AGING IS ASSOCIATED WITH AN ANTI-TUMOR IMMUNE MICROENVIRONMENT IN A GENETICALLY ENGINEERED MOUSE MODEL OF COLORECTAL CANCER

Background: Immune checkpoint blockade has revolutionized cancer treatment by harnessing the patient’s native anti-tumor T cells, but most colorectal cancers do not respond, for unclear reasons. A major challenge in the field is understanding how patient charactistics, such as age, modify cancer cell-mediated antigen presentation and T cell activation. Patient age across cancer types is associated with increased tumor mutational burden, increased expression of immune checkpoint genes, and increased pro-inflammatory signaling. Here, we investigated the effects of aging on immune pathways in an inducible, genetically engineered mouse model of colorectal cancer.

Materials and Methods: We induced Apc-null tumors in the distal colons of young (age 2-3 months) and old (age 18-22 months) Apc^fl/fl;Villin^CreERT2 mice (C57BL/6J background) via colonoscopy-guided injection of 4-hydroxytamoxifen. Tumor size was measured by ex vivo weight. We used flow cytometry to measure the proportions of T cell subtypes, monocytes, macrophages and dendritic cells. Epithelial (Epcam+) cells were isolated from young and old tumors with flow cytometry, and then analyzed with RNA-Seq and ATAC-Seq.
Results: Tumor weight was significantly lower in old mice compared to tumors from young mice (N=6 in each group) (0.36 gr vs 0.46 grams, P=0.04). Compared to tumors from young mice (N=3), tumors from old mice (N=3) demonstrated a significantly higher percentage of dendritic cells ( 36.5 vs. 23.5, P=0.03), CD3+ T cells ( 27.6 vs. 15.4, P=0.005) (Figure 1), and IFN-gamma producing Th17 cells (22.1 vs. 14.9, P=0.02), and lower proportion of Tregs ( 25.4 vs. 45.6, P=0.02). CD4+, CD8+, and exhausted T cell population frequencies were not significantly different between old and young cohorts. Gene ontology (GO) analysis from RNA-Seq data (Figure 2) showed that differentially expressed genes in old vs. young colon tumor epithelial cells were significantly enriched in multiple immune functions, including inflammatory pathways, T-cell function, and antigen presentation (MHC class II). We found increased expression and chromatin accessibility of Ciita (a master regulator of MHC class II) , and increased expression of H2-Ab1 (which encodes the MHC class II protein complex), in cancer epithelial cells from old vs. young mice. Higher expression of MHC-II-related genes in tumor epithelial cells from old vs. young mice was confirmed by flow cytometry and in situ hybridization.

Conclusion: Results from this study demonstrate that aging promotes an anti-tumor immune microenvironment in an inducible, genetically engineered mouse model of colorectal cancer, possibly by enhancing cancer cell-mediated antigen presentation by MHC class II machinery.