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Isolation, culture and identification of colon cancer stem-like cells from LoVo cell line
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Abstract:
Abstract Objective:To isolate, culture and identify the colon cancer stem-like cells with CD44+/EPCAM high phenotype from LoVo cell line, and to observe their biological behaviors and verify the existence of tumor stem cells in LoVo cell line. Methods: CD44+/EPCAM high cells were sorted from LoVo cells cultured in common-serum medium by flow cytometry, and the resultants were further cultured in serum-free medium (SFM) supplemented with growth factors. The proliferation and differentiation of CD44+/EPCAM high cells were observed. The proliferations and cell cycle distributions of CD44+/EPCAM high, EPCAM low, and un-sorted LoVo cells were estimated by MTT and flow cytometry, respectively. Nude mice were implanted with the above 3 different cells, and tumor formation rates of different groups were analyzed. Expressions of CD44 and EPCAM in the second passage of CD44+/EPCAM high cells were determined by immunofluorescence staining. Results: We found that 17.4% of LoVo cells were CD44+/EPCAM high cells, which could steadily proliferate and assemble into tumor cell spheres in SFM supplemented with growth factors. CD44+/EPCAM high cells could differentiate into adherent cells by serum, similar to LoVo cells. The proliferation capacity of CD44+/EPCAM high cells was higher than those of EPCAM low and LoVo cells, and the cell cycle of CD44+/EPCAM high cells was mostly in G0/G1 phase. Tumor formation rate of 500 CD44+/EPCAM high cells was 90% (9/10) in nude mice, with 1×104 EPCAM low cells being 0% (0/10). Moreover, expressions of CD44 and EPCAM in the second passage of CD44+/EPCAM high cells could still be detected. Conclusion: Colon cancer cell line LoVo contains CD44+/EPCAM high stem-like cells, and CD44+/EPCAM high cells can be used in further research of colon cancer stem cells.
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Project Supported:
Project supported by the Natural Science Foundation of Chongqing (No. 2008BB5230)
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