Abstract 5493: Digital spatial profiling of MC38 colon carcinoma following checkpoint inhibition
Authors: Singh Priyanka , Saims Daniel , Barnes Sheri , Draper David 
Published: March 2024

Abstract
Immunotherapy aims to bolster the immune response within the tumor microenvironment (TME). However, checkpoint inhibitors utilized in the treatment of colon carcinoma have demonstrated efficacy only in certain patients. To develop more effective therapeutics, strategies that navigate the heterogeneous nature of the TME must be employed. The MC38 tumor model, known for its immunologically active characteristics and responsiveness to certain immunotherapies, is widely used for preclinical drug development. Furthermore, elevated levels of pan-cytokeratin (PanCK) expression in tumors have shown an association with diminished survival rates among colon carcinoma patients. In this study, we used the GeoMx® DSP platform (NanoString Technologies) to investigate the spatial expression profile of PanCK in MC38 tumors and analyze the impact that PanCK expression exerts on anti-PD-1 induced gene expression in tumor and immune cells. To this end, C57BL/6J female mice were implanted with MC38 cells, enrolled into groups, and treated with anti-PD-1 antibody. Tumor growth was monitored by caliper measurements and anti-PD-1 produced minimal responses with a Day 22 median % Delta T/C value of 88%. For DSP analysis, tumors were collected 10 days post-treatment, formalin fixed for paraffin embedding, and then sectioned for PanCK and CD45 protein visualization. Regions of interest were drawn to create a matrix analysis that defined regions with high or low PanCK expression, that were proximal or distal to CD45+ immune rich regions of the tumor. A segmentation algorithm was applied to examine gene expression within each region using the GeoMx Mouse Whole Transcriptome Atlas. Cluster analysis revealed an enrichment of apoptosis and autophagy gene sets in tumor cells that were proximal to immune rich regions. In the CD45+ segment, immune cells adjacent to high PanCK-expressing cells contained 318 genes with significantly increased expression levels when compared to low PanCK-expressing cells, and only 11 genes with decreased expression levels. The upregulated genes were enriched for numerous biomarkers of T cell recruitment and activation, including cd3, cd8, cxcr3, ccl5, cxcl9, icos, and cd27. Next, we conducted a linear mixed model analysis to examine the impact of PanCK expression on anti-PD-1 induced gene regulation. Notably, an enrichment of genes that code for T cell exhaustion biomarkers was observed in immune cells near PanCK high expressing cells, including ctla4, slamf7, tigit, and lag3. In summary, the most pronounced transcriptomic changes occurred within immune cells situated near tumor cells displaying high PanCK expression and suggest that PanCK expression correlates directly with T cell recruitment and activation but also exhaustion. Furthermore, these data emphasize the significance of spatial analysis as a valuable component of preclinical research, as demonstrated in MC38 tumor model.
Citation Format: Priyanka Singh, Daniel Saims, Sheri Barnes, David Draper. Digital spatial profiling of MC38 colon carcinoma following checkpoint inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5493.