Society: AGA

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.

<b>Figure 1. Aging is associated with expansion of the intratumoral T cell population.</b> Representative quantification of the percentage of CD45+, CD3+ T cells in colorectal tumors from old versus young mice (N=3); P<0.01.

Figure 1. Aging is associated with expansion of the intratumoral T cell population. Representative quantification of the percentage of CD45+, CD3+ T cells in colorectal tumors from old versus young mice (N=3); P<0.01.

<b>Figure 2. Aging is associated with upregulation of immune signaling and antigen processing pathways in colorectal tumor epithelial cells</b>. Gene Ontology (GO) pathways (Biological Process category) enriched for up-regulated genes in sorted epithelial cells from old mice compared to sorted epithelial cells from young mice. The top GO term items with the most significant enrichment (FDR < 0.05) were selected.

Figure 2. Aging is associated with upregulation of immune signaling and antigen processing pathways in colorectal tumor epithelial cells. Gene Ontology (GO) pathways (Biological Process category) enriched for up-regulated genes in sorted epithelial cells from old mice compared to sorted epithelial cells from young mice. The top GO term items with the most significant enrichment (FDR < 0.05) were selected.

Paul Wade

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