CILP2: From ECM Component to a Pleiotropic Modulator in Metabolic Dysfunction, Cancer, and Beyond

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript of Zheqiong Tan and colleagues aims to provide a comprehensive review of CILP2 across a wide spectrum of human diseases, including cancer, aswell as cardiovascular, metabolic, and fibrotic diseases. While the topic is timely and potentially valuable, the current version of the manuscript suffers from conceptual fragmentation, which substantially limits its clarity, accessibility and impact. In its present form, the review reads largely like a catalogue of disease associations rather than as a synthesis that extracts recurring biological principles underlying CILP2 function. As a result, the central message remains unclear, and the reader is left without a coherent framework for understanding why CILP2 appears in such diverse pathological contexts. The figures do not align well with the corresponding text sections and do not give detailed information.

I believe the manuscript could be substantially strengthened by restructuring around shared mechanisms rather than disease entities, and by developing a clearer conceptual narrative supported by more informative figures.

Author Response

Comment 1: This manuscript of Zheqiong Tan and colleagues aims to provide a comprehensive review of CILP2 across a wide spectrum of human diseases, including cancer, aswell as cardiovascular, metabolic, and fibrotic diseases. While the topic is timely and potentially valuable, the current version of the manuscript suffers from conceptual fragmentation, which substantially limits its clarity, accessibility and impact. In its present form, the review reads largely like a catalogue of disease associations rather than as a synthesis that extracts recurring biological principles underlying CILP2 function. As a result, the central message remains unclear, and the reader is left without a coherent framework for understanding why CILP2 appears in such diverse pathological contexts. The figures do not align well with the corresponding text sections and do not give detailed information.

I believe the manuscript could be substantially strengthened by restructuring around shared mechanisms rather than disease entities, and by developing a clearer conceptual narrative supported by more informative figures.

Response 1: Thank you for your valuable suggestions. We agree with all your comments, and the revisions have been made in the manuscript as outlined below.

• We have restructured the manuscript based on the biological functions of CILP2. We have comprehensively rewritten the section on “The functions and mechanisms of CILP2 in diseases”(line 118-469). It now consists of five subsections, each dedicated to detailing the mechanisms and functions of CILP2 in ECM modification, metabolic reprogramming, neurodevelopment, EMT, and TME remodeling. Furthermore, we have created three new schematic diagrams to visually summarize the roles and mechanisms of CILP2 in ECM modification, metabolic reprogramming, and TME remodeling.
• As the revised manuscript is reorganized around the shared mechanisms that explain CILP2's pleiotropic roles across different diseases, the central message of the manuscript is now clear. We also rewrote the Conclusion part (line 470-489) and explicitly stated “why CILP2 appears in such diverse pathological contexts”as: “CILP2 emerges as a pivotal molecular node across diverse pathologies due to its dual role as both a structural ECM modulator and a pleiotropic signaling regulator. Its broad expression and stress-responseive upregulation—triggered by metabolic and oxidative cues—enable it to act as a molecular integrator across diverse disease contexts. Structurally, domains like TSP-1 and Ig C-2 of CILP2 might facilitate its interactions with ECM components such collagen VI, ECM receptors, and signaling molecules (e.g., Wnt3a, ACLY), linking it to ECM remodeling as well as the regulation of critical signaling pathways. Beyond structural roles, CILP2 also engages in PPARγ/CD36 and Akt/EMT signaling axes in marcrophage and PDAC, respectively. In cancer, CILP2 functions as an oncoprotein that remodels the TME, correlating with immunosuppressive genes upregulation and increased infiltration of CAFs, TAMs and Tregs, while activating TGF-β and ECM-receptor interaction pathways. Furthermore, genetic variations of CILP2 link it to metabolic disorders and osteosarcoma risk, while its consistent dysregulation in patient serum and tissues underscores its translational potential as a diagnostic and prognostic biomarker. Collectively, CILP2 represents a central molecular node whose structural adaptability, signaling plasticity, and genetic association explain its pervasive role in metabolic, fibrotic, neurodevelopmental, and neoplastic diseases, positioning it as a compelling target for diagnostic and integrated therapeutic strategies”. 

Reviewer 2 Report

Comments and Suggestions for Authors

This review is very reasonable considering it's a small field of research and an even smaller gene family of just 2 members, which aren't even too closely related.

But the review nicely covers the emerging research field and the scientific ground. There are 74 papers in PubMed on CILP2, many of them mention CILP2 only in passing (e.g., as a member of a gene signature found here or there), and only 14 are from cancer studies, with maybe just 5 of them that really focus on CILP2. So the question emerges: is there even enough of a "research field" to cover? The answer is YES, but only if we cover all the diseases CILP2 is somehow associated with. In many cases, these are no more than associations, including genetic associations, mainly from the older studies. Functional associations are predominant only in the more recent publications. 

The references covered in the article are accodingly organized: they adress all of the different (metabolic) diseases CILP2 has been associated with, and cancer is only a small section of these. The references included here seem comprehensive and also include the higher impact papers. WHich arent too many, in this area. Many of the references are recent (2024) but there are also the "fundational" older papers from 1998 to 2011 which are cited here. This provides a complete, comprehensive coverage ogf this small but emerging field of research. There is a good balance between the older genetic association studies and the more recent mechanistic or functional studies. But still, its not a huge amount and yet suufficient number of rereferences to justify a review article - the first of its kind, as far as I can tell. 

There is also a good narrative: The paper has a clear logistical structure. It starts with the basic biology and then moves to the important edges, like the metabolic diseases it has been associated with (although we often ddont know the functin), and finally, it moves to some clinical significance - which is mostly "future aspects". Table 1 is a very good summary of all the diseaeses CLIP2 is allegedly associated with, and figures 2 and 3 give a good overview of the pathways and diseases it plays a role in. The authors also construct a stroy in which the gene/protein moves from just a bystander component of the Tcm rto a more critical and multifunctional modulator of multiple (diseaserelevant) processes. 

 Some of the sections (e.g., the one on cancer) are very dense with abbreviations, which doesn't improve the reading pleasure. And the transition from one topic to another (or one chapter to the next) isn't always very smooth; this could be improved. There is only a small amount of grammatical errors and nothing that cannot be fixed upon the editorial process/production. 

There is a reasonable "conclusions section (Section 6) but it doesn't really have anything like a "future aspects" part in it. Instead, we find a few "future aspects" thoughts scattered throughout the manuscript, for example, 

the review could benefit from a more explicitly labeled "Future Perspectives" section. Currently, future directions are scattered throughout, for example, in chapter 4, the question of what may be the core regulators of CILP2 expression? How are we going to investigate them? And how do we turn CILP2 into a useful, informative biomarker; how do we standardize it and how do we identify its putative receptors? That should all be bundled at the end of the discussion. Creating a dedicated "Future Perspectives" subsection. 

The authors also dont go deeper into the many potential limitations of the studies they are reviewing here. Much of the data is actually from purely correlational databases like TCGA; we dont know if it means anything, especially if its just based on RNA and on bulk sequencing. And how would we address (and close) the gap between the few functional studies, and the clinical translation/application? Is the gene of interest for pathologists ? 

The authors could also discuss the alleged functions of CLIP2 within the tumor microenvironment more extensively. Which genes express CILP2? Which cells may have a receptor (if there is such a thing). Does CILP2 play any role in immune cell infiltration in cancers, for example?

Author Response

Comment 1: This review is very reasonable considering it's a small field of research and an even smaller gene family of just 2 members, which aren't even too closely related.

But the review nicely covers the emerging research field and the scientific ground. There are 74 papers in PubMed on CILP2, many of them mention CILP2 only in passing (e.g., as a member of a gene signature found here or there), and only 14 are from cancer studies, with maybe just 5 of them that really focus on CILP2. So the question emerges: is there even enough of a "research field" to cover? The answer is YES, but only if we cover all the diseases CILP2 is somehow associated with. In many cases, these are no more than associations, including genetic associations, mainly from the older studies. Functional associations are predominant only in the more recent publications.

The references covered in the article are accodingly organized: they adress all of the different (metabolic) diseases CILP2 has been associated with, and cancer is only a small section of these. The references included here seem comprehensive and also include the higher impact papers. Which arent too many, in this area. Many of the references are recent (2024) but there are also the "fundational" older papers from 1998 to 2011 which are cited here. This provides a complete, comprehensive coverage of this small but emerging field of research. There is a good balance between the older genetic association studies and the more recent mechanistic or functional studies. But still, its not a huge amount and yet suufficient number of rereferences to justify a review article - the first of its kind, as far as I can tell.

Response 1: Thank you for your thoughtful and encouraging comment. We sincerely appreciate your recognition of the challenges and value in synthesizing this emerging field. You have precisely captured the current state of CILP2 research—a field defined not by volume but by compelling, cross-disciplinary connections that warrant a cohesive synthesis.

Your assessment aligns perfectly with our core rationale for this review. As you noted, while the absolute number of deeply focused CILP2 studies is limited, its consistent appearance across a strikingly diverse spectrum of pathologies—from cardiovascular and metabolic diseases to fibrosis and cancer—creates a critical mass of associative and, increasingly, functional data. This very dispersion across disease contexts highlights a significant knowledge gap: the lack of a unified mechanistic framework to explain CILP2's pleiotropic roles.

Our review was conceived to fill this specific gap. By aggregating and analyzing all available evidence—from the foundational genetic associations to the latest functional studies—we aim to transform these fragmented observations into a coherent narrative. We have comprehensively rewritten the section on “The functions and mechanisms of CILP2 in diseases” (line 118-469). It now consists of five subsections, each dedicated to detailing the mechanisms and functions of CILP2 in ECM modification, metabolic reprogramming, neurodevelopment, EMT, and TME remodeling. The goal is to move beyond a simple catalogue of associations and toward identifying the shared biological principles, such as its role in ECM modification and metabolic reprogramming, that underlie its function in different tissues.

Moreover, we have been consistently conducting research on CILP2 in recent years, and our findings are forthcoming for publication. Indeed, the current research landscape of CILP2 presented in this review has provided us with valuable insights and inspiration, which was also our original intention in writing this review.

 

Comment 2: There is also a good narrative: The paper has a clear logistical structure. It starts with the basic biology and then moves to the important edges, like the metabolic diseases it has been associated with (although we often don’t know the function), and finally, it moves to some clinical significance - which is mostly "future aspects". Table 1 is a very good summary of all the diseaeses CLIP2 is allegedly associated with, and figures 2 and 3 give a good overview of the pathways and diseases it plays a role in. The authors also construct a stroy in which the gene/protein moves from just a bystander component of the ECM to a more critical and multifunctional modulator of multiple (diseaserelevant) processes.

Some of the sections (e.g., the one on cancer) are very dense with abbreviations, which doesn't improve the reading pleasure. And the transition from one topic to another (or one chapter to the next) isn't always very smooth; this could be improved. There is only a small amount of grammatical errors and nothing that cannot be fixed upon the editorial process/production.

Response 2: Thank you for your suggestions. We have carefully addressed the issues you raised to improve the readability and flow of the manuscript. We have gone through sections with a high density of abbreviations and ensured that each abbreviation is clearly defined upon its first use. In addition, we have revised the transitions between topics and chapters to create a smoother and more logical progression. Specifically, we have added brief linking sentences at the end of sections and the beginning of new ones to better guide the reader through the conceptual framework of the review (e.g., line 106-109, 132, 152, 205, 257, 297, 426, 459).

 

Comment 3: There is a reasonable "conclusions section (Section 6) but it doesn't really have anything like a "future aspects" part in it. Instead, we find a few "future aspects" thoughts scattered throughout the manuscript, for example,

The review could benefit from a more explicitly labeled "Future Perspectives" section. Currently, future directions are scattered throughout, for example, in chapter 4, the question of what may be the core regulators of CILP2 expression? How are we going to investigate them? And how do we turn CILP2 into a useful, informative biomarker; how do we standardize it and how do we identify its putative receptors? That should all be bundled at the end of the discussion. Creating a dedicated "Future Perspectives" subsection.

Response 3: Thank you for your excellent suggestion. We have revised the manuscript by adding a new subsection titled "Future Perspectives" after Conclusion (line 490-524). This subsection now systematically integrates and expands upon the previously scattered points to outline clear and actionable research avenues. It specifically addresses the key questions you highlighted, including: First, mechanistic studies must define its structure-function relationships by identifying protein interactors and cellular receptors. Second, research needs to map its upstream regulatory network, clarifying the transcriptional control and signaling pathways that govern its expression. Finally, for clinical translation, future work should focus on standardizing CILP2 as a reliable biomarker and developing targeted therapeutic strategies, such as specific inhibitors or combination therapies.

 

Comment 4: The authors also don’t go deeper into the many potential limitations of the studies they are reviewing here. Much of the data is actually from purely correlational databases like TCGA; we don’t know if it means anything, especially if its just based on RNA and on bulk sequencing. And how would we address (and close) the gap between the few functional studies, and the clinical translation/application? Is the gene of interest for pathologists ?

Response 4: Thank you for highlighting these important limitations. We have pointed out the challenges in clinical translation in “Clinical significance and challenges” part (line 353-469). We also expanded the “Future Perspectives” sections to directly address this gap. The clinical translation of CILP2 as a biomarker faces several challenges as the following: Firstly, lack of standardization in detection methods remains a major barrier. Althogh CILP2 ELISA kits and immunohistochemiscal antibodies are commercially available, they have not been validated for clinical use, limiting consistent measurement and interpretation across laboratories. Secondly, the establishment of specificity versus sensitivity is complicated by the absence of disease-specific diagnostic thresholds, while confounding variables—such as obesity, aging, and metabolic comorbidities—may influence CILP2 levels and have not been adequately accounted for in existing studies. Finally, while CILP2 shows strong correlative associations with disease outcomes, causality and clinical utility remain uncertain, particularly in oncology where most evidence derives from bioinformatic analyses and retrospective cohorts. Therefore, prospective validation in well-designed clinical studies is essential to define actionable cut-offs, and ultimately enable its integration into routine diagnostic and prognostic workflows.

 

Comment 5: The authors could also discuss the alleged functions of CLIP2 within the tumor microenvironment more extensively. Which genes express CILP2? Which cells may have a receptor (if there is such a thing). Does CILP2 play any role in immune cell infiltration in cancers, for example?

Response 5: Thank you for your comments. We have significantly expanded the "TME remodeling" subsection (line 269-311) and integrated related discussions into the "Future Perspectives" section. We also indicated the potential role of CILP2 in tumor microenvironment in Figure 5. In silico analysis showed that high expression of CILP2 was positively correlated to immuno-suppressing genes and the infiltration of TAMs, CAFs, Tregs in cancer. Conversely, CILP2 is negatively associated with antitumor immune subsets including CD4+ T and Th17 cells. These evidence indicated CILP2's multifaceted role in shaping the tumor microenvironment. However, the specific receptors for CILP2 in cancer and its detailed molecular mechanisms within the tumor microenvironment remain unelucidated. This gap is precisely the focus of our ongoing research efforts.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been substantially strengthened by restructuring around shared mechanisms and by more informative figures.  It remains unclear to me how Cilp2 can have all these functions and how intra- and extracellular roles are explainable. 
There are still some smaller things to correct such as:
line 139 mechanistically ?
line 192 CLIP2

Author Response

Comments 1: The manuscript has been substantially strengthened by restructuring around shared mechanisms and by more informative figures. It remains unclear to me how Cilp2 can have all these functions and how intra- and extracellular roles are explainable.

Response 1: Thank you for your valuable comments. We sincerely appreciate your positive feedback on our revised version. For the question “how CILP2 can perform such diverse functions and reconcile its intracellular and extracellular roles”, we have further expanded our discussion (lines 83-94, 471-479, 500-514) and rewrote the Conclusion and the first paragraph of Future Perspectives (highlighted in green) to clarify this in the latest revised manuscript.

Based on the current evidence, CILP2’s multifunctionality could be explained by its structure. First, the presence of a TSP-1 domain in CILP2 raises the possibility that it may be able to anchor to other matrix constituents. Although the receptors or binding proteins of CILP2 have not been fully studied, the functions of the TSP-1 domain in other proteins, such as ADAMTS4 and CILP1, have been extensively studied. The TSP-1 domain was reported to act as attachment sites, protein-binding sites, and glycosaminoglycan (GAG)-binding sites in various cell types [1-4], providing the potential structural basis for the interaction of CILP2 with other ECM proteins or receptors. These interactions may enable CILP2 to participate in various biological processes—from ECM assembly to signal modulation—depending on the cellular context and binding partners.

Second, as a secreted glycoprotein, CILP2 could act in a paracrine or autocrine manner, thereby regulating intra- and extracelluar functions. For example, CILP2 secreted by brain endothelial cells binds to Wnt3a in neural precursor cells to regulate neurogenesis, while in muscle, cellular CILP2 interacts with Wnt3a to suppress myogenesis.

In addition, as the protein-protein interactions and autocrine or paracrine feedback loops of CILP2 have not been fully elucidated so far, the ongoing research of our group is focusing on these gaps. Indeed, the current research landscape of CILP2 presented in this review has provided us with valuable insights and inspiration, which was also our original intention in writing this review.

Reference

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2. Tan K, Duquette M, Liu JH, Dong Y, Zhang R, Joachimiak A, et al. Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication. J Cell Biol. 2002;159(2):373-82.
3. Tortorella M, Pratta M, Liu RQ, Abbaszade I, Ross H, Burn T, et al. The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage. J Biol Chem. 2000;275(33):25791-7.
4. Bernardo BC, Belluoccio D, Rowley L, Little CB, Hansen U, Bateman JF. Cartilage intermediate layer protein 2 (CILP-2) is expressed in articular and meniscal cartilage and down-regulated in experimental osteoarthritis. J Biol Chem. 2011;286(43):37758-67.

Comments 2: There are still some smaller things to correct such as:

line 139 mechanistically ?

line 192 CLIP2

Response 2: Thank you for pointing out these errors. We have corrected them (hilighted in green) in the revised manuscript. We also reviewed the entire article and corrected some other grammatical and spelling errors (lines 121, 123, 142, 148, 167, 169, 177, 181, 192, 195, 196, 239, 283, 307, 422, and 430).