Immortalization of Human Carcinoma-Associated Fibroblasts from Primary Colorectal Carcinomas

Abstract

Introduction: Reciprocal interplay bewteen epithelial and stromal cells contributes to the initiation, progression and metastasis of colorectal cancer. The importance of carcinoma-associated fibroblasts (CAFs) in tumor develoment is highlighted by the observation that nontumorigenic prostate epithelial cells can form tumors in nude mice when co-implanted with CAF, but not when co-implanted with fibroblasts from benign prostatic hypertrophy. The precise role of colorectal CAFs in tumor-stroma interactions has been difficult to study, in part because of the difficulty of obtaining primary CAFs in sufficient quantity for experiments. Once isolated, the CAFs can be passaged for a limited number of times before they undergo senescence. Further characterization of human colorectal CAFs would provide the basis for stromal-specific therapeutic targeting in colorectal cancers. The purpose of this study was two-fold: 1) immortalize human CAFs derived from colorectal cancers in order to begin to investigate their influence on human colorectal cancer cells and 2) document gene-specific changes attributable to the immortalization process. Methods: Freshly dissected and dissociated cells were cultured from a resected colon cancer specimen under an IRB-approved protocol. The patient had not received prior chemoradiotherapy. The cells were cultured on collagen I and the purity of the fibroblast culture was verified by immunohistochemistry. Fibroblasts are positive for vimentin and/or α-smooth muscle actin, but negative for cytokeratin. CAFs were infected with lentiviral vectors containing a CMV promotor upstream of SV40 Large T antigen and human telomerase. Total RNA was extracted using RNAqueous (Ambion) from triplicate cultures of transformed (t)CAFs and parental (p)CAFs. The purity and concentration of the RNA samples were determined using Agilent 2100 Bioanalyzer. Samples were hybridized to Affymetrix Gene Chips (HGU133 Plus 2.0). Analyses using the Partek Genomics Suite included routine quality controls, raw data background subtraction and normalization. Differentially expressed candidate genes were identified using the Benjamani-Hochberg multiple hypothesis comparisons (ANOVA, p<0.01 and p<0.005) to decrease the false discovery rate. Ingenuity Pathways analyses were performed to identify differences between the t- and p-CAFs. Results: ANOVA identified 2779 and 648 genes that were significant at the p≤0.01 and 0.005 level, respectively. The major pathways representing the genes differentially expressed bewteen the t- and p-CAFs include: 1) the transcriptional regulator TP53 2) growth factor TGFB1, 3) mitogen-activated protein kinase pathways (p38 and p-extracellularly regulated kinase), and 4) the transcriptional regulator nuclear factor-κB. As expected, one canonical pathway that was altered by the transformation, were genes involved in cell cycle and G1/S checkpoint regulation. Conclusions: The immortalization and subsequent careful characterization of human colorectal CAFs provides an invaluable tool for further study of stromal-epithelial interactions in colorectal carcinogenesis and metastasis. Although genes related to the cell cycle has been significantly altered, future studies are required to verify that the t-CAFs alone are non-tumorigenic in nude mice, similar to the p-CAFs. Additionally, immortalized normal fibroblasts derived from the normal a colonic mucosa will need to be generated as an important control.