Characterization of tumor microenvironment phenotypes as potential biomarkers for immune infiltration

Background:   Improved biomarkers are needed to identify immune permissive tumor microenvironment (TME) phenotypes that correlate with tumor infiltrating CD8+ T cells (TILs) and immunotherapy response. Myofibroblastic and inflammatory cancer-associated fibroblasts (myCAFs and iCAFs) are CAF subtypes involved in remodeling extracellular matrix and immunomodulation. Versican (VCAN), a matrix proteoglycan, and its proteolysis product, versikine (Vkine), regulate immune infiltration in the CRC TME. Here, we evaluate VCAN proteolysis and CAF phenotypes together as potential biomarkers for immune infiltration.  Methods:    Immunohistochemistry was performed to evaluate the below CAF markers, as well as VCAN and Vkine, in 636 primary and metastatic patient tumor samples. CD8+ TILs were quantified per high-powered field (HPF). All other stains were scored on a scale of 0-3 based on stromal abundance and intensity. MyCAF (alphaSMA and TAGLN) and iCAF marker (PDPN and ICAM1) scores were averaged and categorized as previous. VCAN status was categorized as previous. Results:  Of the VCAN proteolytic weak (VPW) cancers, 25.9% were myCAF high, 9.6% were iCAF high, 31.9% were neither high, and 32.7% were both high. Of the VCAN proteolytic predominant (VPP) cancers, 31.9% were myCAF high, 16% were iCAF high, and 30.3% were both high. Of the VCAN/Vkine low (VVL) cancers, 18.5% were myCAF high, 14.1% were iCAF high, and 14.1% were both high. In VPW cancers, the mean CD8+ TILs/HPF were relatively low across all CAF phenotypes with the neither high phenotype having the greatest TILs (5.7/HPF). In VVL cancers, the mean CD8+ TILs/HPF in the both high (3.2) and myCAF high (4.5) phenotypes were less than that observed with the iCAF high (7.7) and neither high (12.5) phenotpyes. For VPP cancers, the mean CD8+ TILs/HPF in myCAF high cancers (10) is significantly lower (p=0.045) than in all other phenotypes (neither high 24.2, iCAF high 16.5, both high 23.4). Conclusion: The absence of CAFs or the presence of high iCAF correlates with enhanced T-cell infiltration in VVL and VPW metastatic cancers. For VPP cancers, the presence of a high myCAF phenotype does not suppress T-cell infiltration, though it is reduced relative to cancers with an absence of CAFs or presence of iCAFs. The combination of CAF phenotype and versican status should be further assessed as a biomarker for immune therapy response.