| Objective Human adrenocortical carcinoma cell line SW-13 was used to construct subcutaneous transplanted model, peritoneal metastasis model and lymphatic metastasis model in nude mice, investigating the tumorigenicity, invasion and metastasis of SW-13. while establishing a standard culture environment of SW-13 in vitro and stem cell culture environment,which detected the expression of cancer stem cell surface markers CD44, CXCR4, CD 133 and ABCG2 in these five different microenvironments, and sorted cancer stem cells surface markers of adrenocortical carcinoma. This provided the basic theory for separating clinical adrenocortical carcinoma cancer stem cells, and provided new ideas for targeted therapy.Methods1. Established the model of SW-13 transplanted subcutaneously, peritoneal metastasis and lymph node metastasis in nude mice, it was to explore the cell line SW-13's ability of tumorigenesis and invasion. 2. Constructing different culture environment in vitro of cell line SW-13, the expression of CD44, CXCR4, CD133 and ABCG2 were detected by immunohistochemistry, immunofluorescence and flow cytometry method in five microenvironments of SW-13 cell in vivo and in vitro, which provided an initial basic research for selecting specific cancer stem cells surface markers in adrenocortical carcinoma.3. Collecting specimens of adrenocortical carcinoma, the expression of CXCR4 was observed in adrenocortical carcinoma samples to explore the possibility of CXCR4 as adrenocortical carcinoma cancer stem cell surface marker.Results1. Establishment cell line SW-13 transplantation and metastasis model: The tumor formation rate was 100% in nude mice three kinds models. It was found tumorigenicity after inoculating about 5 days in subcutaneous transplanted model; metastasis happened at the time point of the third week post-injection in peritoneal metastasis model, tumor cells mainly transferred to mesenteric lymph nodes, they could also be found in kidney, pancreas.The lymphatic metastasis happened at the 4th week in lymphatic metastasis model. By HE staining, it was easy to catch sight of pathological mitotic figure, and we also found that tumor cells violated blood vessel, infiltrated skeletal muscle and bone lamella, there were cancerous nodes in kidney and pancreas, only a little lymph structure remained when vast majority was damaged seriously. Ki-67 protein was high expression in tumor tissue by immunohistochemistry.2. The expression of CSC associated markers in different microenvironments:immunofluorescence staining showed CD44 were highly expressed in SW-13 cell, CXCR4 had weakly expression, CD 133 and ABCG2 were negative on SW-13 cells in standard culture medium, FCM showed the frequencies are 98.22±1.78%(CD44),40.97±2.15%(CXCR4), 5.06±1.32%(CD133), and 2.91±3.80%(ABCG2). Immunofluorescence staining showed CD44 were highly expressed in stem cell culture environment, CXCR4 had weakly expression, CD133 and ABCG2 were negative, FCM found the frequencies of CD44, CXCR4, CD133 and ABCG2 in stem cell culture environment were 44.34%±0.93%,1.45%±1.27%,11.10±1.56% and 1.24±1.36%. Cancer cells in subcutaneous transplanted tumor highly expressed CD44 and CXCR4, CD 133 was lowly expressed, ABCG2 was negative yet. FCM indicated the frequencies were 95.32±2.36%(CD44),45.82±9.53% (CXCR4),0.02±0.03%(CD133),2.44±2.10%(ABCG2). Immunohistochemistry staining showed CD44 and CXCR4 were highly expressed in the cancer cells in popliteal fossa lymph nodes within lymph node metastasis modle, yet CD 133 was lowly expressed and ABCG2 was negative expressed. FCM showed the frequencies of cancer cells were 88.54±7.02% (CD44),62.47±6.98%(CXCR4),0.87±1.26%(CD133),4.00±2.10% (ABCG2). Immunohistochemistry staining showed CD44 and CXCR4 were highly expressed in the cancer cells in peritoneal metastasis model, yet CD133 was lowly expressed and ABCG2 was negative expressed. FCM showed the frequencies of cancer cells were 90.68±2.36%(CD44),45.82±9.53%(CXCR4), 0.02±0.03%(CD133),2.44±2.10%(ABCG2)3. The expression of CXCR4 in clinical specimens:CXCR4 had strongly positive expression in adrenocortical carcinoma (14 in 15 cases), weak expression in adrenocortical adenoma (4 in 16 cases), CXCR4 did not express in adrenal hyperplasia (0 in 11 cases) and normal adrenal tissue (0 in 10 cases) There was significant statistically difference (P= 0.000). Conclusion1. It was successful to construct subcutaneous transplanted model, peritoneal metastasis model and foot pad of lymphatic metastasis model of human adrenocortical carcinoma cells (SW-13) in nude mice.2. In three constructing microenvironment in vivo, SW-13 cell line had obvious pathological mitotic figure, high invasive, and Ki67 protein was high expression in subcutaneous and metastatic tumors.There may be some cancer stem cells in cell line SW-13, which could be used for the research of expression regulation of markers in cancer stem cells.3. The expression of cancer stem cell related markers of SW-13 cells in vivo and vitro micro-environment were that CD44>CXCR4>CD133>ABCG2, CD44 had high sensitivity but poor specificity, but CXCR4 had better sensitivity and specificity, CD133 and ABCG2 had good sensitivity but low specificity, the results suggested that CXCR4 could be used as cancer stem cells marker for further screening.
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