Abstract 5110: Cancer-associated fibroblast phenotypes vary across colorectal cancers and correlate with CD8+ T-cell infiltration
Authors: Johnson Katherine A. , Emmerich Philip B. , Pasch Cheri A. , Clipson Linda , Matkowskyj Kristina A. , Deming Dustin A. 
Published: September 2020

Abstract
BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment (TME). This has been correlated with changes in cancer biology, including tumor progression and treatment response. CAFs exhibit divergent phenotypes differentiated by their expression profiles and function. Across cancer types, two CAF subsets have been consistently identified: myofibroblastic and non-myofibroblastic. Here we examine podoplanin (PDPN), α-smooth muscle actin (αSMA), and collagen as potential CAF subtype markers in the stromal compartment of colorectal cancers (CRCs). These markers were compared with versican (VCAN), an immunoregulatory proteoglycan associated with an immune excluded TME.
METHODS: A tissue microarray containing representative tumor and normal tissue from 122 CRC patients was stained using Masson's Trichrome (collagen) or immunohistochemistry for αSMA, PDPN, VCAN and versikine (Vkine, a proteolytic fragment of VCAN). Quantification of the stains (0 to 3+) was performed by at least two observers in consultation with a surgical pathologist. Smooth muscle from the muscularis propria was excluded from quantification in the αSMA stain. CD8 staining was quantified as the number of tumor infiltrating CD8+ lymphocytes (TILs) per high power field (HPF). Separate tissue cores were sequenced for 107 of these patients using the Qiagen Comprehensive Cancer Panel. Mutations were identified using Strelka; SNPs and indels were cross-referenced to ClinVar.
RESULTS: Stromal staining for αSMA, PDPN, and collagen varied across CRC sections. The abundance and intensity of staining was independent of disease stage. Those cancers with 3+ collagen or αSMA had reduced TILs (collagen 0-1+: 8.0 TILs/HPF vs 2-3+: 3.0, p=0.02; αSMA 0-1+: 7.4 vs 2-3+: 3.6, p=0.04). There was a similar trend observed for PDPN (0-1+: 6.5 vs 2-3+: 3.5, p=0.1). VCAN proteolysis predominant (VPP; VCAN 0-1+ and Vkine 2-3+) samples, previously reported to associate with higher CD8+ TILs, had lower collagen vs VCAN proteolytic weak (VPW; mean collagen score 1.6 vs 2.2, p=0.002) and lower αSMA (VPP: 1.7 vs VPW: 2.1, p=0.02). PDPN was not associated with proteolysis (p=0.4). In VPP tumors, stromal marker abundance did not correlate with CD8+ TILs; however, in VPW tumors, CD8+ TILs were higher in low PDPN (4.1 vs 1.5 TILs/HPF, p = 0.02) and low αSMA tumors (4.8 vs 1.6; p = 0.009). The trend was similar for low collagen tumors (3.7 vs 1.9, p = 0.1). Collagen was more abundant in TP53-mutant cancers (p=0.03, Chi-Square Test).
CONCLUSIONS: The stromal microenvironment of CRC is highly variable across patients with diverse CAF densities and phenotypes. The makeup of this microenvironment has potential clinical impacts, as it correlates with immune cell infiltration. Further investigation into CAF biology in the TME could reveal therapeutic targets to alter immune surveillance of cancers.
Citation Format: Katherine A. Johnson, Philip B. Emmerich, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Dustin A. Deming. Cancer-associated fibroblast phenotypes vary across colorectal cancers and correlate with CD8+ T-cell infiltration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5110.