Background: Incidences of early age onset colorectal cancer (CRC) (those diagnosed before age 50) have been increasing by 2% every year since the 1990s, with the rate expecting to double by 2030. Although there has been some research done on differences in mutational profiles between patients diagnosed before and after age 50, little research has been done to understand the immune and stromal environments of early age onset (EAO) CRC. As identified by previous literature, there are two distinct phenotypes of cancer associated fibroblasts (CAFs): myofibroblastic (myCAFs) and non-myofibroblastic (non-myCAFs). Here, we evaluate both CAF environments as well as immune infiltrating cells in context of EAO CRC. Methods: A total of 153 CRC patient samples were obtained with matching adjacent normal tissue. Of these, 60 patients had EAO CRC. Tissue slides were stained via immunohistochemistry (IHC) for the CAF subtype markers αSMA, FAP, PDPN, and MMP2, by Masson's Trichrome for collagen, and then quantified on an intensity scale from 0-3. MyCAF and non-myCAF scores were calculated by averaging the scores of αSMA and collagen, or FAP, PDPN, and MMP2, respectively. Once these scores were determined, they were split into low (average score <2) and high (average score ≥2) groups. CD4 and CD8 IHC stains were quantified as the number of tumor infiltrating lymphocytes (TILs) per high power field (HPF) in the epithelial compartment. Results: Cancers with a low myCAF and non-myCAF score display the highest average number of CD4+ (10.6) and CD8+ (10.3) TILs across both age groups. Furthermore, cancers with both myCAF and non-myCAF high scores had reduced average CD4+ and CD8+ TILs when compared to both CAF scores being low (p value: 0.018 for CD8+ TILs, <0.001 for CD4+ TILs). Also, cancers that show myCAF high and non-myCAF low scores show the overall lowest average CD8+ TILs. Comparing the age groups directly, there are significantly higher CD4+ TILs in the 50+ age group in all CAF phenotypes except non-myCAF high (p-values 0.01 myCAF low, 0.05 myCAF high, 0.007 non-myCAF low, 0.36 non-myCAF high). However, for CD8+ TILs, the EAO CRC group trends towards higher CD8+ TILs in the myCAF high (3.7 vs 2.1) and non-myCAF high cancers (5.7 vs 3.3), but lower CD8+ TILs in myCAF low (6.8 vs 10.5) and non-myCAF low cancers (4.0 vs 8.4) when compared to the 50+ age group. Conclusions: Here we demonstrate that there are differences in the stromal and immune microenvironments between both age groups of CRC. We indicate that increased myCAF and non-myCAF scores are associated with T cell exclusion. However, the extent of T cell exclusion is different in EAO CRC compared to patients diagnosed after age 50. The reasoning for the difference in T-cell exclusion remains unknown and this implicates the importance for further research into the stromal and immune microenvironments of EAO CRC. Citation Format: Anna Lippert, Katherine A. Johnson, Philip B. Emmerich, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyi, Wei Zhang, Dustin A. Deming. Impact of cancer associated fibroblast phenotypes on the infiltration of t-lymphocytes in early age onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3166.

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 <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.

Background: Cancer-associated fibroblasts (CAFs) are major regulators of the immune microenvironment and therapeutic response in colorectal cancer (CRC). Neutralizing their role in modulating the immune landscape could be the key to enhancing immunotherapy success. Imatinib, dasatinib, and nilotinib are tyrosine kinase inhibitors with several kinase targets. Methods: Tissue microarrays spanning 153 patients were stained for αSMA, TAGLN, PDPN, ICAM1, and CD8. CD8 stains were quantified as number of tumor infiltrating lymphocytes per high powered field (TILs/HPF) in the epithelial compartment. All other stains were quantified by intensity on a 0-3+ scale. Scores for αSMA and TAGLN were combined into a myCAF score, and PDPN and ICAM1 into an iCAF score. myCAF gene expression signatures derived from a re-analysis of scRNA-seq data previously done by our lab were entered into the LINCS database to discover potential drugs to reverse the phenotype. Primary cancer associated fibroblasts were derived from patient tumor samples, then treated with clinically relevant concentrations of imatinib, dasatinib, or nilotinib for 96 hours. RNA was isolated and RT-qPCR was performed to quantify the myCAF genes ACTA2, COL11A1, and TAGLN, and the iCAF genes ICAM1, PDPN, IL1R1, CXCL1 and CXCL2. TGFB1 expression was also measured. Expression was normalized to untreated cells and GAPDH expression levels. Results: Cancers with high expression of myCAF markers but low expression of iCAF markers had the most CD8+ TILs (average 10.2; median 1.5; range 0-73), while cancers with low myCAF scores and high iCAF scores had the least (average 1.5; median 0; range 0-19; p < 0.01). Reversing the myCAF signature relative to iCAFs in the LINCs database revealed nilotinib as a top hit. Treatment with imatinib did not significantly alter the expression of myCAF genes (control vs. max dose: p = 0.06 for ACTA2, COL11A1 p =0.2, TAGLN p = 1), while treatment with dasatinib significantly increased these genes (ACTA2 1.4x higher, p < 0.001; COL11A1 2.6x higher, p < 0.01; TAGLN 1.5x higher, p < 0.001). Only treatment with nilotinib significantly decreased myCAF genes (ACTA2 2.2x lower, p <0.001; COL11A1 1.3x lower, p =0.05; TAGLN 1.9x lower, p < 0.01). All three drugs decreased iCAF gene CXCL1, and all but dasatinib decreased CXCL2. All three drugs significantly decreased TGFB1, a potential functional marker for altering myCAF phenotype (dasatinib 1.1x lower, p = 0.1; imatinib 1.6x lower, p < 0.001; nilotinib 1.5x lower, p < 0.05). Conclusions: myCAFs may be major actors in immune exclusion in the microenvironment, and the reversal of the myCAF phenotype may be a target for treatment with immunotherapy. Nilotinib, but not imatinib or dasatinib, is effective at decreasing expression of myCAF genes. Further research is warranted into the mechanisms of this drug on altering expression and whether these trends continue in vivo. Citation Format: Katherine Anne Johnson, Anna L. Lippert, Sean G. Kraus, Grace E. McGrath, Philip B. Emmerich, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Wei Zhang, Dustin A. Deming. Effects of tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib on cancer-associated fibroblast phenotypes in 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 3533.

Background: The incidence of colorectal cancer (CRC) in those of pre-screening age continues to rise. A deeper understanding of the differences in tumor biology in early age onset (EAO) cancers is needed. Differences in the tumor microenvironment have been understudied in this setting. We previously have identified versican (VCAN), a large matrix proteoglycan, as an important factor in lymphocyte exclusion in CRC. Here we explore the impact of the mutation profile and VCAN status on the infiltration of CD8+ and CD4+ lymphocytes in EAO CRC. Methods: Cancer tissues from 153 patients with CRC were stained via immunohistochemistry for CD4, CD8, and VCAN. CD4 and CD8 stains were quantified by number of positive-staining tumor-infiltrating lymphocytes per high powered field (TILs/HPF). VCAN stains were quantified on an intensity scale from 0-3+. 121 of these patients had tissue that was sequenced using the Qiagen Comprehensive Cancer targeted sequencing panel, and variations were called using Strelka. Patients were split by age into early-onset (EAO; age at diagnosis <50) and later-onset (LAO; age at diagnosis 50 or later). Results: Mutations in BRAF and APC were significantly more common in LAO cancers (p<0.05 for both). TP53 mutations were correlated with significantly lower CD4+ and CD8+ TILs/HPF in the LAO cohort (12.6 CD4+ TILs/HPF for TP53-wild type [WT] vs 3.5 for TP53-mutant, p<0.001; 10.0 CD8+ TILs/HPF for WT vs 3.2 for mutant, p<0.01), but trended opposite, though not statistically significant, in the EAO group (2.4 vs 5.9 CD4+ TILs/HPF; 3.0 vs 5.9 CD8+ TILs/HPF). PIK3CA mutations were correlated with an increase in CD8+ TILs in the LAO cohort (5.3 vs 11.9 CD8+ TILs/HPF; p<0.05) but a decrease in CD4+ TILs in the EAO cohort (4.9 vs 0.8 CD4+ TILs/HPF; p<0.05). TP53-WT tumors are disproportionately low in VCAN in the LAO cohort (p<0.01). Differences in VCAN accumulation could account for the changes in CD8+ and CD4+ T lymphocyte infiltration across the age cohorts and TP53 mutation status. Conclusions: The impacts of TP53 and PIK3CA mutations on immune infiltration in CRC differ in EAO compared to later onset cancers, indicating that the processes controlling immune infiltration vary between age cohorts. Such differences in immune infiltration may be linked to changes in microenvironmental factors such as VCAN. These data warrant further investigation into the relationship between tumor microenvironment and mutation profiles in EAO CRC. Citation Format: Katherine A. Johnson, Philip B. Emmerich, Anna L. Lippert, Cheri A. Pasch, Linda Clipson, Wei Zhang, Kristina A. Matkowskyj, Dustin A. Deming. Impact of the mutation profile and versican status on lymphocyte infiltration in early age onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2734.

Background: Fibroblast Activation Protein (FAP) is a protein expressed by cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) of epithelial cancers. There is potential interest in the use of FAP as a way to selectively target cancers, including colorectal cancer (CRC). Here we examine the expression of FAP in human colorectal cancers across stages and compare that expression to expression in normal colon and liver tissue. Additionally, we examine the correlation of FAP expression with key CRC clinical and biologic features to aid in potentially determining which CRC patients would be the most likely to benefit from FAP-directed therapies. Methods: CRC and adjacent normal tissue samples spanning all stages of disease were collected across 243 patients. Immunohistochemistry (IHC) was used to evaluate FAP, MMP2, αSMA, and PDPN expression and quantified on an intensity scale from 0-3+. These scores were split into low (average score <2) and high (average score >/=2) groups. IHC was used to determine mismatch repair status by staining for MLH1, MSH2, MSH6, and PMS2. Separate samples from these patients were sequenced with the Qiagen Comprehensive Cancer Panel and analyzed for APC, KRAS, BRAF, PIK3CA, and TP53 mutations. Results: Of 149 normal colon tissue sections, 99% had low FAP expression and 94% of those had no FAP expression. Of the 87 normal liver tissue sections, 99% had low FAP expression. In CRC sections, 58% had high FAP expression. Those patients >50 years of age had cancers with a higher rate of FAP expression (p=0.02). There was no correlation with FAP expression and sex, stage of disease, or sidedness. In cancers with BRAF V600 mutations, 91% have high FAP expression (x2 = 0.02). There is a trend for higher FAP expression in cancers possessing APC, KRAS, PIK3CA, or TP53 mutations (x2=NS). In correlating FAP with other CAF markers, MMP2 had the greatest correlation (Spearman’s Rho = 0.56). FAP is moderately associated with αSMA and PDPN (Spearman’s Rho = 0.33, 0.38). Conclusion: FAP is an exciting therapeutic target for CRC due to its cancer selectivity. Here, we demonstrate that FAP marks a rather independent subset of CRCs, though there is particular interest in the BRAF V600 mutant cancers given the high expression and poor prognosis of this subtype. Further investigation is needed to further develop FAP-directed therapies. Citation Format: Anna L. Lippert, Katherine A. Johnson, Cheri A. Pasch, Wei Zhang, Kristina A. Matkowskyj, Aaron M. LeBeau, Dustin A. Deming. Fibroblast activation protein (FAP) expression as a target for colorectal cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2357.