Molecular characterization of cancer associated fibroblasts in colorectal cancer

Abstract

Background: Recent studies have shown that cacner stromal cells organize the tumor microenvironment and affect the cancer progression. In particular, cancer associated fibroblasts (CAFs), a major component of the tumor stroma, play a key part in tumor progression by communication with cancer cells in many solid tumors to promote tumor growth, invasion and metastasis. However, much of mechanisms of cancer progression by CAFs still remains poorly understood. Method(s): CAFs and NFs (normal fibroblasts) were isolated from freshly resected specimens from patients with colorectal cancer (CRC) for primary culture. CAFs were established from the tumor tissue, and NFs were from non-tumor tissue at least 5 cm away from the margin of tumor. RNA sequence was performed using RNA extracted from these fibroblasts to obtain gene expression profiles. We analyzed the profiles and compared gene expression differences between CAFs and NFs with the gene ontology (GO) analysis, pathway analysis and proteinprotein interaction (PPI) network analysis. Moreover, proliferation assay and migration assay were performed in vitro for evaluating function of the isolated genes. Result(s): Gene expression profiles were obtained from 5 pairs of CAFs and NFs. In the analysis of gene expression profiles, 283 upregulated genes and 301 downregulated genes in CAFs were identified (p < 0.05, Fold change>2 or < 0.5). GO and pathway analysis revealed that the gene sets related to Wnt signal pathway were highly expressed in CAFs, besides TGFbeta signal pathway considered as trigger of differentiation to CAFs from normal fibroblasts. Expression levels of Wnt2 and Wnt5A were especially increased in CAFs compared to other Wnt family genes. In addition, VCAM1 was remarkably upregulated in CAFs and PPI network analysis showed that VCAM1 had many interactions with other genes. Therefore, VCAM1 may be involved in some significant signal transduction. Experiments in vitro using medium including recombinant Wnt2 protein or condition medium of CAFs treated with siRNA for Wnt2 demonstrated that CRC cell proliferation and migration was enhanced by Wnt2. Conclusion(s): Wnt2 protein secreted from CAFs promotes the CRC progression. Wnt2, Wnt5A and VCAM1 in colorectal CAFs may play important roles in regulating CRC progression.