CD40 Pathway and IL-2 Expression Mediate the Differential Outcome of Colorectal Cancer Patients with Different CSF1R c.1085 Genotypes

Round 1

Reviewer 1 Report

In this experimental work titled “CD 40 pathway and IL-2 expression mediate the differential outcome of colorectal cancer patients with different CSF1R 3 c.1085 genotypes” Dr. Yeh and colleagues detailed the possible mechanism(s)  underlying better prognosis of patients carrying the CSF1R c.1085 variant They do so by interrogating a CRC patient cohort by means of  a gene  expression array containing 395 immune-related genes.  They found that higher expression of CD40LG was found in the tumor tissues of patients with CSF1R c.1085 variant. In parallel with and related to this, they found increased infiltrate  of CD3+CD40L+ T cells   and a trend toward higher IL-2 levels in the CRC tissue from CSF1R 21 c.1085 variant carrying patients.

This work sits on previous clinical evidence showing a better prognosis for CSF1R 21 c.1085 variant carrying patients. It does confirm the previous work and adds some mechanistic insights, which may be of translational relevance for patient stratification purposes. The findings of Dr. Yeh and colleagues adds on the already investigated changes in M2 polarization of Tumor associated macrophages carrying the CSF1R 21 c.1085 variant and expand this evidence by looking at T-cell infiltrate, an important and concurrent determinant of therapy response.

This work shows a logical, straight-forward design. The text is generally clear, the figures self-explanatory. There is a number of issues to address, in order to fully unleash the translational potential of this work and to better support the authors’’ conclusions

General.  Figure 2a. Have the authors explored the IL-34 signaling and its role in this experimental system?

Figure 3a. Can the authors provide CSF1R staining by IHC since the gene expression data seems to indicate different levels of CSF1R mRNA. Further,  It is unclear whether there is higher CD40 expression on the CSF1R c-1085A>G macrophages?

Figure 4c. The difference in IL-2 content is not statistically significant but trending. This may be explained with the different stage of the lesions distributed in the A-A and A-G groups. Therefore, it would be possibly informative to test for circulating IL-2 levels (system) or for IL-2 secreted by ex-vivo cultures representative.

The authors should anyway detail on this result and provide possible explanations.

Author Response

1. General. Figure 2a. Have the authors explored the IL-34 signaling and its role in this experimental system?

Response:

Thanks for the comment. IL-34 and colony-stimulating factor 1 (CSF-1) share CSF-1 receptor, but the binding and subsequent intracellular signaling show differences (J Leukoc Biol, 2021;110(4):771-796). In our prior studies, CSF-1 was used to stimulate CSF-1R signaling and our data showed macrophages with different CSF1R c.1085 genotypes displayed a different response to CSF-1 stimulation. However, we did not explore the role of IL-34 in this experimental system and IL-34 gene was not included in this Immune Response Research Assay.

2. Figure 3a. Can the authors provide CSF1R staining by IHC since the gene expression data seems to indicate different levels of CSF1R mRNA. Further, it is unclear whether there is higher CD40 expression on the CSF1R c-1085A>G macrophages?

Response:

1. Thanks for the comment. Based on the gene expression data, we hypothesized CD40 ligand/CD40 interaction and subsequent immune responses mediated the different clinical outcome of CRC patients with different CSF1R1085 genotypes. Therefore, we applied immunofluorescent and immunohistochemical staining to confirm the findings of gene expression. Interestingly, the gene expression data also showed higher CSF1R mRNA expression in CRCs with CSF1R c.1085 genotype A_A compared to genotype A_G. It’s very likely that CRCs with CSF1R c.1085 genotype A_A had higher CSF-1R protein expression than those with genotype A_G. CSF-1R is primarily expressed on macrophages and it has been known that CSF-1R signaling is important to maintain macrophages in an M2-like state. Higher proportion of M2 macrophages associated with advanced stage and poor clinical outcome has been reported in many different types of cancer (Trends Immunol, 2002;23(11):549-55). The underlying mechanism mediating this correlation had been extensively studied in vitro and in vivo (Cancer Cell, 2015;27(4):462-72). Lower CSF-1R expression, less tumor-associated macrophages and less M2-like macrophages in tumors with CSF1R genotype A_G also provide an explanation why patients with this germline variant had better clinical outcome. The CSF-1R expression in macrophages by IHC staining is worth further explored to confirm this hypothesis. We have added this information in the “Discussion” of revised manuscript (line 268-281, page 8).
2. CD40 is the receptor found on macrophages. The binding of CD40 to CD40 ligand on T cells activates the macrophages and induces a variety immune and inflammatory responses. Our gene expression data showed CD40 expression is slightly higher in CRCs with CSF1R genotype A_G compared to genotype A_A (log₂ fold of change: 0.04849 and p=0.772221) and our prior study demonstrated the number of macrophages was significantly higher in tumor tissues with the genetic background of CSF1R1085 genotype A_A compared to genotype A_G (Clin Cancer Res, 2017;23(20):6021-30). The higher CD40 expression and lower macrophages in CRCs with CSF1R c.1085 genotype A_G suggest the level of CD40 expression might be higher in macrophages with CSF1R c.1085 genotype A_G than genotype A_A. Further studies are still needed to confirm this hypothesis. The above information has been added in the “Discussion” of revised manuscript (line 239-247, page 7).

3. Figure 4c. The difference in IL-2 content is not statistically significant but trending. This may be explained with the different stage of the lesions distributed in the A-A and A-G groups. Therefore, it would be possibly informative to test for circulating IL-2 levels (system) or for IL-2 secreted by ex-vivo cultures representative. The authors should anyway detail on this result and provide possible explanations.

Response:

Thanks for the comment. The difference in IL-2 level is not statistically significant but trending. This may be explained by the case number and different stage of the lesions distributed in different groups of CSF1R genotyping. We agree with the reviewer’s comment that it would be very informative to test for circulating IL-2 levels or IL-2 secretion by ex-vivo cultures which will be explored in the future. We have added this in the part of Discussion of revised manuscript (line 256-259, page 7).

Author Response File: Author Response.docx

Reviewer 2 Report

Thanks to immunotherapy and other treatment solutions colorectal cancer is today discussed chronic disease. Article “CD 40 pathway and IL-2 expression mediate the differential 2 outcome of colorectal cancer patients with different CSF1R 3 c.1085 genotypes” presents new data on the colorectal cancer primary and secondary resistance to immunotherapy. As authors mention understanding of the tumor immune microenvironment is essential to overcome the resistance   and establish efficient cancer immunotherapies. The data are well presented and illustrated by authors. I hope that the data will have clinical impact and the results will help to individualize the therapy of colorectal cancer.

I recommend the article for publication.

Author Response

Thanks for the comments and suggestions.