Abstract 99: Versican regulates T cell abundance within the colorectal cancer microenvironment

Abstract

Abstract Background: Clinical benefit from immunotherapy is seen in a minority of patients with metastatic colorectal cancer (CRC). Identifying how the tumor microenvironment (TME) influences immune infiltration and treatment response is an unmet need. Versican (VCAN), a chondroitin sulfate proteoglycan, common in the CRC TME has immunoregulatory properties and can be cleaved releasing an immunostimulatory product, versikine (Vkine). Here we evaluate the impact of the abundance of VCAN and Vkine on CD8+ T cell infiltration in CRC. Methods: Samples include 306 primary colon tumors, an additional 176 primary CRCs with matched liver metastases (175 samples), and 328 normal samples (238 colon and 90 liver). Samples were stained via immunohistochemistry (IHC) for VCAN, Vkine, and CD8. CD8+ tumor infiltrating lymphocytes (TILs) were counted per high powered field (HPF, 400X magnification) and presented as averages. Intensity of VCAN and Vkine expression were scored from 0-3+. Samples designated a 0/1 were considered low accumulation and samples scored as a 2/3+ were designated high. Samples scored low for VCAN but high for Vkine were designated VCAN proteolytic predominant (VPP) and all other combinations considered VCAN proteolytic weak (VPW). To create an in vitro model, the mouse MC38 cell line was CRISPR engineered to over-express VCAN. Conditioned media was then concentrated and added to a collagen matrix in a transwell with activated T cells. The number of cells and their subtype that migrated through the matrix were evaluated with flow cytometry. Results: VCAN accumulation was highest in cancerous stroma with 58% of cancers being VCAN high and only 14% of adjacent normal tissues (p<0.001). Vkine accumulation was similar between groups, with 67% of tumors and 54% of adjacent normal samples scored as Vkine high. The VPP phenotype was least common in tumor tissues with only 27% of tumor samples designated VPP versus 43% of adjacent normal samples (p<0.001). VCAN high tumors had significantly fewer TILs with 4 TILs/HPF vs 13 in the VCAN low tumors (p<0.001). High levels of Vkine correlated with greater TIL abundance (Vkine high: 10 TILs/HPF, Vkine low: 5 TILs/HPF; p=0.02). Versican proteolysis had the strongest correlation with TIL abundance in cancers (VPP: 18 TILs/HPF, VPW: 5 TILS/HPF; p<0.001). In vitro, the addition of VCAN containing media into transwell matrices reduced both CD4+ and CD8+ T cell migration through the matrix (Control: 4277 CD8+ T cells, VCAN: 2603 CD8+ T cells; p<0.001 and Control: 7770 CD4+ T cells, VCAN: 5340 CD4+ T cells; p=0.007). Conclusions: VCAN accumulation in human CRCs is common and correlates with reduced TIL abundance, whereas VCAN proteolysis correlates with increased TILs. In vitro, VCAN demonstrated the ability to reduce T cell migration. Future studies will explore the mechanism by which VCAN abundance leads to decreased T cell infiltration and the importance of this as an immunotherapy biomarker. Citation Format: Sean Kraus, Philip Emmerich, Mitch Hayes, Kristina Matkowskyj, Wei Zhang, Cheri Pasch, Fotis Asimakopoulos, Dustin A. Deming. Versican regulates T cell abundance within the colorectal cancer microenvironment [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 99.