Abstract 1281: Endothelial cells promote the cancer stem cell phenotype of human colorectal cancer cells

Abstract

Abstract Background: Cancer stem cells (CSC), which possess the properties of self-renewal, multi-potential differentiation and tumor initiation, can arise either from normal stem cells or due to the influence of the tumor micro-environment. This study was done to determine the potential role of endothelial cell (EC) derived paracrine factors on promoting the CSC phenotype in human colorectal cancer (CRC) cells. Methods: RFP-labeled human CRC cells HCT116 were co-cultured with RF24 ECs (immortalized HUVECs) under low serum conditions. After FAC-sorting for the CRC cells, the CSC population was analyzed by 1) the Aldefluor assay, 2) sphere forming ability, and 3) Western blot analysis for the CSC markers CD133 and CD44. In addition, low serum condition media obtained from either ECs or parental CRC cells (control), was added to CRC cells to determine its effect on the CSC phenotype. In order to characterize the apoptotic effect of EC derived factors on CRC cells, we performed Annexin V staining by FACS, PARP cleavage, Caspase 3 cleavage, and Bcl2 levels by Western blotting. Using MTT assay, we determined the effect of EC derived factors on chemo-sensitivity of CRC cells exposed to 5-FU. Results: Co-culturing of CRC cells with ECs markedly increased the ALDH-positive population from 5% to 18%, and the sphere forming ability by more than 4-fold in CRC cells. In addition, CRC cells also displayed increased CD133 (6-fold) and CD44 (4-fold) protein levels. Furthermore, this effect could be mimicked simply by co-culturing CRC cells with conditioned media obtained from ECs. Similarly, treatment of CRC cells with conditioned medium from ECs significantly increased the ALDH-positive population from 4% to 8%, sphere forming ability >3-fold, and the expression of CD133 (7-fold) and CD44 (6-fold). Conditioned medium from ECs, concomitantly decreased spontaneous apoptosis in CRC cells as demonstrated by a 3-fold decrease in Annexin V-positive population (21% to 7%), down-regulation of cleaved PARP and Caspase 3, and up-regulation of Bcl2. CRC cells exposed to EC conditioned medium also displayed decreased sensitivity to 5-FU [>10-fold increase of the IC50 from 2.0 μg/ml to 22.3 μg/ml (p<0.05)]. Conclusions: Exposure of CRCs to ECs increased CSC properties, and decreased spontaneous apoptosis. Since, this effect can also be mimicked by the conditioned media, additional experiments to identify and validate the soluble factors that mediate this effect are in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1281.