Contribution of MMP14-expressing cancer-associated fibroblasts in the tumor immune microenvironment to progression of colorectal cancer

Abstract

Matrix metalloproteinase 14 (MMP14) expression is implicated in progression of colorectal cancer, but its role in the tumor microenvironment (TME) has been unclear. The relevance of MMP14 to colorectal cancer progression was explored by analysis of transcriptomic data for colorectal adenocarcinoma patients (n = 592) in The Cancer Genome Atlas. The role of MMP14 in the TME was investigated in a retrospective analysis of tumor samples from 86 individuals with stage III colorectal cancer by single cell-based spatial profiling of MMP14 expression as performed by 12-color multiplex immunohistochemistry (mIHC). Analysis of gene expression data revealed that high MMP14 expression was associated with tumor progression and implicated both cancer-associated fibroblasts (CAFs) and tumor-associated macrophages in such progression. Spatial profiling by mIHC revealed that a higher percentage of MMP14⁺ cells among intratumoral CAFs (MMP14⁺ CAF/CAF ratio) was associated with poorer relapse-free survival. Multivariable analysis including key clinical factors identified the MMP14⁺ CAF/CAF ratio as an independent poor prognostic factor. Moreover, the patient subset with both a high MMP14⁺ CAF/CAF ratio and a low tumor-infiltrating lymphocyte density showed the worst prognosis. Our results suggest that MMP14⁺ CAFs play an important role in progression of stage III colorectal cancer and may therefore be a promising therapeutic target.

Keywords: M2 tumor-associated macrophages (M2-TAMs); cancer-associated fibroblast (CAF); colorectal cancer; matrix metalloproteinase 14 (MMP14); multiplex immunohistochemistry (mIHC).

Figure 1

Analysis of the colorectal adenocarcinoma cohort (n = 592) of the TCGA database according to MMP14 expression. (A) Kaplan-Meier curves for disease-specific survival in patients divided according to the median value of MMP14 expression level. Vertical bars denote censoring. (B) The top 20 genes whose expression level was correlated with that of MMP14 ranked according to Spearman’s correlation coefficient. Genes related to stromal tissue or M2-TAMs are highlighted in green and yellow, respectively. (C) The top 20 genes whose expression was associated with that of MMP14 ranked according to log₂ of the fold change in normalized expression value for MMP14-high relative to MMP14-low tumors. FDR q values were also calculated. Genes are highlighted as in (B). (D) The top five hallmark gene sets whose expression was up-regulated in the MMP14-high group as revealed by GSEA. FDR q values were calculated with GSEA software. (E) GSEA plot of enrichment for the gene signature related to epithelial-mesenchymal transition for MMP14-high versus MMP14-low tumors. (F) The top three immune cell signatures associated with MMP14 expression by CIBERSORTx analysis. In the dot plots for the immune cell signatures (top), each dot represents one patient and the median value and interquartile range are indicated. The mean rank difference values and adjusted P values calculated by multiple Wilcoxon rank-sum tests with Bonferroni correction are also shown (bottom).

Figure 2

Multiplex immunohistochemistry (mIHC) and strategy for quantitative evaluation of cell populations. (A) Representative images of mIHC. Images for hematoxylin staining (upper) and multicolor images (lower) are shown. The area within the red box is shown at higher magnification in the other corresponding images. Seven markers are shown in the top two multicolor images, with CD8 (green), CD68 (white), CD163 (pink), and pan-cytokeratin (panCK, orange) being shown in the lower left image and CD8 (green), CD31 (red), αSMA (blue), and panCK (orange) in the lower right. (B) Definition of cell lineages according to marker expression in the present study. (C) Gating strategy for mIHC-based single-cell population analysis.

Figure 3

Prognostic impact of MMP14-expressing cells. (A) Percentage of MMP14-expressing cells among cell subsets as determined by mIHC analysis. Each dot represents one patient, and the median value and interquartile range are shown for each plot. (B) Kaplan-Meier curves for RFS according to the median values of the MMP14⁺ CAF/CAF ratio (left) or the MMP14⁺ tumor cell/tumor cell ratio (right).

Figure 4

Association of M2-TAM distribution in tumors with MMP14 expression in CAFs and survival outcome. (A) Representative mIHC images showing the relation between MMP14-expressing CAFs and the spatial distribution of M2-TAMs. The regions within the yellow squares are shown at higher magnification in the corresponding images to the right. The upper and lower sets of images correspond to patients with a high or low MMP14⁺ CAF/CAF ratio, respectively. (B) Kaplan-Meier curves for RFS according to the median values of M2-TAM density in ISA (left) or TN (right) regions.

Figure 5

Kaplan-Meier curves for RFS based on the combination of the IS-Immunoscore and MMP14⁺ CAF/CAF ratio. The median value was used as the cutoff for the ratio.