The present study was undertaken to develop a new method for enriching cancer stem cells (CSCs) from the human adrenal cortical carcinoma (ACC) cell line SW-13. Given that the existence of CSCs in ACC causes resistance to conventional chemotherapies, treatment with cyclophosphamide was used for in vivo selection of CSCs in a BALB/c nude mouse tumor xenograft model established using the ACC cell line SW-13. The characteristics of CSCs in three generations of tumor xenografts were assessed for single-cell colony formation, flat colony formation, and cell sphere formation in serum-free suspension culture. The formation rates of single-cell colonies, flat colonies, and cell spheres were significantly higher for tumor xenograft cells treated with cyclophosphamide than for untreated engrafted tumor cells. Flow cytometry to examine expression of the CSC markers C-X-C chemokine receptor type-4 (CXCR4; CD184) and ATP-binding cassette sub-family G member-2 (ABCG2; CDw338) revealed markedly higher levels of CXCR4 and ABCG2 in cyclophosphamide-treated xenograft tumor cells compared to untreated tumor cells. Together, these results indicate that cyclophosphamide treatment of tumor xenograft cells caused enrichment of CSCs with a strong capability for self-renewal and proliferation. In this method, the administration of cyclophosphamide selectively kills cancer cells without toxicity to CSCs and thereby provides a practical approach for achieving the enrichment of CSCs in ACC.
Keywords: ABCG2; Adrenocortical carcinoma; CXCR4; cancer stem cell; chemotherapy; cyclophosphamide.