CX3CR1-Dependent Macrophages Drive Ovarian Cancer Progression Through MMP-2 and TGF-β Production

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study presents a well-designed and conceptually strong investigation into the role of the CX3CL1–CX3CR1 axis in regulating macrophage behavior within the tumor microenvironment. The use of Cx3cr1 knockout mice, combined with in vivo tumor models and mechanistic analyses, provides a solid experimental framework and offers meaningful insights into how chemokine signaling contributes to tumor progression. Overall, the data are compelling and the manuscript is clearly written. To further strengthen the conclusions and enhance the specificity of the proposed mechanism, several points merit consideration.

1. In Figure 4, the authors demonstrate reduced F4/80⁺ macrophage infiltration in tumors derived from Cx3cr1 knockout mice. To more definitively attribute this effect to a tumor microenvironment–specific mechanism rather than to systemic differences in macrophage abundance, it would be helpful to include data showing that F4/80⁺ macrophage levels are comparable between wild-type and Cx3cr1 knockout mice in non-tumor tissues or under normal physiological conditions. 

2. In Figure 5, the rationale for introducing MMP2 warrants further clarification. Although MMP2 is a well-known mediator of extracellular matrix remodeling, its specific relationship to the CX3CL1–CX3CR1 axis is not fully explained in the current manuscript. 

3. The manuscript would benefit from a more explicit discussion of how macrophage-derived MMP2 and TGF-β each contribute to tumor progression. 

4. The study does not present experimental data demonstrating M2 macrophage markers. Nevertheless, macrophages are labeled as “M2” in the schematic figure. What is the basis for this designation? 

5. The mouse model used in this study is a global Cx3cr1 knockout model. Is there evidence to exclude the possibility that the observed effects are mediated by Cx3cr1 deficiency in non-macrophage cell populations?

Author Response

Reviewer 1

1. In Figure 4, the authors demonstrate reduced F4/80⁺ macrophage infiltration in tumors derived from Cx3cr1 knockout mice. To more definitively attribute this effect to a tumor microenvironment–specific mechanism rather than to systemic differences in macrophage abundance, it would be helpful to include data showing that F4/80⁺ macrophage levels are comparable between wild-type and Cx3cr1 knockout mice in non-tumor tissues or under normal physiological conditions. 

 

In accordance with the instructive comment, we agree that the reduced F4/80⁺ macrophage infiltration in tumors from Cx3cr1-deficient mice should be distinguished from a possible systemic difference in basal macrophage abundance. To address this point, we analyzed unstimulated peritoneal exudate cells from wild-type and Cx3cr1 knockout mice by flow cytometry under steady-state conditions. As a result, the proportion and absolute number of macrophages were comparable between the two groups. These findings indicate that Cx3cr1 deficiency does not affect basal macrophage abundance under physiological conditions, and support the interpretation that the reduced macrophage accumulation observed in tumors mainly reflects a tumor microenvironment–dependent mechanism rather than a systemic reduction in macrophages. We have added these data to the revised manuscript.

On the other hands, Flow cytometric analysis of unstimulated peritoneal exudate cells demonstrated that macrophage numbers were comparable between wild-type and Cx3cr1-deficient mice under physiological conditions, suggesting that the reduced macrophage accumulation in tumors of Cx3cr1-deficient mice was not due to a systemic deficiency of macrophages (line 335-339).

 

2. In Figure 5, the rationale for introducing MMP2 warrants further clarification. Although MMP2 is a well-known mediator of extracellular matrix remodeling, its specific relationship to the CX3CL1–CX3CR1 axis is not fully explained in the current manuscript. 

 

In accordance with the comment, we agree that the rationale for focusing on MMP-2 was not sufficiently explained. MMP-2 was selected as a representative effector molecule because of its well-established role in extracellular matrix remodeling and peritoneal dissemination in ovarian cancer. In addition, previous studies have suggested that tumor-associated macrophages can produce MMP-2 and thereby promote tumor invasion. In the present study, we demonstrate that MMP-2 expression is markedly reduced in Cx3cr1-deficient mice in parallel with decreased macrophage infiltration, suggesting that the CX3CL1–CX3CR1 axis indirectly regulates MMP-2 levels through recruitment of MMP-2–producing macrophages. Thus, we have clarified this rationale in the revised manuscript (line 417-424).

 

3. The manuscript would benefit from a more explicit discussion of how macrophage-derived MMP2 and TGF-β each contribute to tumor progression. 

 

We thank the reviewer for this insightful comment. We agree that the distinct contributions of macrophage-derived MMP-2 and TGF-β to tumor progression should be more clearly articulated. MMP-2 primarily contributes to extracellular matrix degradation, thereby facilitating tumor cell invasion and peritoneal dissemination. In contrast, TGF-β exerts pleiotropic effects, including promotion of tumor cell invasiveness, activation of stromal fibroblasts, and suppression of antitumor immune responses. We have added a more explicit discussion of these distinct roles in the revised manuscript (line 402-413).

 

4. The study does not present experimental data demonstrating M2 macrophage markers. Nevertheless, macrophages are labeled as “M2” in the schematic figure. What is the basis for this designation? 

 

Thank you for this important comment. We agree that our study does not provide direct experimental evidence demonstrating M2 macrophage polarization. Therefore, the designation of macrophages as “M2” in the schematic figure was not sufficiently supported by our data. However, our previous study demonstrated (Ref. 20 and 30) that CX3CR1 was expressed on M2 macrophages, indicating that CX3CR1 could be one of the markers of M2 macrophages. In response to this concern, we have removed the “M2” labeling from the figure in the revised manuscript to avoid overinterpretation (Graphical Abstract and line 452-461).

 

5. The mouse model used in this study is a global Cx3cr1 knockout model. Is there evidence to exclude the possibility that the observed effects are mediated by Cx3cr1 deficiency in non-macrophage cell populations?

 

Thank you for this important point. We agree that the use of global Cx3cr1 knockout mice does not allow us to formally exclude the contribution of CX3CR1 deficiency in non-macrophage cell populations. However, several lines of evidence in our study support the interpretation that macrophages are the primary mediators of the observed effects. First, Cx3cr1 deficiency markedly reduced the accumulation of F4/80⁺ macrophages in tumor tissues. Second, double immunofluorescence analysis demonstrated that MMP-2 and TGF-β were predominantly produced by F4/80⁺ macrophages. Third, the reduction in tumor burden and ascites formation in Cx3cr1-deficient mice was accompanied by decreased expression of these macrophage-derived factors, suggesting a functional link between macrophage recruitment and tumor progression. Nevertheless, we cannot completely exclude the potential contribution of CX3CR1 expressed on other cell types, including monocytes, dendritic cells, or even tumor cells. Future studies using macrophage-specific conditional knockout models or bone marrow chimera experiments will be necessary to definitively address this issue.　We have added this limitation to the Discussion section (line 502-508).

Reviewer 2 Report

Comments and Suggestions for Authors

General comments

The authors address a highly relevant interaction between tumor progression and macrophage infiltration mediated by the CX3CL1/CX3CR1 axis in ovarian cancer. While the study tackles an important biological question and the experimental approach is largely convincing, several aspects require clarification before the manuscript can be considered for publication.

Methods section

• It remains unclear whether the recombinant CX3CL1 used in the migration and proliferation assays corresponds to the full-length or soluble form of CX3CL1. This should be clarified.

• Given the pronounced heterogeneity of ovarian cancer and the inclusion of different subtypes, a brief summary of clinical characteristics (e.g., FIGO stage, age, TNM) would help contextualize the findings.

• The number of injected cells in the syngeneic mouse model is inconsistent between the Methods section and the main text. Since the initial cell count is a critical experimental parameter, this discrepancy should be resolved.

Results section

• The authors state that 17 human EOC samples and 5 healthy ovarian samples were analyzed. However, the figure 1 displays 4 benign tumors and 4 serous adenocarcinomas. While subsampling is acceptable given ovarian cancer heterogeneity, the text must align with the figures. It also remains unclear why the remaining cases are not shown for either qPCR or IHC. A clear rationale should be provided, or the additional data should be included in the supplementary material.

• Comparisons between healthy ovarian tissue and ovarian cancer should be interpreted with caution, as most ovarian cancers originate from the outer epithelial layer. Bulk RNA analysis of whole tissue inevitably includes multiple cell types. This limitation should be briefly acknowledged in the Discussion.

• The nomenclature for murine versus human genes and proteins is inconsistent. Please clearly indicate species origin throughout.

• The qPCR data are described as normalized to Actb, which is appropriate; however, the normalization strategy (e.g., ΔΔCt, fold change) is not specified and should be clarified in the Methods.

• Enhanced CX3CL1 and CX3CR1 expression in tumor tissue compared with benign neoplasms is shown by qPCR, and protein expression is assessed by IHC. It remains unclear whether all samples were stained. If available, quantitative IHC data demonstrating increased CX3CL1/CX3CR1 expression would substantially strengthen the conclusions, particularly given that these are membrane-associated proteins.

• The distribution of CX3CL1 and CX3CR1 staining in IHC is not described. CX3CR1 appears predominantly stromal, which does not fully support the proposed tumoral expression. Additionally, were CX3CR1-positive macrophages detected by IHC?

• Interpretation of expression differences between EOC and benign tissue would benefit from inclusion of publicly available datasets (e.g., TCGA) as external validation and to increase sample size.

• In the FACS analysis of CX3CR1 expression (figure 2), the red curve represents CX3CR1 staining, but the identity of the black curve is unclear and should be specified in the figure legend. I also suggest labeling of the axis.

• Proliferation assays were performed with 2.5 nmol/L recombinant CX3CL1, whereas migration assays used 5 and 10 nmol/L. Please clarify the rationale for using different concentrations. Could higher CX3CL1 concentrations also induce proliferation?

• The survival benefit observed in CX3CR1 knockout mice is striking (figure 3). Given the extensive peritoneal dissemination typical of the ID8 model, a brief description of metastatic sites in knockout versus wild-type mice would be helpful. In the provided images, the peritoneal wall appears tumor-free, yet tumor burden is reported as 50% of wild-type levels. Please clarify tumor localization and specify which tumor regions were used for downstream analyses.

• The authors report marked reductions in Mmp2 and Tgf-β expression. While mRNA levels decrease by approximately 50%, IHC signals appear nearly absent. Please comment on this discrepancy. A short discussion of additional cellular sources of Mmp2 and TGF-β would be valuable.

• Colocalization of F4/80 with Mmp2 and TGF-β in murine tumors is shown by ICC. However, interpretation is difficult without a nuclear marker. F4/80 is a membrane protein, yet appears cytoplasmic, while Mmp2 and TGF-β are secreted proteins that may transiently localize intracellularly. Please clarify the staining patterns and discuss to what extent true colocalization can be inferred.

Discussion

• The authors present the CX3CL1/CX3CR1 axis as a major mediator of macrophage infiltration in ovarian cancer, which is supported by the data. However, it remains unclear why other chemokine axes (e.g., CXCR3, CCR5/CCR21) do not compensate for CX3CR1 loss. A brief discussion of this point would strengthen the manuscript.

• The CX3CL1/CX3CR1 axis likely also affects recruitment of other immune populations such as T cells and NK cells. If experimental data are unavailable, this limitation should be acknowledged.

• Although the authors aim to move beyond the conventional M1/M2 paradigm, macrophage characterization relies mainly on Mmp2 and TGF-β. Including at least one M1-associated marker (e.g., Il-6) or tempering conclusions would improve rigor.

• Several statements in the Discussion remain vague and would benefit from clarification:

  • Line 336: Please specify what constitutes the “new angle” of interpretation.

  • Line 345: How might macrophage-derived Mmp2 and TGF-β be specifically targeted, given their multiple cellular sources?

  • Line 358: Please clarify the distinction between the M1/M2 paradigm and current concepts of macrophage plasticity.

  • Line 376: Reference 33 only refers to lung cancer. 

Author Response

Reviewer 2

Methods section

• It remains unclear whether the recombinant CX3CL1 used in the migration and proliferation assays corresponds to the full-length or soluble form of CX3CL1. This should be clarified.

 

Thank you for your feedback. The recombinant murine CX3CL1 (manufactured by R&D Systems) used in this study is, according to manufacturer information, the extracellular domain of CX3CL1 in its soluble form. It has been reported that CX3CL1-mediated migration induction is primarily carried out by the soluble form; therefore, the CX3CL1 stimulation in this in vitro experiment evaluated the action of soluble CX3CL1, not the membrane-bound form. While CX3CL1 is known to function in both membrane-bound and soluble forms, the main objective of this study was to evaluate the effects of CX3CL1-mediated cell migration response and tumor progression. In this regard, soluble CX3CL1 is widely used as an appropriate ligand. Therefore, we believe this does not fundamentally affect the conclusions of this study. We will add this point to the Materials and Methods section (line 117).

 

• Given the pronounced heterogeneity of ovarian cancer and the inclusion of different subtypes, a brief summary of clinical characteristics (e.g., FIGO stage, age, TNM) would help contextualize the findings.

 

As suggested by the reviewer, a table of brief summaries has been added (Table 1, 164-166).

 

• The number of injected cells in the syngeneic mouse model is inconsistent between the Methods section and the main text. Since the initial cell count is a critical experimental parameter, this discrepancy should be resolved.

 

Thank you for pointing out this inconsistency. We apologize for the confusion. The correct number of injected cells in the syngeneic mouse model is 5 × 10⁶ cells per mouse. We have revised the Methods section and the main text to ensure consistency (line 307).

 

Results section

• The authors state that 17 human EOC samples and 5 healthy ovarian samples were analyzed. However, the figure 1 displays 4 benign tumors and 4 serous adenocarcinomas. While subsampling is acceptable given ovarian cancer heterogeneity, the text must align with the figures. It also remains unclear why the remaining cases are not shown for either qPCR or IHC. A clear rationale should be provided, or the additional data should be included in the supplementary material.

 

As the reviewer pointed out, we added Table 1 to provide clearer data (line 164-166).

 

• Comparisons between healthy ovarian tissue and ovarian cancer should be interpreted with caution, as most ovarian cancers originate from the outer epithelial layer. Bulk RNA analysis of whole tissue inevitably includes multiple cell types. This limitation should be briefly acknowledged in the Discussion.

 

Thank you for this important comment. We agree that comparisons between healthy ovarian tissue and ovarian cancer should be interpreted with caution, as bulk RNA analysis includes multiple cell types and most ovarian cancers arise from the epithelial layer. We have now acknowledged this limitation in the Discussion section of the revised manuscript (line 413-416).

 

• The nomenclature for murine versus human genes and proteins is inconsistent. Please clearly indicate species origin throughout.

 

Thank you for pointing out this issue. We apologize for the inconsistency in nomenclature. We have carefully revised the manuscript to ensure that murine and human genes and proteins are clearly distinguished throughout, following standard nomenclature conventions. Gene and protein names have been corrected according to standard guidelines (e.g., mouse genes in italics with initial capital letters and human genes in all capitals).

 

• The qPCR data are described as normalized to Actb, which is appropriate; however, the normalization strategy (e.g., ΔΔCt, fold change) is not specified and should be clarified in the Methods.

 

Thank you for this helpful comment. We apologize for the lack of clarity. The qPCR data were normalized to Actb using the ΔΔCt method and are presented as relative fold changes. This information has now been added to the Materials and Methods section (line 223-231).

 

• Enhanced CX3CL1 and CX3CR1 expression in tumor tissue compared with benign neoplasms is shown by qPCR, and protein expression is assessed by IHC. It remains unclear whether all samples were stained. If available, quantitative IHC data demonstrating increased CX3CL1/CX3CR1 expression would substantially strengthen the conclusions, particularly given that these are membrane-associated proteins.

 

Thank you for this important comment. We confirm that all samples were subjected to IHC staining for CX3CL1 and CX3CR1. However, quantitative analysis of the IHC data was not performed in the present study because sample number was small. We agree that quantitative evaluation would further strengthen the conclusions, particularly for membrane-associated proteins, and this will be addressed in future studies.

 

• The distribution of CX3CL1 and CX3CR1 staining in IHC is not described. CX3CR1 appears predominantly stromal, which does not fully support the proposed tumoral expression. Additionally, were CX3CR1-positive macrophages detected by IHC?

 

We thank the reviewer for this important comment.

First, regarding the distribution of CX3CL1 and CX3CR1 in immunohistochemistry, CX3CL1 staining was mainly observed in tumor epithelial cells, whereas CX3CR1 staining was detected predominantly in stromal regions rather than uniformly in tumor cells. Actually, some of infiltrating immune cells within the tumor microenvironment expressed CX3CR1. We have now clarified this point in the revised Results section (line 260-266).

Importantly, our interpretation does not rely solely on IHC to support tumoral CX3CR1 expression. As shown in our manuscript, CX3CR1 expression in tumor cells was confirmed using the murine ID8 cell line by both immunohistochemistry and flow cytometry (Fig. 2B, C), supporting that tumor cells can express CX3CR1 . Therefore, we consider that CX3CR1 is expressed in both tumor cells and stromal components, reflecting its dual role in ovarian cancer progression.

Regarding macrophages, CX3CR1-positive macrophages were indeed detected. Immunohistochemical analysis demonstrated F4/80⁺ macrophage infiltration in tumor tissues, and double-color immunofluorescence analysis clearly showed co-localization of CX3CR1 with F4/80⁺ macrophages (Fig. 4C), indicating that tumor-associated macrophages are a major CX3CR1-expressing population in the tumor microenvironment.

We have revised the manuscript to better describe the spatial distribution of CX3CR1 and to explicitly state the presence of CX3CR1⁺ macrophages in tumor tissues (line 329-340).

 

 New Fig. 1G; Scale bar, 50 μm

 

• Interpretation of expression differences between EOC and benign tissue would benefit from inclusion of publicly available datasets (e.g., TCGA) as external validation and to increase sample size.

 

We thank the reviewer for this valuable suggestion.

We agree that validation using publicly available datasets such as The Cancer Genome Atlas would further strengthen the interpretation of expression differences between EOC and benign tissues and increase the robustness of our findings.

In the present study, our primary aim was to evaluate CX3CL1 and CX3CR1 expression at both the mRNA and protein levels using clinical samples and experimental models. However, we acknowledge that the relatively small sample size is a limitation.

We have now added this point as a limitation in the Conclusions section and will incorporate analyses of publicly available datasets, including TCGA, in future studies to validate and extend our findings (line 566-570).

 

• In the FACS analysis of CX3CR1 expression (figure 2), the red curve represents CX3CR1 staining, but the identity of the black curve is unclear and should be specified in the figure legend. I also suggest labeling of the axis.

 

We thank the reviewer for this helpful comment.

The black curve represents the isotype control used as a negative control for CX3CR1 staining. We have now clarified this point in the figure legend (Fig. 2C, line 296-299).　In addition, the axes have been labeled appropriately in the revised figure to improve clarity.

 

• Proliferation assays were performed with 2.5 nmol/L recombinant CX3CL1, whereas migration assays used 5 and 10 nmol/L. Please clarify the rationale for using different concentrations. Could higher CX3CL1 concentrations also induce proliferation?

 

We thank the reviewer for this insightful comment.

The concentrations of recombinant CX3CL1 were selected based on preliminary experiments and prior reports indicating that migration responses generally require higher chemokine concentrations than proliferation assays. Therefore, lower concentrations were used for proliferation assays, whereas higher concentrations were applied in migration assays to adequately assess chemotactic responses.

Regarding the possibility that higher concentrations of CX3CL1 may also induce proliferation, we cannot completely exclude this possibility. However, in our experimental setting, CX3CL1 did not significantly enhance proliferation at the tested concentration. Evaluation of a broader range of concentrations, including higher doses, would be of interest and will be addressed in future studies.

 

• The survival benefit observed in CX3CR1 knockout mice is striking (figure 3). Given the extensive peritoneal dissemination typical of the ID8 model, a brief description of metastatic sites in knockout versus wild-type mice would be helpful. In the provided images, the peritoneal wall appears tumor-free, yet tumor burden is reported as 50% of wild-type levels. Please clarify tumor localization and specify which tumor regions were used for downstream analyses.

 

We thank the reviewer for this important comment.

In the ID8 intraperitoneal model, tumor nodules were predominantly observed on peritoneal surfaces, including the mesentery, diaphragm, and peritoneal lining of abdominal organs in both wild-type and Cx3cr1-deficient mice. While the representative image may give the impression that the peritoneal wall is tumor-free, this reflects regional variability in tumor distribution rather than the absence of dissemination. Overall tumor burden, including the number and size of disseminated nodules throughout the peritoneal cavity, was markedly reduced in Cx3cr1-deficient mice compared with wild-type mice.

For downstream analyses, tumor tissues were collected from representative intraperitoneal nodules, primarily from the mesentery and peritoneal surfaces, in a consistent manner across experimental groups.

We have now clarified tumor localization, the basis of tumor burden evaluation, and the sampling strategy in the revised Methods (line 184-190) and Results sections (line 312-317).

 

• The authors report marked reductions in Mmp2 and Tgf-β expression. While mRNA levels decrease by approximately 50%, IHC signals appear nearly absent. Please comment on this discrepancy. A short discussion of additional cellular sources of Mmp2 and TGF-β would be valuable.

 

We thank the reviewer for this important observation.

The apparent discrepancy between the approximately 50% reduction in mRNA levels and the more pronounced decrease in IHC signals may reflect differences in sensitivity and dynamic range between these methods. While quantitative PCR detects total transcript levels in whole tumor tissue, immunohistochemistry is a semi-quantitative method that highlights protein localization and may visually emphasize reductions in specific high-expressing cell populations. In our model, MMP-2 and TGF-β are predominantly produced by infiltrating macrophages; therefore, the marked reduction in CX3CR1⁺ macrophage accumulation in Cx3cr1-deficient mice likely results in a substantial decrease in protein-positive areas, even if residual expression persists at the mRNA level from other cell types.

We also acknowledge that, in addition to macrophages, other cell populations such as tumor cells and stromal fibroblasts can contribute to MMP-2 and TGF-β production.

We have revised Discussion section (line 426-443).

 

• Colocalization of F4/80 with Mmp2 and TGF-β in murine tumors is shown by ICC. However, interpretation is difficult without a nuclear marker. F4/80 is a membrane protein, yet appears cytoplasmic, while Mmp2 and TGF-β are secreted proteins that may transiently localize intracellularly. Please clarify the staining patterns and discuss to what extent true colocalization can be inferred.

 

We thank the reviewer for this important comment.

We agree that the absence of a nuclear marker limits precise interpretation of cellular localization and strict assessment of colocalization. In our double immunofluorescence analysis, F4/80 staining, although a membrane marker, can appear partly cytoplasmic due to sectioning and signal spread, whereas MMP-2 and TGF-β, as secreted proteins, may show intracellular staining during synthesis and secretion processes. Therefore, complete spatial overlap at the subcellular level is not necessarily expected.

Accordingly, our intention was not to demonstrate strict subcellular colocalization, but rather to indicate that MMP-2 and TGF-β signals are predominantly associated with F4/80⁺ macrophage populations at the cellular level.

We have now clarified this point in the revised manuscript and acknowledged the limitation regarding the absence of nuclear staining (line 365-373).

 

Discussion

• The authors present the CX3CL1/CX3CR1 axis as a major mediator of macrophage infiltration in ovarian cancer, which is supported by the data. However, it remains unclear why other chemokine axes (e.g., CXCR3, CCR5/CCR21) do not compensate for CX3CR1 loss. A brief discussion of this point would strengthen the manuscript.

 

We thank the reviewer for this important comment.

We agree that multiple chemokine axes are involved in macrophage recruitment, and compensation by other pathways cannot be excluded. However, our results demonstrate that CX3CR1 deficiency leads to a significant reduction in macrophage accumulation, suggesting that the CX3CL1–CX3CR1 axis plays a non-redundant or dominant role in this model. One possible explanation is that CX3CL1, as a membrane-bound and soluble chemokine, uniquely mediates both adhesion and migration, thereby facilitating efficient retention and accumulation of macrophages within the tumor microenvironment.

In addition, it is possible that other chemokine axes contribute to macrophage recruitment but are not sufficient to fully compensate for the loss of CX3CR1 signaling in this context.

We have now added a brief discussion addressing this point in the revised manuscript (line 483-490).

 

• The CX3CL1/CX3CR1 axis likely also affects recruitment of other immune populations such as T cells and NK cells. If experimental data are unavailable, this limitation should be acknowledged.

 

We thank the reviewer for this important comment.

We agree that the CX3CL1–CX3CR1 axis may also regulate the recruitment of other immune cell populations, including T cells and NK cells. In the present study, we focused primarily on macrophages, and we did not perform a comprehensive analysis of other immune subsets. Therefore, we cannot exclude the potential contribution of these cell populations to the observed phenotypes.

We have now acknowledged this point as a limitation in the revised manuscript (line 506-525).

 

• Although the authors aim to move beyond the conventional M1/M2 paradigm, macrophage characterization relies mainly on Mmp2 and TGF-β. Including at least one M1-associated marker (e.g., Il-6) or tempering conclusions would improve rigor.

 

We thank the reviewer for this insightful comment.

We agree that a more comprehensive characterization of macrophage phenotypes, including the assessment of M1-associated markers such as Il-6, would strengthen the rigor of the study. In the present work, we focused on functional readouts, specifically MMP-2 and TGF-β production, to define a CX3CR1-dependent macrophage subset. However, we acknowledge that this does not fully capture the spectrum of macrophage polarization.

Accordingly, we have tempered our conclusions and clarified that our findings identify a functionally defined macrophage population rather than comprehensively characterizing M1/M2 phenotypes. We have also noted this limitation in the Conclusions section (line 542-549).

 

• Several statements in the Discussion remain vague and would benefit from clarification:
• Line 336: Please specify what constitutes the “new angle” of interpretation.
• Line 345: How might macrophage-derived Mmp2 and TGF-β be specifically targeted, given their multiple cellular sources?
• Line 358: Please clarify the distinction between the M1/M2 paradigm and current concepts of macrophage plasticity.
• Line 376: Reference 33 only refers to lung cancer. 

 

Line 336: We thank the reviewer for this helpful comment.

We agree that the phrase “new angle” was vague. In this study, the novelty lies in identifying a CX3CR1-dependent macrophage subset defined by its functional output—specifically the production of MMP-2 and TGF-β—rather than by conventional M1/M2 polarization markers.

We have revised the manuscript to clarify this point (line 452-461).

 

Line 345: We thank the reviewer for this important point.

We agree that directly targeting MMP-2 and TGF-β is challenging due to their production by multiple cell types. In this context, our study suggests that targeting upstream regulatory pathways, such as the CX3CL1–CX3CR1 axis, may represent a more selective approach by preferentially limiting the recruitment and accumulation of macrophages that serve as major sources of these mediators.

In addition, emerging strategies such as cell type–specific drug delivery systems or macrophage-targeted therapies may allow more selective modulation of macrophage-derived factors while minimizing effects on other cellular sources.

We have added a brief discussion addressing this point in the revised manuscript (line 526-532).

 

Line 358: We thank the reviewer for this important comment.

The classical M1/M2 paradigm categorizes macrophages into two polarized states: M1 macrophages, which are pro-inflammatory and anti-tumorigenic, and M2 macrophages, which are anti-inflammatory and pro-tumorigenic. However, current understanding emphasizes that macrophages exhibit substantial plasticity and exist along a dynamic continuum of activation states, shaped by microenvironmental cues rather than fixed categories.

In this study, we aimed to move beyond this simplified dichotomy by focusing on functional characteristics, specifically the production of MMP-2 and TGF-β, to define a CX3CR1-dependent macrophage subset.

We have clarified this distinction in the revised manuscript (line 542-549).

 

Line 376: Previous studies have demonstrated that CX3CR1⁺ immune cell populations contribute to antitumor responses in certain cancer types, such as lung cancer [33]. However, given the differences in tumor microenvironments across cancer types, these findings may not be directly extrapolated to ovarian cancer and should be interpreted with caution.

We have modified line 476-478.

Reviewer 3 Report

Comments and Suggestions for Authors

The work is very clear and comprehensive. I only ask the authors to discuss the role played by other components of the tumor environment (such as fibroblasts, adipocytes, etc.) in the CX3CL1–CX3CR1 axis interaction.

Author Response

Reviewer 3

The work is very clear and comprehensive. I only ask the authors to discuss the role played by other components of the tumor environment (such as fibroblasts, adipocytes, etc.) in the CX3CL1–CX3CR1 axis interaction.

 

We thank the reviewer for this insightful comment.

We agree that, in addition to macrophages, other components of the tumor microenvironment, such as fibroblasts and adipocytes, may also contribute to CX3CL1–CX3CR1 axis–mediated interactions. These cell types are known to produce chemokines and growth factors and may influence tumor progression through crosstalk with immune cells.

We have now added a brief discussion addressing this point in the revised manuscript (line 483-490).

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript investigates a potentially important and translationally relevant topic, though it needs some improvements before possible publication in the journal.  

1. Lines 89-91: While the introduction states the role of CX3CR1 in ovarian cancer is "insufficiently characterized," this gap could be defined more sharply. The reader is left wondering what exactly is unknown. Stating what exactly is unknown.
2. The methods do not describe how "CX3CR1-dependent macrophages" were generated or isolated for any in vitro experiments. If co-cultures or macrophage-conditioned media experiments were performed, the procedure for obtaining these macrophages (e.g., differentiated from bone marrow, sorted from tumors) is completely missing.
3. Section 2.5. Was this study approved by the Institutional Review Board (IRB) or the Ethics Committee of Wakayama Medical University Hospital? An approval number must be provided. Secondly, the description "10 patients" is insufficient. Please provide a table or a detailed description of the cohort, including age, stage (FIGO), grade, histological subtype (e.g., high-grade serous, endometrioid), and any relevant clinical outcomes. This information is essential for interpreting the gene expression data.
4. Section 2.10. This method is prone to bias and lacks rigor. How were the 10 fields selected? Were they chosen randomly, or were they "hot spots" with the highest density?
5. Section 4. The concept of a "dual role" of CX3CR1 (tumor cell vs. macrophage) is stated repeatedly (Lines 347, 371-372, 404-405, 410-411). The therapeutic potential of CX3CR1 inhibitors is also mentioned multiple times (Lines 366-377, 400-406, 407-414). The final paragraph of the Discussion (Lines 407-414) is almost an exact replica of the preceding one (Lines 400-406). The authors should consolidate these points.
6. Overall, significant language revision is required throughout the manuscript, with emphasis on using correct biomedical terms, required for effective scientific communication.  

Author Response

Reviewer 4 

1. Lines 89-91: While the introduction states the role of CX3CR1 in ovarian cancer is "insufficiently characterized," this gap could be defined more sharply. The reader is left wondering what exactly is unknown. Stating what exactly is unknown.

 

We thank the reviewer for this helpful comment.

We agree that the knowledge gap was not sufficiently defined. In this study, the key unresolved issue is not simply whether CX3CR1 is expressed in ovarian cancer, but how the CX3CL1–CX3CR1 axis functionally contributes to tumor progression, particularly through the regulation of tumor-associated macrophage recruitment and their effector functions, such as MMP-2 and TGF-β production.

We have revised the Introduction to clarify this point (line 93-98).

 

1. The methods do not describe how "CX3CR1-dependent macrophages" were generated or isolated for any in vitro experiments. If co-cultures or macrophage-conditioned media experiments were performed, the procedure for obtaining these macrophages (e.g., differentiated from bone marrow, sorted from tumors) is completely missing.

 

We thank the reviewer for this important comment.

We apologize for the lack of clarity. In this study, we did not generate or isolate “CX3CR1-dependent macrophages” for in vitro experiments, nor did we perform co-culture or macrophage-conditioned media assays. Our conclusions regarding CX3CR1-dependent macrophages are based on in vivo observations, including immunohistochemistry and double immunofluorescence analyses demonstrating the presence and functional characteristics of CX3CR1⁺ macrophages in tumor tissues.

We have revised the manuscript to clarify this point and to avoid any misunderstanding regarding in vitro macrophage experiments (line 184-190).

 

 

1. Section 2.5. Was this study approved by the Institutional Review Board (IRB) or the Ethics Committee of Wakayama Medical University Hospital? An approval number must be provided. Secondly, the description "10 patients" is insufficient. Please provide a table or a detailed description of the cohort, including age, stage (FIGO), grade, histological subtype (e.g., high-grade serous, endometrioid), and any relevant clinical outcomes. This information is essential for interpreting the gene expression data.

 

1. Institutional Review Board Statement: No. 1509
2. As the reviewer pointed out, we added Table 1 (line 164-166).

 

1. Section 2.10. This method is prone to bias and lacks rigor. How were the 10 fields selected? Were they chosen randomly, or were they "hot spots" with the highest density?

 

We thank the reviewer for this important comment.

We agree that field selection is a potential source of bias. In this study, the 10 high-power fields were selected in a systematic and unbiased manner across the entire tumor section, rather than focusing on “hot spots” with the highest macrophage density. Field selection was performed by an examiner blinded to the experimental groups to minimize selection bias.

We have clarified this point in the revised Methods section (line 210-213).

 

1. Section 4. The concept of a "dual role" of CX3CR1 (tumor cell vs. macrophage) is stated repeatedly (Lines 347, 371-372, 404-405, 410-411). The therapeutic potential of CX3CR1 inhibitors is also mentioned multiple times (Lines 366-377, 400-406, 407-414). The final paragraph of the Discussion (Lines 407-414) is almost an exact replica of the preceding one (Lines 400-406). The authors should consolidate these points.

 

We thank the reviewer for this important comment.

We agree that the description of the “dual role” of CX3CR1 and its therapeutic implications was repetitive. In the revised manuscript, we have consolidated these points to avoid redundancy and improve clarity. Specifically, we streamlined the discussion of CX3CR1 functions in tumor cells and macrophages into a single coherent paragraph and removed overlapping statements regarding therapeutic potential. The final paragraph of the Discussion has also been revised to eliminate duplication and provide a more concise summary of the study’s significance.

 

1. Overall, significant language revision is required throughout the manuscript, with emphasis on using correct biomedical terms, required for effective scientific communication.  

 

We agree that careful language revision is essential for effective scientific communication. The manuscript has been thoroughly revised to improve clarity, consistency, and the use of appropriate biomedical terminology. In addition, the revised version has been carefully edited by a professional editing service (Editage, No. HGNYD_36) to ensure accuracy and readability.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors provided appropriate responses to all comments.

Author Response

Thank you for your constructive and helpful advice.

Reviewer 4 Report

Comments and Suggestions for Authors

None

Author Response

Thank you for your constructive and helpful advice.