Abstract. Cancer-associated fibroblasts have been proposed to play a role in promoting carcinogenesis and tumor progression. To our knowledge, no direct evidence concerning fibroblasts in the genesis of cholangiocarcinoma (CCA) has previously been presented. This study aims to assess the value of activated fibroblasts with high alpha-smooth muscle actin (·-SMA) expression as an indicator for survival in CCA patients. The immunohistochemistry results indicated a high expression of ·-SMA in CCA fibroblasts which had a statistically significant correlation with larger tumor size (P=0.009) and shorter survival time (P=0.013). The effect of CCA-associated fibroblasts (Cfs) on non-tumorigenic biliary epithelial cells (H-69) and CCA cell lines was investigated in vitro and compared to the effect of non-tumorigenic liver fibroblasts (Lfs). The increased proliferation effect of Cfs having high ·-SMA on H-69 and 4 CCA cell lines compared to Lfs that expressed low ·-SMA was observed. Cell cycle analysis indicated that Cf-derived conditioned-medium and direct Cf-epithelial cell contaction could drive epithelial cells into S+G2/M phases. These results indicate that fibroblasts in CCA stroma express high ·-SMA and can be a prognostic indicator for poor patient survival. CCA fibroblasts have proliferative effects which may directly effect tumor promotion and progression of biliary epithelial cells. This warrants further investigation of fibroblasts as alternative therapeutic targets in CCA patients. IntroductionCarcinomas develop from cells of epithelial origin that have undergone genetic mutations and consequently result in the dysregulation of normal growth-controlling mechanisms. Research on the genesis of several carcinomas has been focused mainly on the study of tumor cells and considered the tumorigenesis as an independent process governed by the genes carried within the cancer cells themselves. However, changes in tumor stromal cells surrounding the epithelial malignancy have been observed (1). Tumor stroma is composed mainly of fibroblasts with a minority of inflammatory, smooth muscle and endothelial cells. Changes in these stromal cells have been postulated to enhance several tumorigenic phenotypes of the epithelial cells. It is well accepted that epithelial and stromal cells exchange a reciprocal molecular dialogue that ensures organ homeostasis for proper development and function (2,3). Malignant transformation of epithelial cells disrupts such homeostasis causing changes in tissue architecture, adhesion, cell death and proliferation.Cholangiocarcinoma (CCA), a carcinoma of bile duct epithelium, is a serious health problem in South Asian countries including Thailand, Lao People's Democratic Republic, Vietnam, Cambodia and South China (4). Moreover, the incidence of CCA has been reported to be increasing in America and Europe (5,6). In Thailand the endemic area of CCA is in the Northeastern part of the country which strongly relates to the high incidence of a liver fluke, Opisthorchis viverrini infection (7). While ...