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Carcinoma-associated fibroblasts (CAF) promote tumor progression of pre-neoplastic epithelial cells. To investigate the basis of this phenomenon, we compared the properties of fibroblasts cultured from normal human prostate (NHPF) to prostate CAF. NHPF and CAF were assayed for growth potential, cell death and proliferative capacity by measuring population doubling time, cell cycle distribution and capability to form colonies in soft agar. Resistance to genotoxic (UV radiation: 0-50 J/cm2) and chemotoxic (0-200 nM Taxol) agents were compared between CAF and NHPF by measuring cell viability and cell cycle analysis. Transforming growth factor beta1 (TGF-beta1) immunoreactivity was assessed in non-malignant and malignant prostatic tissue. No detectable differences were found when comparing CAF and NHPF with respect to population doubling time, cell cycle distribution and response to genotoxic and chemotoxic agents. The mean number of colonies in soft agar was 120.5 for CAF vs. 18.2 for NHPF (p < 0.05). Because TGF-beta1 and matrix metalloproteinase (MMP)-9 have been associated with growth of fibroblasts in soft agar and tumor promotion, we measured the expression of these factors in NHPF and CAF by ELISA. There was no difference in expression of MMP-9; however, TGF-beta1 was expressed in higher concentrations in CAF than in NHPF (p < 0.0014). Furthermore, TGF-beta1 expression was higher in the carcinoma-associated stroma of prostate cancer tissue than stroma of non-malignant prostatic tissue. Increased capability of CAF as compared to NHPF to form colonies in soft agar may be due to a higher expression of TGF-beta1 and correlates with the ability of CAF to promote malignant progression of prostate epithelial cells., (Copyright 2004 Wiley-Liss, Inc.)

The present study demonstrates that fibroblasts associated with carcinomas stimulate tumor progression of initiated nontumorigenic epithelial cells both in an in vivo tissue recombination system and in an in vitro coculture system. Human prostatic carcinoma-associated fibroblasts grown with initiated human prostatic epithelial cells dramatically stimulated growth and altered histology of the epithelial population. This effect was not detected when normal prostatic fibroblasts were grown with the initiated epithelial cells under the same experimental conditions. In contrast, carcinoma-associated fibroblasts did not affect growth of normal human prostatic epithelial cells under identical conditions. From these data, we conclude that in this human prostate cancer model, carcinoma-associated fibroblasts stimulate progression of tumorigenesis. Thus, carcinoma-associated fibroblasts can direct tumor progression of an initiated prostate epithelial cell.

The microenvironment influences the progression of an epithelial malignancy. To examine the effect of fibroblasts on epithelial cells by direct cell-cell contact in vitro, a coculture system was designed to assess cell death and proliferation of two cell populations when grown together. We used a green fluorescent dye to stain fibroblasts and distinguish them from unstained epithelial cells by a flow cytometer. We show that tumor cell death is 5-fold less when cocultured with normal human prostatic fibroblasts than when cultured alone. In contrast, proliferation of tumor cells was similar when cocultured with normal human prostatic fibroblasts or when grown alone. The reduction in tumor cell death during coculture appears to play a significant role in promoting tumor formation. Combination of prostatic fibroblasts with LNCaP xenografts formed large tumors at a high frequency with a low apoptotic index in vivo, whereas, LNCaP xenografts alone formed small infrequent tumors with a high apoptotic index. Therefore, prostatic fibroblasts promote tumor formation by retarding the apoptotic pathways in tumor cells.

The present study demonstrates that fibroblasts associated with carcinomas stimulate tumor progression of initiated nontumorigenic epithelial cells both in an in vivo tissue recombination system and in an in vitro coculture system. Human prostatic carcinoma-associated fibroblasts grown with initiated human prostatic epithelial cells dramatically stimulated growth and altered histology of the epithelial population. This effect was not detected when normal prostatic fibroblasts were grown with the initiated epithelial cells under the same experimental conditions. In contrast, carcinoma-associated fibroblasts did not affect growth of normal human prostatic epithelial cells under identical conditions. From these data, we conclude that in this human prostate cancer model, carcinoma-associated fibroblasts stimulate progression of tumorigenesis. Thus, carcinoma-associated fibroblasts can direct tumor progression of an initiated prostate epithelial cell.