Genomics and proteomics approaches to the study of cancer-stroma interactions

Abstract

Background: The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.

Methods: The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.

Results: We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR.

Conclusions: A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.

Figure 1

Immunofluorescence analysis of cytokeratin and vimentin in stromal fibroblasts and Hep-2 cell line. (A and D) Absence of immunoreactivity in sections incubated with control nonimmune mouse serum. Stromal fibroblasts (B and E) and Hep-2 cell line (C and F) were positive for vimentin and cytokeratin, respectively. (G): Densitometric analysis of immunofluorescence reaction to vimentin and cytokeratin in stromal fibroblasts and Hep-2 cell line. Scale bar, 20 μm.

Figure 2

Growth curve of Hep-2 cell line. Hep-2 cells were cultured in complete medium, treated with self-conditioned medium (HCM) or with conditioned medium from fibroblast cultures (FCM) and collected 1, 3, 5 and 7 days after medium replacement. Data are means ± s.d. of two independent experiments in duplicates. *P < 0.05. Error bars indicate S.D.

Figure 3

Immunohistochemistry reaction with AnxA5 antibody showed the presence of gold particles on the cytoplasm of apoptotic cells. Hep-2 cells (A) without treatment and (B) treated with conditioned medium from fibroblast culture (FCM) show AnxA5 immunoreactivity. Apoptotic cells immunolabeling for AnxA5 can be seen in Hep-2 cells treated with FCM (arrows). Staining with haematoxylin. Scale bar, 20 μm.

Figure 4

Real-time PCR gene expression in a conditioned medium-treated neoplastic cell line and in primary tumors. (A) Expression of ARID4A, CALR, DAP3, GNB2L1, PRDX1, RNF10, SQSTM1 and USP9X genes in Hep-2 cells treated with conditioned medium from fibroblast cultures. (B). ARID4A gene expression in 47 laryngeal and oral tongue carcinomas. Relative quantitation of target gene expression for each sample was calculated according to Pfaffl [50]; GAPDH was used as the internal reference and control sample as the calibrator. Values were Log2 transformed (y-axis) so that all values below -1 indicate down-regulation in gene expression while values above 1 represent up-regulation in tumor samples compared to normal samples.

Figure 5

Enlarged 2-DE gels of proteins from conditioned medium-treated Hep-2 cells and stromal fibroblasts. Five proteins (arrows), tubulin beta (A-B), alpha enolase (C-D), aldolase A (E-F), glyceraldehyde-3-phosphate dehydrogenase (G-H) and heterogeneous nuclear ribonucleoprotein C (I-J) were down-regulated in Hep-2 cell line treated with fibroblast conditioned medium (A, C, E, G and I) and two proteins (K-L), vimentin (arrow on left) and actin (arrow on right), were underexpressed in fibroblasts treated with Hep-2 cell line conditioned medium (K).