Medthods Of Enrichment Of Adrenocortical Carcinoma Cancer Stem Cells

Background The theory of tumor stem cells have proposed that the composition of tumor cells are heterogeneous, tumors account for small proportion of cells with the ability of self-renewal, differentiation potential and high-tumorigenic, these cells called tumor stem cells or cancer stem cells. Studies have shown that cancer stem cells can be enriched bycultured in serum-free suspension medium, defected by chemotherapy drugs, transplanted into NOD/SCID mice. Then separated and purified them by cancer stem cell markers to get high purity cancer stem cells for research their biological characteristics and surface markers.Objective 1, Detected the reproduction of SW-13 cells in vitro.2, Explore whether cancer stem-like cells of adrenal cortical carcinoma can be enriched through cultured in serum-free suspension medium in vitro, defected by chemotherapy drugs adriamycin, transplanted into NOD/SCID mice.3, Research the expression of cancer stem cell genes CD44, CD 133, CXCR4 and ABCG2 in adrenal cortical carcinoma cancer stem-like cells.Method 1, Tested the cell growth curve of adrenal cortical carcinoma cell line SW-13 cells in vitro and colony formation of SW-13 cells in plates and in soft agar.2, SW-13 cells cultured in serum-free suspension medium, interfere in F12/DMEM 1:1 culture with 12ug/ml adriamycin, were injected into nude mice to enrich adrenal cortical carcinoma cancer stem-like cells.3, collected those cells and detected the expression of cancer stem cell markers CD44, CD133, CXCR4 and ABCG2 on them.Results 1, the reproduction of SW-13 cells was strong in vitro, had high colony formation rate in flat and soft agar.2, SW-13 cells can form spherical cell mass in serum-free suspension medium, resistance to adriamycin and form tumors in nude mice. These three methods can enrich adrenal cortical carcinoma cancer stem-like cells.3, Adrenal cortical carcinoma cancer stem-like cells with high expression of CD44, low and stable expression of ABCG2, the expression of CD 133 and CXCR4 were difference in three environments. SW-13 cells in stem cell medium in vitro, FCM showed the frequencies were 97.07± 2.58%(CD44),3.99±3.01%(CD133),23.01±12.06%(CXCR4), and 1.12±1.65%(ABCG2). SW-13 cells in standard culture with adriamycin in vitro, FCM indicated the frequencies were 57.64±7.48% (CD44),7.16+2.83% (CD133),24.58±10.56%(CXCR4),0.31±0.75% (ABCG2). SW-13 cells in subcutaneous transplanted tumors in nude mice, FCM found the frequencies were 94.46%±2.31% (CD44),1.36%±1.88%(CD133),77.10%±13.92% (CXCR4) and 0.53%±0.52% (ABCG2).Conclusion Adrenal cortical carcinoma cell line SW-13 cells appeared in some characteristics of cancer stem-like cells. Adrenal cortical carcinoma cancer stem-like cells can be enriched by serum-free suspension medium, adriamycin intervention and transplanted into nude mice. By detecting and analyzing the expression of cancer stem cell markers in SW-13 cells in these three micro-environments created by enrichment strategies, we found that CD44 may be malignant markers of adrenal cortical carcinoma, CXCR4 may play an important role in tumor-forming and metastasis of adrenal cortical carcinoma, CD133 and ABCG2 may serve as candidate markers for sorting adrenal cortical carcinoma cancer stem cells.