Abstract 3198: Validation and analysis of cancer associated fibroblast subtype markers in metastatic colorectal cancer
Authors: Lippert Anna L. , Johnson Katherine A. , Pasch Cheri A. , Kraus Sean G. , Emmerich Philip B. , Clipson Linda , Matkowskyj Kristina A. , Zhang Wei , Deming Dustin A. 
Published: June 2022

Abstract
Background: Cancer Associated Fibroblasts (CAFs) are a significant component of tumor stroma, and have an important impact on immune infiltration in the tumor microenvironment (TME). Two major subtypes of CAFs have been previously identified by literature: myofibroblastic (myCAF) and inflammatory (iCAF). Our lab has identified subtype markers for each CAF phenotype and previously analyzed a sampling of 153 colorectal cancer (CRC) patients. Here, we validate these subtype markers and investigate CAF phenotypes in metastatic colorectal cancer patients.
Methods: Dual immunofluorescence on formalin fixed paraffin embedded tissue sections was performed to analyze co-staining between combinations of myCAF markers, αSMA and TAGLN, and iCAF markers, PDPN and ICAM1. Slides were imaged using a fluorescent microscope. Also, tissue microarrays sampling 212 CRC patients spanning all stages of disease, 90 with matched metastatic cores, were stained via immunohistochemistry (IHC) for the CAF subtype markers then quantified on an intensity scale from 0-3+. iCAF and myCAF marker scores were averaged to get a composite score for each, then split into low (average score &lt;2) and high (average score ≥2) groups. CD8 IHC stains were quantified as the number of tumor infiltrating lymphocytes (TILs) per high power field (HPF) in the epithelial compartment.
Results: Significant co-staining was observed between iCAF markers PDPN and ICAM1, as well as myCAF markers αSMA and TAGLN. Co-staining did not occur, or was minimal, between combinations of myCAF and iCAF markers. There is not significant different in abundance of iCAFs or myCAFs in primary site cores of patients with metastatic versus non-metastatic disease (p = 0.67 for iCAF, p = 0.57 for myCAF). Of matched primary and metastatic samples able to be scored, 43.3% of samples had a decrease in iCAF score from primary to metastatic site while only 18.8% increased. Overall, 34.4% of samples had a decrease in score of more than 1 and only 2.2% of samples had an increase of more than 1. However, the percentage of samples that had a decrease in myCAF score was 32.2% while 22.2% increased. In all primary cores of patients with metastatic disease, there was higher average CD8+ TILs in those with high iCAF scores compared to those with low iCAF scores (12.0 vs 5.5, p=0.03). There was not a significant difference in average CD8+ TILs in those with high myCAF scores compared to those with low myCAF scores (9.3 vs 7.1, p=0.7).
Conclusions: Here, we validate the myCAF markers TAGLN and αSMA, as well as, iCAF markers ICAM1 and PDPN by demonstrating co-staining between CAFs of the same subtype and exclusion between different subtypes. These data indicate that that CAF phenotype correlates with CD8 T cell infiltration into the TME. iCAFs correlate with immune infiltration and myCAFs with immune exclusion.
Citation Format: Anna L. Lippert, Katherine A. Johnson, Cheri A. Pasch, Sean G. Kraus, Philip B. Emmerich, Linda Clipson, Kristina A. Matkowskyj, Wei Zhang, Dustin A. Deming. Validation and analysis of cancer associated fibroblast subtype markers in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3198.