Disparities in Tumor Microenvironment Between Primary and Metastatic Colorectal Cancer: Impact on Immune Infiltration and Survival

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Dziąg-Dudek et al describes the differences in the tumor microenvironment between primary and metastatic CRC samples in terms of abundance of immune cells. Although the topic is relevant, several points must be addressed to improve the study.

Major points:

1. The sample size is too small (n=30). This substantially limits the statistical power of the study and the conclusions that can be drawn from it. Although it is understandable that the authors do not have access to a wider cohort, they should further discuss this limitation in the discussion section: how this sample size can affect the reliability of results, the fact that they could not draw any conclusion regarding sex-specific differences, etc. Furthermore, the authors should avoid generalization of their findings for this reason.
2. A major concern is the high proportion of missing clinical data. In particular, a high percentage of samples do not have information about the M stage, and KRAS status and tumor budding is unknown in 80% or more samples. The authors do not explain why these data are missing. Moreover, given the relevance of these parameters in CRC, the authors should clarify this point.
3. In this regard, the authors analyze the correlation of some markers with KRAS mutational status and tumor budding, even though they have very limited information about these parameters. Thus, this analysis clearly lacks statistical robustness and validity and should be interpreted with caution.
4. Of the 30 samples included in the study, 29 of them correspond to liver metastasis and only 1 to pancreas metastasis. Thus, it is not appropriate to generalize conclusions to site-specific correlations. The authors should explicitly refer to liver metastasis and justify the inclusion of the pancreatic metastasis case, potentially considering its exclusion from the study.
5. The reported median follow-up time is 49.6 months, but with a very wide range (2.9-109.6). This large variability is not addressed in the manuscript, and the authors should clarify whether these highly different follow-up times reflect withdrawal of participants, death or other causes.
6. The current presentations of tables 3a and 3b make it very difficult to interpret the results, since the aim of the manuscript is comparing primary and metastatic tissues. I would suggest considering merging both tables into a single one, comparing these tumors. Alternatively, graphical representations such as boxplots would also allow the visualization of the variability of the samples, which is particularly important given the small sample size.
7. Tables 4, 5, and 6 are also difficult to follow. Presenting the correlations in separate tables based on correlations with % vs mm² does not add clarity. I would suggest showing the correlations using a heatmap to summarize them in a single figure, or at least using one table with different headings depending on the marker analyzed.
8. The discussion section is quite long and not sufficiently integrated with the results presented. Some interpretations are overstated especially taken into account the small sample size and the high proportion of missing data. Furthermore, the authors should discuss how there is an increased immune infiltration in metastatic tissue but metastases show an immunosuppressive microenvironment.
9. For all tables, the number of significant digits should be the same, to keep consistency throughout the manuscript.

Minor points:

1. The introduction section could be improved by including several studies that have already analyzed primary tumors vs metastatic ones. In fact, several of them are already mentioned in the discussion section. By adding a brief comment summarizing these studies, the readers will be given more context to better understand the present work.
2. Antibodies information should also include catalog number, not only the clone.
3. Typos and nomenclature inconsistencies should be corrected throughout the manuscript (eg “aSMA” vs “mSMA”, “PD-L1” vs “PDL-1”, etc.).
4. The variable Time to metastasis in Table 2 is not clear in the table.
5. The images in Scheme 1 would be more useful if they appear earlier in the manuscript.

Author Response

Please find attached a document containing our responses and detailed explanations to all of the reviewer’s comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The conducted analyses indicated that primary and metastatic tumors differ in terms
of their tumor microenvironment and immune cell infiltration. Metastatic lesions were characterized by a predominance of cells with an immunosuppressive phenotype. The study further demonstrated that PD-L1 expression is elevated in metastatic tumors compared to primary lesions. I have few questions

Kindly highlight the novelty of your work in introduction and abstract section.

Kindly highlight the selection criteria

I suggest you to define your hypothesis in one section.

I suggest you to add one schematic cell signaling picture about presented cancer biology

Kindly discuss current challenges section and how your work may help to overcome it. 

Kindly add recent references. 

Kindly add future perspective of your work with proposed methodology.

 

 

 

Author Response

Please find attached a document containing our responses and detailed explanations to all of the reviewer’s comments.

Author Response File: Author Response.pdf