Title: Identification of the "Collagen-Macrophage" sub-category of patients with colorectal
cancer as an extension of the CMS4 subtype with THBS2 as a therapeutic target
Version: 3 Date: 23 Feb 2025
Reviewer's report:
Major
1. Please better clarify how the proposed "Collagen-Macrophage (CM)" subclass extends
beyond the CMS4 classification. Please  emphasize the practical implications of this new
subclass in clinical settings.
2. THBS2 is proposed as a key biomarker, however its not clear whether validation across
independent datasets or prospective clinical cohorts is performed or not. If not please add this
as limitations to emphasize the role of external validation for stronger translational relevance.
3. The description of single-cell sequencing methodologies and cell clustering lacks clarity.
Providing more methodological details on normalization, batch correction, and cell annotation
would enhance reproducibility.
4. The manuscript mentions a correlation between the CM-clustering differential gene score
(CSDGS) and TIDE score, but the biological rationale behind this correlation should be
discussed in greater depth.
5. The study suggests that blocking THBS2 improves PD-1 therapy. However, more direct
evidence, such as immune infiltration assays post-treatment, is necessary to substantiate these
claims.
6. Please better clarify how the multiplex immunohistochemistry images have been quantified
and statistically analyzed to strengthen claims regarding the spatial localization of FAP+ CAFs
and SPP1+ macrophages.
7. Please explain how the authors considered potential confounders. Given the heterogeneity
of CRC, factors such as microsatellite instability status, mutation burden, and immune profiles
should be controlled when defining CM classification.
8. Please clarify whether the Kaplan-Meier survival curves are adjusted for clinical covariates
to ensure the association is independent of other prognostic factors or not.
Minor
9. Please improve the annotations, legends of figures (e.g., UMAP plots) to better interpret CM
vs. non-CM differences.
Title: Identification of the "Collagen-Macrophage" sub-category of patients with colorectal
cancer as an extension of the CMS4 subtype with THBS2 as a therapeutic target
Version: 3 Date: 23 Feb 2025
Reviewer's report:
Major
1. Please better clarify how the proposed "Collagen-Macrophage (CM)" subclass extends
beyond the CMS4 classification. Please  emphasize the practical implications of this new
subclass in clinical settings.
2. THBS2 is proposed as a key biomarker, however its not clear whether validation across
independent datasets or prospective clinical cohorts is performed or not. If not please add this
as limitations to emphasize the role of external validation for stronger translational relevance.
3. The description of single-cell sequencing methodologies and cell clustering lacks clarity.
Providing more methodological details on normalization, batch correction, and cell annotation
would enhance reproducibility.
4. The manuscript mentions a correlation between the CM-clustering differential gene score
(CSDGS) and TIDE score, but the biological rationale behind this correlation should be
discussed in greater depth.
5. The study suggests that blocking THBS2 improves PD-1 therapy. However, more direct
evidence, such as immune infiltration assays post-treatment, is necessary to substantiate these
claims.
6. Please better clarify how the multiplex immunohistochemistry images have been quantified
and statistically analyzed to strengthen claims regarding the spatial localization of FAP+ CAFs
and SPP1+ macrophages.
7. Please explain how the authors considered potential confounders. Given the heterogeneity
of CRC, factors such as microsatellite instability status, mutation burden, and immune profiles
should be controlled when defining CM classification.
8. Please clarify whether the Kaplan-Meier survival curves are adjusted for clinical covariates
to ensure the association is independent of other prognostic factors or not.
Minor
9. Please improve the annotations, legends of figures (e.g., UMAP plots) to better interpret CM
vs. non-CM differences.
Please review this paper.
user
Title: Identification of the "Collagen-Macrophage" sub-category of patients with colorectal
cancer as an extension of the CMS4 subtype with THBS2 as a therapeutic target
Version: 3 Date: 23 Feb 2025
Reviewer's report:
Major
1. Please better clarify how the proposed "Collagen-Macrophage (CM)" subclass extends
beyond the CMS4 classification. Please  emphasize the practical implications of this new
subclass in clinical settings.
2. THBS2 is proposed as a key biomarker, however its not clear whether validation across
independent datasets or prospective clinical cohorts is performed or not. If not please add this
as limitations to emphasize the role of external validation for stronger translational relevance.
3. The description of single-cell sequencing methodologies and cell clustering lacks clarity.
Providing more methodological details on normalization, batch correction, and cell annotation
would enhance reproducibility.
4. The manuscript mentions a correlation between the CM-clustering differential gene score
(CSDGS) and TIDE score, but the biological rationale behind this correlation should be
discussed in greater depth.
5. The study suggests that blocking THBS2 improves PD-1 therapy. However, more direct
evidence, such as immune infiltration assays post-treatment, is necessary to substantiate these
claims.
6. Please better clarify how the multiplex immunohistochemistry images have been quantified
and statistically analyzed to strengthen claims regarding the spatial localization of FAP+ CAFs
and SPP1+ macrophages.
7. Please explain how the authors considered potential confounders. Given the heterogeneity
of CRC, factors such as microsatellite instability status, mutation burden, and immune profiles
should be controlled when defining CM classification.
8. Please clarify whether the Kaplan-Meier survival curves are adjusted for clinical covariates
to ensure the association is independent of other prognostic factors or not.
Minor
9. Please improve the annotations, legends of figures (e.g., UMAP plots) to better interpret CM
vs. non-CM differences.
assistant