumulation of lipid molecules by upregulating the expression of ACC and ACLY in colorectal cancer.

Subsequently, to determine the precise substrate targeted by ZDHHC6, which has the ability to control the production of ACC and ACLY, we employed a method of isolating the ZDHHC6-associated protein complex in HCT116 cells using tandem affinity purification, followed by analysis using mass spectrometry. Notably, PPARγ (PPARG) was ranked at the top of the list (Fig.  5 A). In addition, our research revealed that PPARγ is the sole counterpart among many transcription factors associated with lipid metabolism, such as PPARα, PPARδ, and SREBF1 (Fig.  5 B). Furthermore, endogenous PPARγ was found in endogenous ZDHHC6 immunoprecipitates from SNU-C2A, SW48, HT-29, LS1034, and Caco-2 cells (Fig.  5 C). Through in vitro pulldown tests using purified recombinant proteins, it was shown that ZDHHC6 directly interacts with PPARγ, as depicted in Fig.  5 D. Given significant potential correlation of ZDHHC6 with PPARγ, we next integrated the ZDHHC6-interacting proteins obtained from the IP-MS experiment and the upregulated proteins revealed by proteomics analysis, and we identified PPARγ as the protein that interacts with and is regulated by ZDHHC6 using human HCT116, Caco-2, SNU-C2A and HT-29 colon cancer cell lines (Fig.  5 E). Moreover, to further demonstrate the specific target of ZDHHC6 responsible for its specific function, we performed an additional immunoprecipitation-MS (IP-MS) analysis of Flag-ZDHHC6 and His-PPARγ-transfected HCT116, Caco-2, SNU-C2A, and HT-29, respectively, and identified 126 detectable potential ZDHHC6-interacting proteins. We then integrated the ZDHHC6-interacting proteins obtained from the IP-MS experiment and the upregulated proteins revealed by proteomics analysis, and we identified PPARγ as the key protein that interacts with and is regulated by ZDHHC6 (Fig.  5 E). On the other hand, Flag-ZDHHC6 and His-PPARγ were overexpressed in HCT116, Caco-2, SNU-C2A, and HT-29, respectively. Co-immunoprecipitation (Co-IP) experiments demonstrated that ZDHHC6 co-immunoprecipitated with PPARγ, and vice versa (Fig.  5 F). Glutathione S -transferase (GST) pull-down assay suggested that ZDHHC6 interacts directly with PPARγ in transfected Caco-2 and SNU-C2A cells, respectively (Fig.  5 G). This GST pull-down result is consistent with the direct protein interaction of ZDHHC6 and PPARγ in HCT116 cells (Fig.  5 D). The immunofluorescent staining test demonstrated a localization of ZDHHC6 (major in the cytoplasm) and PPARγ (major in the nucleus) mostly within the CRC cells (Fig.  5 H). According to reports, PPARγ is composed of three distinct structural domains: AF-1, DBD, and the hinge region. Pull-down experiments demonstrated a robust association between ZDHHC6 and the DBD fragment of PPARγ, but only a few interactions were observed with the hinge-region fragment (Fig.  5 I). In addition, an examination of the TCGA-CRC and ICGC-CRC databases revealed a substantial correlation between ZDHHC6 and the PPARγ pathway in CRC, as demonstrated in Fig.  5 J. In summary, our findings validate that ZDHHC6 has a unique interaction with PPARγ, a crucial transcription factor involved in lipid metabolism.

ZDHHC6 specifically binds to the lipid metabolism key transcription factor of PPARγ. ( A ) After 24 h of SFB-ZDHHC6 transfection in HCT116 cells, ZDHHC6-interacting proteins were identified by tandem affinity purification and mass spectrometry (MS). This was accomplished by removing S-protein, Flag, and streptavidin binding peptide (SFB). ( B ) ZDHHC6 or IgG antibodies were used to immunoprecipitate HCT116 cell lysates, and PPARγ, PPARα, PPARδ, SREBP1, and ZDHHC6 antibodies were used for western blotting experiments. ( C ) ZDHHC6 or IgG antibodies were used to immunoprecipitate cellular lysates of SNU-C2A, SW48, HT-29, LS1034, and Caco-2 cells, and ZDHHC6 or PPARγ antibodies were used for western blotting experiments. ( D ) GST pulldown assay using GST-PPARγ and purified His-ZDHHC6 in HCT116 cells. ( E ) Schematic of the experimental procedure showing the genes expression in HCT116, Caco-2, SNU-C2A and HT-29 after adenovirus-mediated ZDHHC6 overactivation (Ad ZDHHC6 ). The lower schematic diagram showing the intersection of the results from the proteomics and IP-MS analyses. ( F ) For a duration of 24 h, plasmids expressing Flag-PPARγ or Myc-ZDHHC6 individually or in combination were transfected into HCT116, Caco-2, SNU-C2A and HT-29 cells, respectively. His or Flag antibodies were used for immunoblotting after cellular lysates had been immunoprecipitated with Flag and/or His antibodies. ( G ) GST pulldown assay using GST-PPARγ and purified Flag-ZDHHC6 in Caco-2 and SNU-C2A cells, respectively. ( H ) Assay for immunofluorescence staining demonstrating ZDHHC6 and PPARγ co-expression in HCT116, Caco-2, and SNU-C2A cells. 20 μm. ( I ) In HCT116 cells, vectors containing the hinge-LBD domain, full length (FL), AF-1, DBD, and PPARγ were co-expressed with SFB-ZDHHC6. S-bead pulldown was used to immunoprecipitate cellular lysates. ( J ) Based on GSEA signaling pathway analysis, an assay of the TCGA-CRC and ICGC-CRC datasets showed a significant connection between ZDHHC6 and the PPARγ pathway in CRC. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P  < 0.05 indicates statistical significance

ZDHHC6 palmitoylates and stabilizes PPARγ

Given the strong association between ZDHHC6 and PPARγ, as well as the crucial role of ZDHHC6 as a palmitoyltransferase in various biological processes, we hypothesized that ZDHHC6 may regulate the palmitoylation of PPARγ in response to alterations in lipid metabolism that occur during the progression of CRC. Therefore, to determine the role of palmitoylated PPARγ in the advancement of CRC, we analyzed its expression patterns and changes in response to 2-bromopalmitate (2-BP), a broad inhibitor of protein palmitoylation. Upon incubation of HCT116 cells with 2-BP to decrease the levels of palmitoylated proteins, there was a significant decrease in the expression of PPARγ, both in terms of protein abundance and pan-palmitoylation contents (Fig.  6 A). In contrast, the expression levels of PPARγ in HCT116 cells were increased by the overexpression of palmitoylation by the use of palmostatin B (Palm B), palmostatin M (Palm M), or ABD957, which is an inhibitor of depalmitoylase enzymes. This increase in expression levels was confirmed through colocalization immunofluorescence analysis (Fig.  6 A). In order to assess the impact of 2-BP on the decrease of PPARγ expression, a click chemistry assay was utilized to visualize the palmitoylated PPARγ (Fig.  6 B). The presence of palmitoylation on PPARγ was verified using the streptavidin beads-mediated pulldown and western blotting analysis, as anticipated (Fig.  6 B).

Identification of the palmitoylation site on PPARγ at evolutionarily conserved cysteine residues. ( A ) For a duration of 24 h, HCT116 cells were exposed to 60 µM 2-BP, 1 µM ABD957, 6 µM palmostatin B (Palm B), and 10 µM palmostatin M (Palm M) treatments. The slices that were fixed underwent immunofluorescence labeling using PPARγ (red) and pan-palmitoylation (green). 10 μm scale bars; n  = 5 per group. ( B ) Schematic diagram of the Click-iT assay for palmitoylation measurement of PPARγ. HCT116 cells were treated with 100 µM Click-iT PA and azides for five hours. The resulting lysates were then submitted to Click-iT detection as per the product instructions, and PPARγ antibody western blotting analysis was performed. The indicated group’s expression of PPARγ is indicated by the western blotting bands on the right. ( C ) Using the GPS-Palm program (MacOS_20200219) (The CUCKOO Workgroup, http://gpspalm.biocuckoo.cn/ ) and the MDD-Palm algorithm ( http://csb.cse.yzu.edu.tw/MDDPalm/ ), the palmitoylation site on PPARγ in Homo sapiens (upper) and Mus musculus (lower) is predicted to be located. PPARγ’s lower palmitoylation site contains conserved cysteine residues shared by Rattus norvegicus , Bos taurus , Canis familiaris , Mus musculus , and Homo sapiens . ( D ) After incubating Click-iT PA and azides for five hours on HCT116 cells overexpressing either PPARγ WT or PPARγ C313S mutant, the corresponding cellular lysates were obtained and Click-iT detection was performed in compliance with the product’s instructions. After the palmitoylated proteins were added to the streptavidin-sepharose bead conjugate for pull-down detection, PPARγ and ACTIN antibodies were used in a western blotting examination. While PPARγ C313S was not palmitoylated in top gel, lane 6, or the control groups, it was for PPARγ WT in lane 5. Three separate runs of this experiment were conducted. ( E ) CHX was cultured with HCT116 cells overexpressing either the PPARγ WT or PPARγ C313S mutant for a specific amount of time. PPARγ and ACTIN antibodies were used in immunoblotting detection of the obtained cellular lysates. The relative PPARγ remaining ratio ( n  = 4 per group) is displayed in the right curve graph at the specified time point. ( F ) PPARγ WT or PPARγ C313S mutant overexpression was observed in the upper HCT116 cells. Pan-palmitoylation (green) and PPARγ (red) immunofluorescent labeling were applied to the cell sections. Lower, Ad ZDHHC6  + PPARγ C313S mutant or PPARγ C313S alone were overexpressed in HCT116 cells, respectively. The bar graph displays the intensity of PPARγ fluorescence in each of the indicated groups ( n  = 5 pictures; P  < 0.05 vs. PPARγ C313S + AdControl or PPARγ WT). Scale bars, 20 μm. ( G ) In HCT116 cells, PPARγ-Flag and ZDHHC6-HA plasmids were transfected. Alk16 labeling was used to determine the palmitoylated PPARγ expression contents in the presence or absence of hydroxylamine therapy. ( H ) PPARγ-Flag was used to transfect SNU-C2A cells (WT) or ZDHHC6-deleted SNU-C2A cells, and Alk16 was used to label the cells. Subcellular fraction was extracted, and the levels of PPARγ protein were adjusted to verify that the input cells from the wild type and the knockout cell had the same quantity of PPARγ. Immunoblotting analysis was used to evaluate the palmitoylated PPARγ expression contents in the cell membrane (Mem.), cell cytoplasm (Cyto.), and cell nucleus (Nuc.) components. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P  < 0.05 indicates statistical significance

Furthermore, given the significant role of palmitoylation in the control of PPARγ stability, we were compelled to determine the precise position of palmitoylation on PPARγ. The function investigation involved determining the anticipated location of the palmitoylation site on PPARγ for Homo sapiens and Mus musculus . This was achieved through a combined analysis of the GPS-Palm software (MacOS_20200219) developed by The CUCKOO Workgroup ( http://gpspalm.biocuckoo.cn/ ) and the MDD-Palm algorithm ( http://csb.cse.yzu.edu.tw/MDDPalm/ ). Both methods fortuitously anticipated and furnished the foremost 4 palmitoylation sites for PPARγ, exhibiting distinct confidence intervals and quality ratings. It is worth mentioning that cysteine 313 (C313) in human PPARγ and mouse PPARγ were identified as the most probable and dependable location for protein palmitoylation modification (Fig.  6 C). Furthermore, the cysteine residue location exhibited significant conservation across many species taxa, as depicted in Fig.  6 C. The presence of PPARγ protein in HCT116 and SNU-C2A cells was confirmed by detecting the residual PPARγ WT, C176S, C159S, C156S, and C313S mutants following CHX injection. As predicted, only the C313S mutation showed a significant decrease in the amount of PPARγ, while the other variants did not show any significant changes (Supplementary Fig.  7 A, B). The impact of a sequence of genetic alterations on the buildup of lipids in the specified transfected colorectal cancer (CRC) cells was additionally validated using immunofluorescence analysis (Supplementary Fig.  7 C, D). Substitution of the C313 residue with serine completely inhibited the palmitoylation of PPARγ, as demonstrated by the Click-iT chemistry experiment (Fig.  6 D). The C313S mutant significantly decreased the expression of PPARγ protein profiles without affecting its mRNA levels (Fig.  6 D). The PPARγ with C313S mutant underwent degradation in parallel with CHX treatment, leading to a progressive reduction in the remaining PPARγ levels (Fig.  6 E), which was comparable to the impact of 2-BP administration. Considering the identification of C313 as the primary location for PPARγ palmitoylation, we are curious about the extent to which S-palmitoylation at C16 contributes to the chemical alteration of PPARγ. Hence, a sequence of alkyl-labeled fatty acids, namely Alk14, Alk16, Alk18, and Alk20, were employed to investigate this biological phenomenon. PPARγ can be efficiently labeled with palmitoylation (using Alk16) but shows significantly lower efficiency when labeled with Alk14, stearoylation (Alk18), or Alk20 (Supplementary Fig.  8 A-C). This suggests that C16-captured S-palmitoylation is the primary acyl group involved in the chemical alteration of PPARγ. Having the C313 mutation as the critical site of PPARγ palmitoylation, we next evaluated the functional impact of these mutants on C16-catched S-palmitoylation over the duration of alkynyl palmitic acid (Alk16) therapy. In the CRC cells that were genetically modified, the presence of PPARγ with human C313S effectively eliminated the labeling of palmitoylation (with Alk16). This was confirmed by the streptavidin pull-down study, which showed that C313 was necessary for the S-palmitoylation of the PPARγ protein (Supplementary Fig.  8 A-C). Furthermore, the role of ZDHHC6 as the primary target for PPARγ was established using a comparative study. This analysis demonstrated the anticipated changes in the quantity of PPARγ protein in HCT116 cells when ZDHHC6 was overexpressed using adenovirus, both with and without the PPARγ C313S mutation. This was confirmed by an immunofluorescence assay (Fig.  6 F). In addition, the absence of palmitoylation due to the C313S mutation resulted in a reduction in the simultaneous expression of PPARγ and pan-palmitoylation, as indicated in Fig.  6 F. Based on our previous findings, we have determined the interaction between ZDHHCs and their targeted substrates. The Co-IP test clearly showed a strong direct binding between ZDHHC6 and PPARγ in the ectopic expression of CRC cells (Fig.  5 ). To enhance the visual representation of PPARγ palmitoylation in the presence of ZDHHC6, we employed Alk16 as a metabolic marker to assess the impact of ZDHHC6 on palmitoylated PPARγ and its movement inside cells, similar to what was shown in Supplementary Fig.  8 . The presence of NH 2 OH significantly reduced the levels of palmitoylation on PPARγ, suggesting that the increase in palmitoylated PPARγ primarily occurred on cysteine residues and was caused by the action of ZDHHC6 (Fig.  6 G). Analysis of fractionation in ZDHHC6 wild-type or ZDHHC6 -deficient HCT116 cells indicated that the palmitoylation of PPARγ by ZDHHC6 increased the quantity of the modified PPARγ protein specifically in the cell nucleus, but not in other cellular components (Fig.  6 H). The reliable outcomes of modifying the level of PPARγ through palmitoylation were further validated in HCT116 cells that overexpressed ZDHHC6 (Supplementary Fig.  8 D). The aforementioned results consistently demonstrated that ZDHHC6 plays a crucial role as a primary palmitoyltransferase in the formation of palmitoylated PPARγ. Furthermore, its function exhibited a significant correlation with the advancement of colorectal cancer in human individuals.

Upregulated ZDHHC6-mediated palmitoylated PPARγ promotes its nucleus translocalization

Following the identification of ZDHHC6 as the primary palmitoyltransferase for PPARγ, the subsequent step was examining the impact of ZDHHC6 on the nucleus localization of PPARγ. This enzyme had an impact not only on the stabilization of PPARγ throughout the process of ZDHHC6 knockdown, but also on the palmitoylation of PPARγ (Fig.  7 A-C). In addition, the expression of ZDHHC6 by ectopic expression in HCT116, SNU-C2A, SW48, HT-29, and Caco-2 cells led to an increase in the expression of PPARγ protein and a speeding up of its translocation to the nucleus of the CRC cells (Fig.  7 D, E). The purpose of this investigation was to determine whether ZDHHC6 interacts with PPARγ in additional colorectal cancer cells. This was done in consideration of the determination of the interaction between DHHCs and their targeted substrates. As a matter of fact, the Co-IP test demonstrated that the direct interaction between ZDHHC6 and PPARγ could be wonderfully observed in the ectopic expression of human Caco-2 cells (Fig.  7 F). Furthermore, considering the fact that C313 site has been identified as the primary location for palmitoylation of PPARγ, we pose the question of whether or not the S -palmitoylation that was captured by C16 was the primary contributor to the chemical change of PPARγ. Consequently, to investigate this biological process, a series of alkyl-labeled fatty acylation operations, which included alk-C14, alk-C16, alk-C18, and alk-C20, were utilized. Palmitoylation (alk-C16) labels are able to successfully mark the PPARγ, whereas labels with alk-C14 chain lengths, stearoylation (alk-C18), or alk-C20 labels are significantly less effective in marking the PPARγ (Fig.  7 G, H). The findings of this study suggest that the primary acyl group responsible for the chemical alteration of PPARγ is C16-catched S -palmitoylation. After determining that the C313S site mutation is the most important location for PPARγ palmitoylation, we proceeded to investigate the functional effects of these mutations on C16-catched S -palmitoylation during the course of treatment with alkynyl palmitic acid (alk-C16). It was anticipated that PPARγ with a human C313S site mutation would drastically eliminate palmitoylation (alk-C16) labels, as corroborated by streptavidin pull-down analysis, which further demonstrated that human C313S was necessary for S -palmitoylation of PPARγ protein (Fig.  7 I). Moreover, to further visualize PPARγ palmitoylation in the presence of ZDHHC6, we utilized alk-C16 as a metabolic sign to investigate the effects of ZDHHC6 on palmitoylated PPARγ and its nucleus translocation. This was done in a manner that was comparable to the methodology described above. Incubation with NH 2 OH resulted in a significant reduction of palmitoylation levels on PPARγ, which suggests that the upregulation of palmitoylated PPARγ mostly happened on cysteine and was driven by ZDHHC6 (Fig.  7 J). An additional protocol was made by fractionation analysis in ZDHHC6 wild-type or ZDHHC6 -deficient Caco-2 cells, which indicated that ZDHHC6-mediated palmitoylation of PPARγ increased the quantity of the modified PPARγ protein in the nucleus, but not in the membrane or cytoplasm components (Fig.  7 K). The studies presented above unambiguously demonstrated that ZDHHC6 is a significant palmitoyltransferase that plays a role in the occurrence of palmitoylated PPARγ, and the activity of this enzyme was found to have a favorable correlation with the progression of colorectal cancer.

ZDHHC6-mediated palmitoylated PPARγ enhances its nucleus translocalization. ( A ) ZDHHC6 and PPARγ expression were examined in the ZDHHC6 -deleted HCT116, SNU-C2A and SW48 cells, respectively ( n  = 3 per group). ( B ) ZDHHC6 and PPARγ co-expression in Adsh ZDHHC6 -transfected HCT116 cells, along with the matching fluorescence density as determined by Pearson’s analysis ( n  = 4 per group; P  < 0.05 vs. AdshRNA). The scale bars are 20 μm. ( C ) In ZDHHC6 -deleted HCT116 or ZDHHC6 -deleted SW48 cells, palmitoylation levels and PPARγ expression were analyzed using western blotting assay ( n  = 4 per group). ( D ) Western blotting assay using PPARγ, ACTIN, and HA antibodies, followed by PPARγ overexpressing the HA-tagged ZDHHC6 construct in various CRC cell lines ( n  = 3 per group). ( E ) Immunofluorescence pictures demonstrating the co-expression of PPARγ and ZDHHC6 in ZDHHC6-overexpressed HCT116 cells, together with the matching fluorescence density as determined by Pearson’s analysis ( n  = 4 per group; P  < 0.05 compared to empty vector). The scale bars are 20 μm. ( F ) HCT116 cells underwent IP of HA after co-transfecting with PPARγ and HA-ZDHHC6. ZDHHC6 and PPARγ Mutual Co-IP shows that endogenous ZDHHC6 and PPARγ bind to each other in HCT116 cells. ( G ) Using various alkyl-labeled fatty acylation, such as alk-C14, alk-C16, alk-C18, and alk-C20, the palmitoylation of PPARγ in the indicated cells was detected. By using streptavidin bead pulldown to identify acylated PPARγ, an immunoblotting experiment using PPARγ and ACTIN antibodies ( n  = 6 per group) was performed. ( H ) To identify acylated PPARγ in SW48, LS1034, and HT-29 cells, the same methodology as in (G) was applied. Following that, the lysates ( n  = 6 per group) were subjected to western blotting analysis using PPARγ and ACTIN antibodies. ( I ) Using Click reaction-associated streptavidin pulldown, the palmitoylation levels of Flag-labeled PPARγ WT, PPARγ C313S, PPARγ C156S, PPARγ C176S, and PPARγ C159S mutants were examined. Three individuals per group underwent an immunoblotting experiment using Flag and ACTIN antibodies on the relevant lysates. ( J ) ZDHHC6-HA and PPARγ-Flag were the vectors used to transfect the HCT116 cells. Using alk-C16 labeling, higher, palmitoylated PPARγ levels were demonstrated in both the presence and absence of hydroxylamine therapy. The corresponding fluorescence density and ACLY and PPARγ co-expression in HCT116 WT or HCT116 ZDHHC6 (KO) cells are depicted in the lower representative immunofluorescence images, which were analyzed using Pearson’s method ( n  = 5 per group; P  < 0.05 vs. WT). The scale bars are 20 μm. ( K ) After transfecting the HCT116 WT or HCT116 ZDHHC6 (KO) cells with PPARγ-Flag, the cells were labeled with alk-C16. To verify that the wild type and knockout cell components for input had the same quantity of PPARγ, subcellular fraction was obtained and PPARγ protein levels were adjusted. Western blotting analysis was used to assess palmitoylated PPARγ levels in the cell membrane (Mem.), cell cytoplasm (Cyto. ), and cell nucleus (Nuc.) components. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P  < 0.05 indicates statistical significance

Palmitoylation-stabilized PPARγ suppresses its lysosome degradation

Prior report has demonstrated that the significant rise in PPARγ protein levels effectively halted the process of lysosome degradation in the face of prolonged metabolic challenges and stress [ 36 ]. PPARγ is believed to undergo many posttranslational modifications that interact either positively or negatively to influence its protein destiny during different physiological and pathological states [ 36 , 37 ]. Therefore, we examined the impact of palmitoylated PPARγ on the evolution of its breakdown in the lysosome. The rapid breakdown of the palmitoylation-deficient mutant PPARγ C313S and the buildup of cellular lipids could be reversed by the lysosomal inhibitors NH 4 Cl and Pepstatin A (Pep A), but not by the proteasomal inhibitor MG132 or the autophagy inhibitor 3-Methyladenine (3-MA) (Fig.  8 A). In order to validate the lysosome-dependent mechanism, we utilized 2-BP to hinder the palmitoylation of naturally occurring PPARγ in HCT116 cells (Fig.  8 B). Pep A and NH 4 Cl, but not 3-MA or MG132, were able to enhance the stability of PPAR following depalmitoylation and a decrease in destabilized PPARγ-related lipid accumulation (Fig.  8 C, D).

Palmitoylation obscures an inherent lysosomal sorting signalling in PPARγ. ( A ) Using immunofluorescence analysis through CHX-chase studies in the presence of lysosome inhibitors such as NH 4 Cl and Pepstatin A (PepA), autophagy inhibitor (3-MA), and proteasome inhibitor (MG132), the degradation and residual amount of PPARγ or the PPARγ C313S mutant in HCT116 cells was assessed. n  = 5 per group. The scale bars are 20 μm. ( B ) Immunofluorescence analysis demonstrating the intensity quantification based on the remaining relative level of PPARγ. Three separate independent runs of this experiment produced findings that were comparable. n  = 5 per group. Scale bars: 20 μm. ( C ) In HCT116 cells, the effects of lysosome inhibitor, autophagy inhibitor, and proteasome inhibitor were measured to evaluate the intracellular TG contents and relative levels of PPARγ remaining after (A). P  < 0.05 compared to WT; n  = 5 per group. ( D ) In HCT116 cells, the relative amounts of residual PPARγ and the intracellular TG contents associated with (B) were measured. P  < 0.05 compared to WT; n  = 5 per group. ( E ) The colocalization of PPARγ with Rab11, Rab7b, Lamp1, and 58 K in HCT116 cells treated with 2-BP or DMSO control was statistically determined. P  < 0.05 compared to DMSO; n  = 5 per group. ( F ) Typical immunofluorescence pictures demonstrating the colocalization of endosome recycling marker Rab11b and ectopically produced PPARγ in HCT116 cells after a 2-BP challenge. Each group has n  = 5 per group. Scale bars: 10 μm. ( G ) Typical immunofluorescence pictures demonstrating the colocalization of lysosome marker Lamp1 and ectopically produced PPARγ in HCT116 cells during 2-BP stimulation. Each group has n  = 5 per group. Scale bars: 10 μm. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P  < 0.05 indicates statistical significance

PPARγ, a nuclear receptor, has been found to have a significant impact on glucose metabolism and overall energy balance through its posttranslational alterations [ 38 ]. Upon transportation to the cell nucleus, PPARγ can undergo various modifications such as phosphorylation, ubiquitination, and acetylation [ 39 , 40 ]. These modifications can affect its activity, stability, and interaction with other molecules. Additionally, PPARγ may be internalized into recycle endosomes or degraded through the late endosome-lysosome pathway [ 41 ]. To investigate the impact of (de)palmitoylation on the movement of PPARγ, we inhibited the palmitoylation process of PPARγ and examined its distribution across various subcellular compartments. Consistent with expectations, the use of 2-BP significantly reduced the overlap of PPARγ with recycling endosomes indicated by Rab11, while increasing its overlap with lysosomes labeled by Lamp1 and late endosomes labeled by Rab7b (Fig.  8 E-G). The results consistently showed that depalmitoylation facilitated the degradation of PPARγ through the lysosomal pathway.

ZDHHC6-mediated fatty acid biosynthesis promotes CRC carcinogenesis by upregulating PPARγ

To ascertain the impact of increased production of fatty acids on cell proliferation, we manipulated the expression of PPARγ in cells that either had ectopically produced PPARγ or had been subjected to ZDHHC6 -knockdown. In our study, we examined the process of glucose oxidation converting into fatty acid synthesis. We accomplished this by cultivating cells with glucose that was consistently enriched with carbon-13 ([U- 13 C] glucose). We specifically focused on cells that had reduced levels of PPARγ and overexpressed ZDHHC6. Our findings showed that when ZDHHC6 was overexpressed alone, it greatly enhanced the labeling of fatty acids from glucose tracers. However, when ZDHHC6 was overexpressed along with the co-transfection of shPPARγ, the knockdown of PPARγ significantly reduced the labeling of fatty acids from glucose tracers (Fig.  9 A). In contrast, the introduction of PPARγ-transduction greatly enhanced the process of labeling fatty acids from glucose tracers in HCT116 cells with ZDHHC6 knockdown (Fig.  9 B). Furthermore, the analysis of bodipy green staining and PPARγ co-expression revealed a consistent pattern of lipid buildup in the aforementioned CRC cells (Fig.  9 C, D). To further investigate the relationship between PPARγ and ZDHHC6, we manipulated the expression of PPARγ in cells lacking ZDHHC6 or cells overexpressing ZDHHC6 by co-transfecting them with a mutant form of PPARγ (PPARγ C313S). We conducted an experiment where we cultured cells with consistently labeled [U- 13 C] glucose in two different types of cells: ZDHHC6 -knockout HCT116 cells and cells with ZDHHC6 restoration and Ad PPARG C313S mutant co-transfection. As expected, we observed glucose oxidation leading to fatty acid biosynthesis in both types of cells. However, when PPARγ C313S expression was present, it counteracted the increase in fatty acid synthesis regulated by ZDHHC6 and significantly reduced the labeling of fatty acids from glucose tracers (Supplementary Fig.  9 A). In ZDHHC6 -knockdown HCT116 cells, there is no significant detection of enhanced fatty acid labeling from glucose tracers when PPARγ C313S mutant-transduction is used (Supplementary Fig.  9 B). Furthermore, the immunofluorescence assay revealed a consistent pattern of lipid accumulation in the aforementioned CRC cells, as demonstrated by the co-expression of PPARγ and the bodipy green staining (Supplementary Fig.  9 C, D). These findings indicate that ZDHHC6 enhances the production of fatty acids by stabilizing PPARγ. To additionally confirm the role of ZDHHC6 in promoting tumor formation through the activation of fatty acid production, we performed xenograft tumor tests utilizing the aforementioned cell lines. Significantly, mice that received implantation of ZDHHC6-overexpressing HCT116 cells developed larger tumors in comparison to those implanted with control HCT116 cells. However, HCT116 cells that were simultaneously overexpressed with ZDHHC6 and co-transfected with sh PPARγ or PPARγ C313S mutants exhibited lower rates of tumor growth (Fig.  9 E; Supplementary Fig.  9 E). In addition, mice that had been implanted with HCT116 cells in which ZDHHC6 had been suppressed and PPAR had been overexpressed exhibited higher rates of tumor formation compared to the CRC cells that had only been transfected with sh ZDHHC6 (Fig.  9 F). In addition, the mice carrying the ZDHHC6 knockdown and mutant PPARγ C313S HCT116 cells exhibited significantly suppressed tumor formation compared to the mice with shControl (Supplementary Fig.  9 F). The results demonstrate that increased expression of ZDHHC6 can stimulate the production of fatty acids in living organisms by activating PPARγ, hence promoting the advancement of colorectal cancer.

ZDHHC6-driven lipid biosynthesis contributes to CRC carcinogenesis by upregulating PPARγ. (A, B) In HCT116-related stable cells (Control, ZDHHC6, and ZDHHC6 + shPPARγ) (A) and HCT116-related stable cells (shControl, shZDHHC6, and shZDHHC6 + PPARγ) (B), the percentages of different isotopomers of FFA C16:0 following exposure to [U- 13 C] glucose are shown. Each group has n  = 5. ( C , D ) The relative TG content and PPARγ expression abundance in the aforementioned cell lines from (A) and (B) are displayed in representative immunofluorescence pictures. Each group has n  = 5. The scale bars are 20 μm. ( E ) In null mice, right flanks were injected with ZDHHC6 + shPPARγ, ZDHHC6, and Control, stable cells related to HCT116. Every two days, tumor volumes were measured. Weight and tumor growth curves were measured 22 days following dissection. Each group has n  = 5. ( F ) The right flanks of null mice were injected with shControl, shZDHHC6, and shZDHHC6 + PPARγ, stable cells linked to HCT116. Every two days, tumor volumes were measured. Weight and tumor growth curves were measured 22 days following dissection. Each group has n  = 5. ( G ) Kaplan-Meier curves representing the survival analysis based on TCGA CRC prognostic data for ZDHHC6-positive, PPARγ-positive, and ZDHHC6 & PPARγ co-positive patients. ( H ) Based on the prognosis information from the ICGC CRC database, Kaplan-Meier curves were used to analyze the survival of ZDHHC6-positive, PPARγ-positive, and ZDHHC6 & PPARγ co-positive patients. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P  < 0.05 indicates statistical significance

On top of that, we have found a strong positive correlation between ZDHHC6 and PPARγ, PPARγ and ALCY, and PPARγ and ACC transcript levels in the ICGC CRC and TCGA CRC databases. This correlation confirms the relationship between PPARγ and its associated factors during CRC formation (Fig.  1 and Supplementary Fig.  5 ). Subsequently, we assessed the predictive significance of ZDHHC6 and PPARγ in these datasets of colorectal cancer tissue microarrays (TMA). It is worth mentioning that patients with elevated levels of ZDHHC6 or PPARγ experienced significantly shorter overall survival compared to patients with low levels of ZDHHC6 or PPARγ in the TCGA CRC and ICGC CRC databases (Fig.  9 G, H). To better understand the impact of PPARγ on the prognosis of ZDHHC6-positive CRC patients, we conducted an analysis of the prognostic value of ZDHHC6 in four different CRC databases: CGWB, UCSC, CANEVOLVE, and COSMIC. This analysis included a total of 671 patients, with those who died within 5 months or were followed up for less than 3 months being excluded. Additionally, we examined the correlation between ZDHHC6 and various factors such as pathologic state (I–IV), race, gender, and age in the indicated CRC datasets (Supplementary Fig.  9 G–K). CRC patients exhibiting a single elevated level of ZDHHC6 demonstrated an unfavorable prognosis. Also, individuals with elevated levels of ZDHHC6 have poorer overall survival compared to those with lower levels of this factor. Furthermore, there is a positive association between ZDHHC6 levels and pathological state but no significant correlation with race, age, or gender. Together, we discovered a previously unknown harmful connection between ZDHHC6 and the variables that regulate the production of fatty acids and the balance of lipid oxidation, specifically related to PPARγ, in patients with colorectal cancer.

Disruption of cellular metabolism is a characteristic feature of the advancement of cancer. Alongside increased glycolysis, there are often abnormalities in fatty acid production and lipid oxidation in developing tumors, which are necessary to fulfill their metabolic needs [ 42 , 43 ]. Fatty acid production is less active in quiescent cells, which primarily take up lipids from the extracellular circulation. On the other hand, de novo lipogenesis (DNL), particularly the production of new fatty acids, plays a significant role in providing tumor cells with a source of lipids [ 44 ]. In this study, we have discovered that ZDHHC6 functions as a palmitoyltransferase enzyme that controls the production of fatty acids. Specifically, ZDHHC6 directly adds palmitoyl groups to PPARγ, a protein involved in regulating gene expression. This palmitoylation process stabilizes PPARγ, leading to the activation of ACLY expression and the subsequent development of lipid buildup-related carcinogenesis (Fig.  10 ).

Palmitoylation stabilizes PPARγ by ZDHHC6 via blocking its lysosomal degradation to promotes lipid biosynthesis-associated CRC development. As a palmitoyltransferase enzyme, ZDHHC6 regulates the synthesis of fatty acids. To be more precise, ZDHHC6 directly attaches palmitoyl groups to PPARγ, a protein that controls the expression of genes. By stabilizing PPARγ and blocking its lysosomal degradation, the palmitoylation mechanism triggers the production of ACLY and subsequently leads to the development of lipid buildup-related CRC carcinogenesis

Alteration of the equilibrium in lipid oxidation and lipid synthesis is becoming a more significant contributor to the development of colorectal cancer (CRC) [ 45 ]. This highlights the intricate relationship between cancer metabolism and cellular signaling pathways. The upregulation of enzymes like ACLY is responsible for this metabolic change, which not only supplies essential lipids for the formation of cell membranes but also enhances cancer-causing signaling pathways involved in cell survival, growth, and specialization [ 46 , 47 ]. Moreover, the production of particular lipid species can influence the activation of these pathways, so strengthening a cancer-promoting milieu. Elevated fatty acid synthesis also affects cellular energy production and can modify the tumor microenvironment, so promoting a favorable setting for cancer advancement and spread. Inhibiting this process of lipid production has demonstrated encouraging outcomes in impeding tumor growth and is a tempting target for therapeutic intervention [ 48 , 49 ]. Unraveling the exact ways in which fatty acid production and signaling pathways come together could reveal new possibilities for focused treatments in managing CRC [ 50 ]. The latest findings align with prior studies, although they are even more noteworthy. This intensifies our curiosity about the sample set and motivates us to investigate the underlying factors.

Aberrant expression or activation of associated enzymes leads to alterations in lipidome metabolic balance [ 51 ]. Currently, there is significant research being conducted on the involvement of zinc finger-aspartate-histidine-cysteine (DHHC)-CRD-type palmitoyl acyltransferases (ZDHHCs) in the stabilization of oncoproteins and their impact on the advancement of cancer. Homo sapiens and Mus musculus encode a combined total of 23 ZDHHCs. Multiple lines of evidence suggest that ZDHHCs have crucial functions in the process of lipogenesis [ 35 , 52 ]. ZDHHC2 exhibits aberrant upregulation in renal cell carcinoma and plays a role in lipid production and carcinogenesis by modulating the ZDHHC2-AGK signaling axis [ 53 ]. ZDHHC18 functions as a palmitoyltransferase for MDH2, promoting the formation of ovarian cancer by maintaining mitochondrial respiration and reinstating the growth and clonogenic potential of ovarian cancer cells [ 54 ]. ZDHHC3 exacerbates the development of nonalcoholic steatohepatitis (NASH) and the progression of hepatocellular carcinoma (HCC) associated to NASH by boosting the accumulation of lipids regulated by IRHOM2 and the production of lipids mediated by FASN signaling [ 35 ]. Furthermore, we have discovered a distinct variation in the expression of ZDHHC6 between colorectal cancer (CRC) and normal tissues. Notably, this difference in ZDHHC6 expression exhibits a substantial association with ACLY and the biological processes associated to lipid production. ZDHHC6 acts as an oncogenic protein in the process of tumor formation. It is significantly upregulated in various types of malignancies, including CRC. ZDHHC6 exhibits an oncogenic role in conjunction with AEG-1 under specific circumstances. ZDHHC6 has also been identified as a predictive gene in the human pathology atlas. Prior research has shown the varied impacts of ZDHHC6 on biological mechanisms linked to the advancement of cancer [ 21 ]. ZDHHC6 plays a crucial function in cancer by exerting its impact on the growth and viability of cells. Evidence demonstrates that it stimulates the proliferation of cancerous cells by augmenting the functionality of growth factor receptors, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) [ 55 , 56 ]. Furthermore, ZDHHC6 plays a vital role in palmitoylating the PI3K/Akt pathway, which is essential for cellular longevity and the ability to fight apoptosis [ 57 , 58 ]. In addition, ZDHHC6 has the ability to regulate metastasis, which is a significant factor in cancer-related fatalities [ 14 , 59 ]. It enhances the invasive and migratory properties of cancer cells via controlling the production and function of matrix metalloproteinases (MMPs) and focal adhesion kinase (FAK). ZDHHC6 also participates in epithelial-mesenchymal transition (EMT), a biological mechanism linked to heightened metastatic capability. Moreover, ZDHHC6 has been discovered to influence angiogenesis, the process of creating new blood vessels that are necessary for the development of tumors [ 58 , 60 ]. It enhances the release of pro-angiogenic substances such as vascular endothelial growth factor (VEGF), which stimulates the growth and creation of blood vessels by increasing the number of endothelial cells. Nevertheless, our findings indicate that ZDHHC6 facilitates the production of fatty acids from scratch in colorectal cancer (CRC). Furthermore, the atypical buildup of lipids induced by ZDHHC6 was partially reliant on the heightened expression of ACLY. ACLY, a pivotal enzyme regulating the production of new lipids, enhances the progression of CRC. Furthermore, the heightened expression of ACLY enhances the production of fatty acids and stimulates the formation of tumors. Our investigation revealed that specifically reducing the activity of ACLY significantly suppressed the promotion of tumor growth and the enhancement of fatty acid production induced by overexpression of ZDHHC6.

PPARγ is a crucial transcription factor that controls the production of lipids by increasing the transcription of enzymes involved in lipid synthesis, such as ACLY [ 61 ]. It exhibits significant expression in adipocytes and plays a role in the absorption, production, and retention of lipids. In this study, we discovered that PPARγ plays a role in the upregulation of ACLY by ZDHHC6 in the nucleus. Additionally, we showed that the palmitoylation of PPARγ by ZDHHC6 is essential for stabilizing PPARγ and allowing its incorporation into the nucleus. Increased expression of PPARγ stimulates the production of lipids and the development of tumors, together with the activation of ACLY in colorectal cancer (CRC). Our work discovered that ZDHHC6 may function as a precursor to ACLY. Nevertheless, regardless of the presence or absence of ACLY, ZDHHC6 still enhances the expression of PPARγ. Thus, ZDHHC6 has a role in stabilizing PPARγ regardless of ACLY expression, indicating that ZDHHC6 is involved in numerous regulatory pathways. Furthermore, during transportation to the cell nucleus, PPARγ can undergo several posttranslational changes that can impact its activity, stability, and interaction with other molecules. ZDHHC6’s role in colon cancer lipid metabolism underscores a multifaceted regulatory mechanism that supports tumor growth and survival. Understanding the precise molecular interactions and pathways modulated by ZDHHC6-mediated palmitoylation will be essential for developing targeted therapies. Future research should focus on elucidating these mechanisms and exploring the therapeutic potential of ZDHHC6 inhibition in colon cancer treatment.

In summary, we demonstrate that ZDHHC6 stabilizes PPARγ through enhancing palmitoylation and greatly reducing its lysosomal degradation progress. The Cys-313 location at the DBD domain of PPARγ has been determined to be significant for the palmitoylation of PPARγ by ZDHHC6. PPARγ knockdown, in the meantime, eliminated the increase in ACLY expression and significantly reduced both carcinogenesis and fatty acid production induced by ZDHHC6 overexpression in CRC cells and xenograft tissues. Furthermore, patients who have colorectal cancer (CRC) and exhibit high expression levels of both ZDHHC6 and PPARγ tend to have an unfavorable prognosis and lower overall survival rates. To summarize, we have discovered a previously unknown signaling pathway called ZDHHC6-PPARγ pathway. This signaling is crucial in the process of lipid biosynthesis and CRC carcinogenesis. It also presents a potential target for cancer therapy that focuses on inhibiting fatty acid production.

Data availability

All data and material during the current study are available from the corresponding author on reasonable request.

Abbreviations

Colorectal cancer

Zinc finger DHHC-type palmitoyltransferase 6

American Type Culture Collection

Peroxisome proliferator-activated receptor gamma

Fetal bovine serum

Immunohistochemistry

The cancer genome database

3- 2-bromopalmitate

Hyaluronic acid

Cytomembrane

ATP citrate lyase

Stearoyl-CoA desaturase

Fatty acid synthase

International cancer genome consortium

Gene expression omnibus

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This work was supported by (1) the Start-up fund of Shandong Cancer Hospital (NO. 2020PYA05); (2) the National Science Foundation of Shandong Province (NO. ZR2020MH254) and (3) the Science and Technology Program of Jinan (NO. 202134062).

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Junqi Shan, Xinyu Li and Runqi Sun contributed equally to this work.

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Department of Surgical Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University, Shandong Academy of Medical Sciences, Jinan, Shandong, 250117, China

Junqi Shan, Yao Yao & Yanlai Sun

School of Clinical Medicine, Shandong Second Medical University, Weifang, Shandong, 261000, China

School of Clinical Medicine, Jining Medical University, Jining, Shandong, 272000, China

Key Laboratory of Biorheological Science and Technology, Chongqing University, College of Bioengineering, Ministry of Education, Chongqing University, Chongqing, 400030, PR China

Yan Sun, Qin Kuang & Xianling Dai

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Conceptualization & Methodology, Junqi Shan, Yanlai Sun;

Investigation, Junqi Shan, Xinyu Li, Runqi Sun, Yao Yao, Yan Sun, Qin Kuang, Xianling Dai, Yanlai Sun; Data analysis, Junqi Shan, Xinyu Li, Runqi Sun, Yao Yao, Yanlai Sun; Funding acquisition, Project administration & Supervision, Yanlai Sun; Writing-original draft, Junqi Shan, Xinyu Li, Runqi Sun, Yanlai Sun; Writing review & editing, Junqi Shan, Xinyu Li, Yanlai Sun. All authors have read and approved the final manuscript.

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Shan, J., Li, X., Sun, R. et al. Palmitoyltransferase ZDHHC6 promotes colon tumorigenesis by targeting PPARγ-driven lipid biosynthesis via regulating lipidome metabolic reprogramming. J Exp Clin Cancer Res 43 , 227 (2024). https://doi.org/10.1186/s13046-024-03154-0

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DOI : https://doi.org/10.1186/s13046-024-03154-0

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