Normalizing the Tumor Microenvironment: A New Frontier in Ovarian Cancer Therapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an excellent review.

A few suggestions should be considered:

  The authors mention throughout the text different ways specific ECM components can influence ovarian cancer development. It would be usefu in the summary if these were listed again since this represents major "takehome " messages to emphasize the importance of the ECM in the progression of this cance

  While the authors mention proteoglycan involvement there is little if any discussion of their importance given that hyaluronan is not a proteoglycan!

There have been some interesting recent studies showing that proteases that degrade specific proteoglycans such as ADAMTS5 play a significant role in not only the progression of OC but in its possible therapy as well. It would seem appropriate to site these studies with a brief discussion  

Author Response

POINT-BY-POINT RESPONSE TO REVIEWERS’ COMMENTS

 

Normalizing the Tumor Microenvironment: A New Frontier in Ovarian Cancer Therapy

Corresponding Author: Lei Xu

We thank the reviewers for their helpful comments. We have revised our manuscript to address all of their comments. Our responses are provided below in blue, and the changes made in the revised manuscript are shown in red.

 

Reviewer 1:

This is an excellent review. A few suggestions should be considered:

Response: We thank the reviewer for their encouraging comments and thoughtful suggestions. In response to the reviewer’s comments, we have addressed all points in detail.

 

1. The authors mention throughout the text different ways specific ECM components can influence ovarian cancer development. It would be useful in the summary if these were listed again since this represents major "take-home" messages to emphasize the importance of the ECM in the progression of this cancer

Response: We thank the reviewer for this thoughtful comment. We have now revised the manuscript to include a summary of the main ECM features that influence ovarian cancer progression and chemoresistance:

In the Introduction section, lines 126-133:

“Importantly, these ECM-driven effects can be broadly categorized into (i) biomechanical mechanisms, such as matrix stiffening, solid stress, and physical barrier formation, and (ii) biochemical and signaling mechanisms, including integrin-mediated adhesion, growth factor presentation, and activation of pro-survival and stemness pathways. These mechanisms operate across distinct compartments of the tumor microenvironment, including tumor cells, the vasculature, immune infiltrates, and cancer stem cell (CSC) niches, and are differentially targetable by emerging therapeutic strategies.”

In the Conclusion section, lines 492-520:

“In OC, various extracellular matrix (ECM) molecules drive metastasis and chemoresistance through multiple interconnected mechanisms (Figure 4).

• Collagens, particularly COL1 and COL6, are upregulated in primary and metastatic tumors, stiffen the matrix, enhance cell adhesion, and promote survival signaling, contributing to platinum and taxane resistance.
• Fibronectin interacts with integrins and L1CAM to support tumorsphere formation, EMT, anoikis resistance, mesothelial attachment, and activation of pro-survival Akt and survivin pathways, thereby facilitating invasion and chemoresistance.
• Proteoglycans such as versican form hydrated pericellular matrices in combination with hyaluronic acid, which promote migration, adhesion, and resistance to mechanical stress while activating CD44-mediated signaling, ABC transporter expression, and stemness pathways like Nanog/STAT3, supporting multi-drug resistance.
• Heparan sulfate proteoglycans, including syndecans and perlecan, regulate growth factor bioavailability, cell-ECM adhesion, and drug penetration, sustaining pro-survival and angiogenic signaling under chemotherapy.
• ECM-remodeling enzymes such as ADAMTS5 and LOX mediate ECM cleavage, collagen crosslinking, and matrix stiffening, thereby promoting invasion, metastatic colonization, and chemoresistance.
• Laminin α5 supports proliferation and metastasis via Notch signaling, while interactions with adipocyte-derived factors and mesenchymal stem cells amplify ECM remodeling, EMT plasticity, and metastatic behavior.

Collectively, ECM composition, mechanical properties, and dynamic remodeling establish a protective microenvironment that promotes OC dissemination, survival, and resistance to chemotherapy. Importantly, these same ECM features also govern whether disseminated tumorspheres successfully transition into established peritoneal metastases, positioning ECM normalization as a strategy that selectively disrupts metastatic colonization rather than initial dissemination.”

 

2. While the authors mention proteoglycan involvement, there is little if any discussion of their importance given that hyaluronan is not a proteoglycan!

Response: We thank the reviewer for the insightful comment. We have now added material on the roles of chondroitin/heparan sulfate proteoglycans in ovarian cancer metastasis and chemoresistance. Please see lines 209-233.

“Proteoglycans have also been shown to reinforce ECM-associated pro-tumorigenic signaling pathways and chemoresistance further. Among these, the chondroitin sulfate proteoglycan (CSPG) versican is consistently upregulated in ovarian tumors and metastatic lesions, where it associates with hyaluronan to form a highly hydrated pericellular matrix that promotes tumor cell survival, migration, and adhesion [46,47]. This versican–hyaluronan network enhances OC cell motility and peritoneal attachment via CD44-dependent signaling, facilitating transcoelomic dissemination while simultaneously providing protection from mechanical stress and anoikis [46–48]. Significantly, disruption of versican–hyaluronan interactions using hyaluronan oligosaccharides significantly reduces invasive behavior in OC models, underscoring the functional importance of this proteoglycan axis in disease progression [46]. Beyond metastasis, versican-rich matrices alter integrin and growth factor signaling, creating a microenvironment that favors cell survival and reduces sensitivity to cytotoxic therapies [27,48].

 

Heparan sulfate proteoglycans (HSPGs), including syndecans, glypicans, and perlecan, further contribute to chemoresistance by regulating growth factor bioavailability, cell–ECM adhesion, and drug penetration within the tumor microenvironment. Syndecan-1 expression is increased in ovarian tumors compared with normal ovarian tissue and has been associated with more aggressive disease phenotypes and altered responsiveness to therapy [49]. ECM-associated HSPGs, such as perlecan, bind and present pro-survival and pro-angiogenic growth factors, including FGF and VEGF, thereby reinforcing signaling pathways that promote tumor persistence under chemotherapeutic stress [50,51]. In addition, proteoglycan-rich ECM structures act as physical barriers that impair drug diffusion and sustain integrin-mediated survival signaling, a recognized mechanism of cell adhesion-mediated drug resistance in OC [27,52].”

 

3. There have been some interesting recent studies showing that proteases that degrade specific proteoglycans such as ADAMTS5 play a significant role in not only the progression of OC but in its possible therapy as well. It would seem appropriate to site these studies with a brief discussion  

Response: We thank the reviewer for this important suggestion. We have hence included it as more support for tumor microenvironment normalization. Please see lines 233-244.

“Enzymatic remodeling of proteoglycans, particularly by proteases such as a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), has been shown to affect OC progression and treatment. ADAMTS5 is elevated in ovarian malignancies compared to borderline and benign lesions and is upregulated by OC cells overexpressing the small GTPase Rab25 [53,54]. Furthermore, ADAMTS5 expression is associated with advanced stages of OC and poor prognosis [53,55]. A recent study has shown that Rab25 upregulates ADAMTS5 in OC through NF-κB signaling and is necessary to stimulate OC cell invasiveness through 3D cancer-associated fibroblast (CAF) matrices, which was reduced by ADAMTS5 inhibition [53]. Collectively, these findings highlight proteoglycans as central drivers of ECM-mediated treatment resistance in ovarian cancer and support targeting them to disrupt tumor–stroma crosstalk.”

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Jones and colleagues provide a comprehensive overview of tumor microenvironment (TME) remodeling in ovarian cancer, with emphasis on extracellular matrix (ECM) dynamics and therapeutic strategies targeting fibrosis, particularly TGF-β inhibition and losartan repurposing. The manuscript is well-structured, up-to-date, and clearly highlights the clinical need for TME-normalizing interventions in advanced ovarian cancer. However, key areas require refinement to improve scientific rigor, critical balance, and clarity on the limitations of current evidence and clinical applicability, as shown below:

A) Although the manuscript emphasizes promising strategies such as TGF-β blockade and losartan therapy, challenges like systemic toxicities, heterogeneous patient response, and lack of validated biomarkers should be more thoroughly discussed and integrated throughout, instead of confined mostly to the conclusions section.

B) Most supporting studies rely on HGSOC-specific preclinical models, yet the review generalizes applicability to all ovarian cancer phenotypes. The authors should specify subtype-dependent variability and whether therapies apply beyond high-grade serous disease.

C) Some mechanistic descriptions conflate ECM-mediated mechanical stress with tumor-signaling pathways. Clearer categorization would improve comprehension and highlight where targeting strategies exert effects (e.g., immune compartment, vasculature, CSC niches).

D) While low response rates (10–25%) are cited, more context is needed regarding failed and ongoing clinical trials using checkpoint inhibitors in ovarian cancer to justify the therapeutic rationale for TME normalization.

E) Peritoneal dissemination and spheroid-mediated metastasis are well described, yet the review could better connect this biology to how normalization therapies disrupt metastatic colonization processes.

F) RAS/losartan section: I suggest including a brief overview of dosing regimens or pharmacokinetic aspects relevant for tumor penetration to strengthen translatability claims.

G) I recommend including the recent study by Simão et al. (Cells, 2025; 14:374), which demonstrates how bidirectional signaling between adipose-derived mesenchymal stem cells and ovarian cancer cells promotes ECM remodeling, EMT plasticity, and metastatic behavior. This reference would reinforce the manuscript’s discussion on stromal–tumor crosstalk and further support the relevance of targeting the TME to improve therapeutic response.

H) Finally, the inclusion of a summary diagram of the main concepts of the manuscript would be valuable to strengthen their contribution.

Author Response

POINT-BY-POINT RESPONSE TO REVIEWERS’ COMMENTS

 

Normalizing the Tumor Microenvironment: A New Frontier in Ovarian Cancer Therapy

Corresponding Author: Lei Xu

We thank the reviewers for their helpful comments. We have revised our manuscript to address all of their comments. Our responses are provided below in blue, and the changes made in the revised manuscript are shown in red.

 

Reviewer 2:

Jones and colleagues provide a comprehensive overview of tumor microenvironment (TME) remodeling in ovarian cancer, with emphasis on extracellular matrix (ECM) dynamics and therapeutic strategies targeting fibrosis, particularly TGF-β inhibition and losartan repurposing. The manuscript is well-structured, up-to-date, and clearly highlights the clinical need for TME-normalizing interventions in advanced ovarian cancer. However, key areas require refinement to improve scientific rigor, critical balance, and clarity on the limitations of current evidence and clinical applicability, as shown below:

Response: We thank the reviewer for their encouraging comments and thoughtful suggestions. In response to the reviewer’s comments, we have addressed all points in detail.

 

1. A) Although the manuscript emphasizes promising strategies such as TGF-β blockade and losartan therapy, challenges like systemic toxicities, heterogeneous patient response, and lack of validated biomarkers should be more thoroughly discussed and integrated throughout, instead of confined mostly to the conclusions section.

Response: We thank the reviewer for this important suggestion. We have included more discussions of the limitations throughout the manuscript to address the comments on systemic toxicities, heterogeneous patient responses, and the lack of validated biomarkers.

Lines 192-195: “At the same time, targeting upstream regulators such as TGF-β presents translational challenges due to its pleiotropic and context-dependent roles in tissue homeostasis, epithelial integrity, and immune regulation, raising concerns regarding systemic toxicity and unintended biological consequences.”

Lines 325-334: “Further studies have shown that TGF-β has complex roles at different stages of OC development and in different OC histopathological types, where TGF-β can induce apoptosis in ovarian surface epithelial cells and low-grade serous carcinomas, but stimulates invasiveness and EMT activation in xenografts of human serous borderline tumors and HGSOCs [81]. Together, these studies highlight the potential of TGF-β therapy for patients with OC. However, therapeutic responses to TGF-β blockade are likely to be heterogeneous, influenced by tumor subtype, disease stage, baseline immune contexture, and stromal composition, emphasizing the importance of identifying predictive biomarkers and defining patient populations most likely to benefit.

Lines 468-471: “However, amplifying immune infiltration within previously immune-excluded tumors may also increase the risk of immune-related adverse events, particularly when combined with checkpoint blockade, highlighting the need for careful dose optimization and patient monitoring.”

 

1. B) Most supporting studies rely on HGSOC-specific preclinical models, yet the review generalizes applicability to all ovarian cancer phenotypes. The authors should specify subtype-dependent variability and whether therapies apply beyond high-grade serous disease.

Response: We thank the reviewer for this thoughtful comment. We have now i) clarified the histological types of OC in the referenced preclinical studies, and ii) added discussion of future direction on the application of these therapies, particularly TGF-beta inhibition, in other subtypes of OC models to understand their true clinical utility.

Lines 398-402: “In summary, while TGF-β blockade effectively modulates tumor vasculature and lymphatics in HGSOC models and shows clear synergy with established immunotherapies and chemotherapies, translating these findings into clinical practice requires addressing its multifaceted roles, mitigating potential off-target effects, and being validated in preclinical models representing diverse histological and molecular subtypes of OCs.”

 

1. C) Some mechanistic descriptions conflate ECM-mediated mechanical stress with tumor-signaling pathways. Clearer categorization would improve comprehension and highlight where targeting strategies exert effects (e.g., immune compartment, vasculature, CSC niches).

Response: We thank the reviewer for this suggestion. However, as TGFb and angiotensin signaling pathways are both pleiotropic and affect multiple aspects of tumor progression, we have provided throughout distinctive summaries of mechanisms and how their effects can be targeted independently or cooperatively.

Lines 204-207: “These proliferative effects arise through both mechanotransduction, whereby matrix stiffness and strain activate intracellular sensors such as YAP/TAZ, and ligand–receptor signaling, including laminin-Notch and hyaluronan-CD44 interactions, highlighting multiple, non-redundant routes by which the ECM reinforces tumor growth.”

Lines 262-269: “This illustrates how ECM-induced mechanical stiffening is transduced into intracellular survival signaling via focal adhesions and mechanosensitive pathways, providing a mechanistic bridge between matrix biomechanics and tumor-intrinsic resistance programs. Notably, these same adhesion- and mechanotransduction-dependent survival pathways are engaged during spheroid reattachment and expansion at metastatic sites, indicating that ECM normalization may simultaneously limit chemoresistance and suppress the establishment of secondary lesions derived from disseminated spheroids.”

Lines 307-311: “Within this framework, CSC niches are particularly sensitive to ECM cues, as matrix stiffness, hyaluronan-rich pericellular coats, and integrin engagement cooperatively sustain stemness, drug efflux capacity, and quiescence. Thus, ECM remodeling not only protects bulk tumor cells but also preserves therapy-resistant CSC reservoirs.”

Lines 429-438: “Critically, these effects are predominantly mechanical and vascular, targeting solid stress and perfusion rather than tumor-intrinsic signaling pathways, thereby distinguishing losartan-mediated ECM normalization from direct anti-fibrotic or anti-proliferative signaling inhibitors. Furthermore, these effects also translate into decreased tumor hypoxia, which can otherwise promote chemoresistance and tumor progression, as well as symptomatic alleviation of ascites. In the context of peritoneal dissemination, alleviation of solid stress and matrix density is expected to impair integrin-mediated spheroid adhesion to mesothelial surfaces and reduce the mechanical permissiveness required for metastatic niche formation, thereby disrupting early colonization events rather than initial spheroid survival.”

Lines 450-453: “Nevertheless, responses to renin–angiotensin system inhibition are unlikely to be uniform, as the magnitude of stromal normalization depends on baseline ECM density, vascular compression, and RAS pathway activity within individual tumors.”

Lines 477-481: “Thus, losartan exerts multicompartmental effects, simultaneously acting on the vasculature (improved perfusion), immune compartment (enhanced immune infiltration and activation), and tumor cells (attenuated IGF-1 signaling), while indirectly reshaping CSC-supportive niches through ECM normalization.”

 

1. D) While low response rates (10–25%) are cited, more context is needed regarding failed and ongoing clinical trials using checkpoint inhibitors in ovarian cancer to justify the therapeutic rationale for TME normalization.

Response: As suggested by the reviewer, we have provided more details on the efficacy of these inhibitors in ovarian cancer and have strengthened the rationale for tumor microenvironment normalization.

Lines 349-372: “TGF-β is not only a major driver of fibrotic responses in OC, but also a key mediator of immunosuppression and resistance to immunotherapies like immune checkpoint inhibitors (ICIs), with poor single-agent response rates around 10-25% in OC [86–88]. Multiple trials of PD-1/PD-L1 monotherapy, including JAVELIN, KEYNOTE-028, and KEYNOTE-100, reported only modest response rates with short progression-free survival (1.9–3.5 months), despite acceptable safety profiles [89–91]. These outcomes were not meaningfully improved by combining ICIs with chemotherapy or VEGF inhibition, as evidenced by large phase III trials such as JAVELIN-200 and IMagyn050, which failed to show a survival benefit over standard chemotherapy alone [92–94]. Even dual checkpoint blockade (e.g. nivolumab plus ipilimumab) yielded only incremental improvements in response and progression-free survival, benefiting a minority of patients while remaining limited by toxicity and lack of durability [95–97]. Collectively, these failures reflect fundamental features of ovarian cancer biology, including a predominantly “cold” TME, extensive stromal desmoplasia, low tumor mutational burden, and high expression of immunosuppressive pathways that limit T-cell infiltration and function [98–103]. The ECM and associated stromal components further contribute to immune exclusion and checkpoint resistance by physically restricting lymphocyte access and reinforcing immunosuppressive signaling networks. These effects reflect both mechanical exclusions, whereby dense and stiff matrices limit immune cell infiltration, and paracrine immunosuppression, mediated by TGF-β–driven signaling that dampens T-cell and macrophage effector functions, underscoring the rationale for combined stromal and immune-targeted interventions. Consequently, emerging therapeutic strategies increasingly emphasize TME normalization, including stromal and ECM remodeling and targeting immunosuppressive pathways such as TGF-β, to convert immune-excluded tumors into inflamed, ICI-responsive states.”

 

1. E) Peritoneal dissemination and spheroid-mediated metastasis are well described, yet the review could better connect this biology to how normalization therapies disrupt metastatic colonization processes.

Response: We thank the reviewer for this important suggestion. We have added details on how normalization therapies may disrupt metastatic colonization.

Lines 147-159: “Here, ECM remodeling contributes both mechanically, by enabling collective detachment and protection from anoikis through multicellular aggregation, and biochemically, by engaging integrin- and PDGFRβ-dependent signaling pathways that actively promote tumorsphere survival, cohesion, and metastatic competence. While tumorsphere formation supports survival during peritoneal transit, successful metastatic colonization requires subsequent attachment to mesothelium, invasion, and expansion within secondary niches. These later steps remain highly dependent on ECM density, stiffness, and ligand availability, suggesting that normalization strategies that reduce matrix deposition and mechanical stress may preferentially disrupt spheroid anchoring and early outgrowth without necessarily affecting initial dissemination. Notably, the efficacy of such approaches is likely influenced by the degree of desmoplasia, ascites composition, and stromal activation within the peritoneal cavity, which differ markedly between patients and may evolve during disease progression or following chemotherapy.”

 

1. F) RAS/losartan section: I suggest including a brief overview of dosing regimens or pharmacokinetic aspects relevant for tumor penetration to strengthen translatability claims.

Response: As suggested by the reviewer, we have added specific dose regimens and pharmacokinetics for losartan.

Lines 409-421: “Losartan is an FDA-approved angiotensin II receptor blocker (ARB) that is approved for the treatment of hypertension in adults and children greater than 6 years old. Losartan is rapidly absorbed and undergoes extensive first-pass metabolism. The resulting active metabolite, EXP-3174, has a longer half-life and greater potency, accounting for the majority of the drug’s therapeutic effect [108]. In a preclinical breast cancer study, multiphysics modeling was employed to characterize losartan’s pharmacokinetics/pharmacodynamics, intratumoral penetration, and interstitial transport [109]. Simulating a standard 50 mg oral dose, the model demonstrated that the active metabolite has a half-life of ~6–9 hours. The combined tissue concentration of losartan and EXP-3174 is predicted to remain above therapeutically relevant levels for approximately 24 hours following a single dose [109]. In a pancreatic cancer mouse model, losartan was tested at various doses (10, 20, and 60 mg/kg). After 15 days of treatment, collagen production - assessed by second-harmonic generation imaging – was reduced by 20% (10 mg/kg), 33% (20 mg/kg), and 67% (60 mg/kg) [19].” 

 

1. G) I recommend including the recent study by Simão et al. (Cells, 2025; 14:374), which demonstrates how bidirectional signaling between adipose-derived mesenchymal stem cells and ovarian cancer cells promotes ECM remodeling, EMT plasticity, and metastatic behavior. This reference would reinforce the manuscript’s discussion on stromal–tumor crosstalk and further support the relevance of targeting the TME to improve therapeutic response.

Response: We thank the reviewer for this suggestion, this paper provides great additional context to understanding how stromal-tumor interactions affect ECM changes in ovarian cancer, and as such we have included it for critical discussion.

Lines 173-192: “Adipose tissue also plays an interesting role in OC metastasis, whereby the desmoplastic evolution within the omentum is driven by adipocyte-derived interleukin (IL)-8 and enables metastatic spread [42]. A recent study has also shown that bidirectional extracellular signaling between adipocyte-derived mesenchymal stem cells and OC cells stimulates ECM remodeling, EMT plasticity, and metastatic behavior [43]. This communication is mediated by small extracellular vesicles and upregulates pro-tumorigenic pathways such as TGF-β/Smad and Wnt/β-catenin, and, through in silico analyses, has been shown to be associated with poor prognostic outcomes in patients with OC. Notably, these processes reflect a coordinated interplay between ECM structural remodeling, which alters tissue architecture and mechanical permissiveness for invasion, and ECM-mediated signaling, which drives EMT/MET plasticity through TGF-β, Wnt/β-catenin, and integrin-dependent pathways. Distinguishing these roles is essential, as mechanical normalization strategies primarily affect tissue stiffness and interstitial pressure, whereas signaling-targeted approaches modulate transcriptional programs governing invasion and stemness. Importantly, this distinction also defines therapeutic windows during metastatic progression: mechanical normalization is predicted to impair mesothelial clearance and early spheroid engraftment by reducing permissive tissue compliance, whereas signaling-based interventions more directly suppress EMT-driven invasion and stem-like plasticity required for durable metastatic colonization.”

 

1. H) Finally, the inclusion of a summary diagram of the main concepts of the manuscript would be valuable to strengthen their contribution.

Response: We thank the reviewer for this suggestion. We have now included a summary diagram that conceptualizes the main concepts in the manuscript to strengthen the paper's contribution. Please see the new Figure 4 below.

Figure 4. ECM remodeling drives peritoneal dissemination, metastatic colonization, and treatment resistance in ovarian cancer and is targetable through microenvironment normalization. OC progression is characterized by extensive remodeling of the ECM, including increased collagen deposition, fibronectin, hyaluronan, proteoglycans, and ECM-modifying enzymes, resulting in matrix stiffening, solid stress, and immune exclusion. These changes support tumorsphere survival in ascites, mesothelial attachment, EMT/MET plasticity, therapy resistance, and suppression of anti-tumor immunity. Therapeutic normalization strategies target these processes through distinct mechanisms: TGF-β blockade primarily modulates profibrotic and immunosuppressive signaling pathways, whereas losartan alleviates mechanical stress, improves vascular perfusion, and enhances drug and immune cell delivery. Together, ECM normalization disrupts metastatic colonization, restores treatment sensitivity, and improves therapeutic outcomes in patients with OC.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors efficiently answered all the questions and improved the quality and significance of the paper.