THE CD133-POSITIVE COLON CANCER CELL PHENOTYPE IS MORE INTERACTIVE WITH THE TUMOR MICROENVIRONMENT COMPARED TO CD133-NEGATIVE CELL

摘要

Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133− cells. We hypothesized that CD133+ cells, compared to CD133−, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma –associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and – cells using FACS. The CD133+ cells formed large tumors in NOD-SCID mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133− cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change, and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs. − cells. RT PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs − cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin β8 and fibroblast growth factor receptor 2 (FGFR2). The CAF highly express the respective ligands: SDF-1, vitronectin, and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in [Ca²⁺]I in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension, compared to only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew both more colonies compared to vehicle as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared to CD133−, cells is due to their increased ability to interact with their neighboring CAF.