Proteome Profiling of Cancer-Associated Fibroblasts Identifies Novel Proinflammatory Signatures and Prognostic Markers for Colorectal Cancer

Abstract

Purpose: Cancer-associated fibroblasts (CAF) are essential components of the stroma that play a critical role in cancer progression. This study aimed to identify novel CAFs markers that might contribute to the invasion and the prognosis of colorectal cancer.Experimental Design: The azoxymethane/dextran sodium sulfate mouse model of sporadic colon cancer represents an adequate source for the isolation of CAFs and normal fibroblasts. By using the explants technique, we purified CAFs and normal fibroblasts from colon tissues. Whole-cell extracts and supernatants were subjected to in-depth quantitative proteomic analysis by tandem mass spectrometry. Further validations of upregulated proteins in CAFs were carried out by chemokine microarray and immunohistochemical analyses of mouse and human tissues.Results: Using a fold-change of 1.4 or more, we found 132 and 125 differentially expressed proteins in whole-cell extracts and supernatants, respectively. We found CAFs-associated proinflammatory and desmoplastic signatures. The proinflammatory signature was composed of several cytokines. Among them, CCL2 and CCL8 caused an increase in migration and invasion of colorectal cancer KM12 cells. The desmoplastic signature was composed of 30 secreted proteins. In mouse and human samples, expression of LTBP2, CDH11, OLFML3, and, particularly, FSTL1 was significantly increased in the tumoral stroma, without significant expression in the cancer epithelial cells. The combination of CALU and CDH11 stromal expression showed a significant association with disease-free survival and poor prognosis.Conclusion: We have identified LTBP2, CDH11, OLFML3, and FSTL1 as selective biomarkers of cancer stroma, and CALU and CDH11 as candidate stromal biomarkers of prognostic significance in colon cancer.

LOXL2 Is Highly Expressed in Cancer-Associated Fibroblasts and Associates to Poor Colon Cancer Survival

“…Proteins contributing to desmoplastic lesions are characteristic of tumor type. We observed a limited coincidence with mouse fibroblasts coming from a chemically induced colon cancer (19), among other coincidences we found FN1, TPM2, TNC, COL1A2, IGFBP7, FSTL1, THBS1, COL4A2, BGN, and CSF1. We also observed CDH13, a mesenchymal cadherin similar to CDH11, previously reported in mouse cancer fibroblasts (19).…”

“…CAFs and NFs were obtained from colonic tissues by the explant technique as described previously (19,23,24). See Supplementary Methods for additional information.…”

“…In a proteomic study, colon cancer CAFs were compared with bone marrow precursors to identify tenascin C, ED-A fibronectin, and SDF-1 as deregulated in CAFs (18). Recently, we used a murine model of sporadic colon cancer based on azoxymethane/dextran sodium sulfate to optimize fibroblast purification and characterize alterations in murine CAFs (19).…”

Expression changes of cell-cell adhesion-related genes in colorectal tumors

“…Its function in tumorigenesis has been investigated in gliomas, where it acts as a marker of the mesenchymal phenotype (17), as well as in prostate (18) and breast cancer (19), where its expression is important in metastasis. Recently, the overexpression of CDH11 was identified as a marker of cancer-associated stromal cells, and this stromal expression was found to have prognostic value (20). Increased immunohistochemical expression was also observed in AC epithelial cells (20).…”

“…Recently, the overexpression of CDH11 was identified as a marker of cancer-associated stromal cells, and this stromal expression was found to have prognostic value (20). Increased immunohistochemical expression was also observed in AC epithelial cells (20). It has been hypothesized that, in certain tumors or tumor cell lines, CDH11 acts as a suppressor gene, and its expression is downregulated via a DNA methylation mechanism (21,22).…”