Cancer-Associated Fibroblast Proteins as Potential Targets against Colorectal Cancers

Abstract

In colorectal cancer (CRC), attempts to identify cancer cell-specific markers to guide antibody-mediated therapeutics have failed to uncover markers that are both exclusive to cancer tissues and abundant across CRCs. Alternatively, cancer-associated fibroblasts (CAFs), which are abundant in the tumor microenvironment and upregulate unique surface markers, are not found in healthy tissues. Here, we evaluated the expression patterns of CAF-associated proteins α-smooth muscle actin (αSMA), fibroblast activation protein (FAP), podoplanin (PDPN), matrix metalloproteinase-2 (MMP2), transgelin (TAGLN), and THY1. While αSMA and THY1 were abundant in cancer tissues, high abundance in normal tissues limited their targeting potential. FAP was present in 94.5% of primary and metastatic CRC tissues and absent in 93.7% of adjacent normal colon and liver tissues assessed. These results indicate that FAP is a promising target for antibody conjugates with potential for broad application in CRC. Co-expression analyses showed that CRCs simultaneously expressing high levels of PDPN, MMP2, and THY1 were enriched for immune-related signatures, indicating potential for antibody-mediated immune engagers. Overall, this work highlights the potential of CAF proteins to act as therapeutic targets for novel anticancer agents and become important therapeutic biomarkers.

Keywords: cancer-associated fibroblasts; colorectal cancer; fibroblast activation protein; immunohistochemistry; liver metastasis; tumor markers.

Figure 1

CAF marker expression in CRC patient tissues. Tissue microarrays with patient CRC tissues were stained with CAF markers and scored 0–3+. (A) Representative staining and scoring for αSMA, FAP, and THY1 in CRC tissues across tissue microarrays. Scale bar = 50 µm. (B) Frequency of marker-absent (score 0), marker-low (score 1), and marker-high (score 2–3) CRC tissue samples for each CAF marker (αSMA, collagen, FAP, MMP2, PDPN, TAGLN, and THY1) (C) Correlations between marker expression scores across all CRC tissues were evaluated by Pearson’s Correlation tests performed on all pairs of CAF markers. Strength of correlation was binned by correlation coefficient, where a correlation coefficient (r) ≥ 0.6 indicated a strong positive correlation, 0.3 ≤ r < 0.6 indicated a weak positive correlation, −0.3 ≤ r < 0.3 indicated no correlation, −0.6 ≤ r < −0.3 indicated a weak negative (inverse) correlation, and r < −0.6 indicated a strong negative (inverse) correlation. Asterisks denote statistical significance (p < 0.05).

Figure 2

FAP expression specifically labels CRC tissue. (A) Frequency of marker-absent (score 0), marker-low (score 1), and marker-high (score 2–3+) in CRC patient adjacent normal tissue samples for each CAF marker (αSMA, collagen, FAP, MMP2, PDPN, TAGLN, and THY1). (B) Correlations between marker expression scores in adjacent normal tissues across all CRC patients were evaluated by Pearson’s Correlation tests performed between all pairs of CAF markers. Strength of correlation was binned by correlation coefficient as in Figure 1C. Asterisks denote statistical significance (p < 0.05) (C) Ratio of the number of patients with marker-high cancer tissues to the number of patients with marker-high adjacent normal. For each marker, only patients with scorable cancer and normal tissues were considered (αSMA: n = 118, collagen: n = 155, FAP: n = 123, MMP2: n = 132, PDPN: n = 121, TAGLN: n = 109, THY1: n = 123) (D) Percentage of mCRC patients with negative staining in adjacent normal colon and liver tissue that had positive-staining colon cancer (pink) or metastases to liver (red). (E) Percentage of mCRC patients with negative staining in adjacent normal colon and liver normal tissue that had high staining in colon cancer (light blue) or liver metastases (dark blue).

Figure 3

FAP expression across clinical subtypes of CRC. (A) Proportion of patients with EAO (light blue) or LAO (dark blue) disease and scorable FAP-absent, FAP-low, and FAP-high primary cancer tissues. (B) Proportion of male (pink) or female (red) patients with scorable FAP-absent, FAP-low, and FAP-high primary cancer tissues. (C) Proportion of patients with scorable FAP-absent, FAP-low, and FAP-high primary cancer tissues across CRC disease stage. (D) Proportion of patients with FAP-absent, -low, and -high liver metastasis tissue given absent, low, or high FAP expression in their primary cancer tissue. (E) Primary cancer tissue FAP expression (absent, low, and high) across patients with left-sided, right-sided, and rectal primary tumors (F) FAP expression (absent, low, and high) across all CRC samples with high CD8⁺ T cell infiltration (CD8⁺ TILs > 5/HPF) compared to those with low CD8⁺ T cell infiltration (CD8⁺ TILs < 5/HPF).

Figure 4

FAP expression across molecular CRC subtypes. (A) FAP expression in primary cancers tissues across APC, BRAF, KRAS, PIK3CA, and TP53 mutant and wild-type CRCs. (B) Percentage of dMMR and pMMR cancers with FAP-absent, -low, and -high primary cancers.

Figure 5

CD8⁺ T cell infiltration varies with collagen abundance in the CRC TME. (A) Representative CD8⁺ T cell staining in FAP-low and FAP-high expressing primary CRC (top row). Representative CD8⁺ T cell staining in collagen-low and collagen-high expressing primary CRC (bottom row). (B) CD8⁺ TILs/HPF across CAF marker expression in all CRC tissues. ** p < 0.01 according to Wilcoxon rank sum test.

Figure 6

CAF markers are often co-expressed and associated with upregulation of inflammatory gene sets. (A) Groups of COADREAD cohort CRCs with intersecting and individual CAF marker expression. (B) GSEA comparing differential gene expression between intersecting or individual groups containing at least 5 CRCs vs. all other CRCs within the COADREAD dataset. Heatmap indicates –log10(FDR q-value) for each hallmark gene set by each group, where only statistically significant FDR q-values (q ≤ 0.05) are reported. Those groups with an FDR q-value > 0.05 for any given gene set were assigned a –log10(FDR q-value) of 0.