Study Of Cancer Stem-like Cells And Their Metastatic Subpopulation From The Small Cell Lung Cancer Cell Line H446

Objective:1、To investigate whether the uPAR+cells are stem-like cells in the cell line H446of SCLC.2、To demonstrate if the uPAR+cells could be further divided into the uPAR+CXCR4+cells and uPAR+CXCR4+cells and the latter are migrating cancer stem cells.Methods:1. uPAR+cells and uPAR+cells were sorted from SCLC H446by flow cytometry and the following experiments are conducted to demonstrate stem cell characters of uPAR+cells:①xenograft tumor implantation in nude mice,②sphere forming assays in serum-free medium,③differentiation capacity of uPAR+cells,④invasive assays by Transwell assays.2. The expression of CXCR4we examined in uPAR+cells by flow cytometry. Labeling uPAR-FITC and CXCR4-PE to confirm that uPAR+CXCR4+subcolone cells exist in uPAR+cells and then uPAR+CXCR4+and uPAR+CXCR4+cells were sorted. Migrating capacity of these two group cells was compared by Transwell assays in vitro. By injecting the cells mentioned above into Balb/c mouse to build a mouse model for evaluation of tumor metastasis, it was demonstrated that uPAR+CXCR4+cells are metastatic cancer stem cells.3. Observing the expression and distribution of uPAR and CXCR4in SCLC tissues by immunohistochemistry and analyzing its relationship with clinical parameters.Results:1. uPAR+cells possessed stem cell properties(1) uPAR+cells possessed high capacity of tumorigenicity. uPAR+cells and uPAR-cells were sorted by FACS,103cells were injected into Balb/c mouse and transplanted tumor can be detected after injecting uPAR+cells after one week but the uPAR+cells not. Three weeks later, mice were sacrificed and tumors (3mm diameter) derived from primary xenografts were transplanted into secondary mice and the serial transplantatons were done likewise. During the in vivo passaging. uPAR+tumor cells maintained the stability of the tumorigenesis, and the expression of uPAR was not changed. uPAR+tumors grew rapidly after the third generation tumors.(2) uPAR+cells were capable of self-renewal.103sorted uPAR+and uPAR-cells were cultured in serum-free conditions. After3days of culture, in uPAR+group, we observed ball-like sphere which contain ten tumor cells per sphere. After7days these spheres grew bigger, ranging from dozens to hundreds cells per sphere. The uPAR+group didn’t form spheres and cells died after7days.(3) uPAR+cells had differentiation potential. uPARTand uPAR+cells were isolated by FACS and cultured in the presence of10%FCS medium. After3days of culture, these cells lost uPAR expression and acquired the CD56and CK expression. Further. uPAR+and uPAR+cells were seeded on top of the matrigel solution in the presence of2%FCS and50ng/ml VEGF. The uPAR+cells showed a substantial formation of capillary structures when cultivated for one week, morever, immunofluorescence staining with endothelial antibodies showed that these cells expressed CD31. In contrast, the uPAR cells showed few capillaries and didn’t express CD31. The expression of factor Ⅷ was detected in xenografts of nude mice.(4) uPAR+cells possessed high capacity of invasion. Much more uPAR cells invaded through the matrigel than uPAR-cells. Immunofluorescence staining showed that E-cadherin expression was downregulated in uPAR+cells but upregulation of fibronectin, and vimentin was observed. These findings suggestd that uPAR plays a functional role in the invasive capacity of uPAR+cells and EMT may attribute to the invasive phenotype and metastasis capacity of the uPAR-cells.2. Isolation and identification of MCSC subclone of uPAR+CXCR4+cells(1) By labeling with uPAR-FITC and CXCR4-PE antibodies and flow cytometry analysis of H446cells, the percentages of uPAR+CXCR4+and uPAR+CXCR4+in H446were1.3%and7.1%. uPAR+cells were composed of uPAR+CXCR4+and uPAR+CXCR4+cells.(2) Results of Transwell assays We compared the migration capacity of uPAR+CXCR4+and uPAR+CXCR4+cells by Transwell assay. In the migration model system in vitro without SDF-la, the percentages of cells migrated into the bottom chamber had no significant differences between these two groups. When SDF-1α was added in the lower chamber. significantly greater numbers of uPAR+CXCR4+cells migrated into the bottom chamber. To provide further evidence that inhibition of CXCR4+could abrogate tumor migration, we performed the experiment using CXCR4antagonist AMD3100. Indeed, it was found that treatment of concomitant AMD3100significantly reduced tumor migration ability. uPAR+CXCR4+cells demonstrated higher migratory capacity than uPAR+CXCR4-cells, which closely related to the function of CXCR4.(3) Results of migration capacity in vivo A thousand uPAR+CXCR4+and uPAR+CXCR4-cells were injected into Balb/c mouse, after one week, both of these two cells formed primary tumor but with no significant differences in tumor growth rate. Eight weeks later, mice were sacrificed and lung metastasis was observed in the group injected with uPAR+CXCR4+cell.3. Expression of uPAR and CXCR4in SCLC tissuesExpression of uPAR was detected in17of50cases of SCLC by immunohistochemistry. uPAR-cells were generally infrequent and were distributed in a scattered pattern but tended to exist in foci near the invasive front of the carcinoma. Strong expression of CXCR4was seen in all50cases. Mean tumor volume of uPAR positive group was markedly bigger than that of uPAR negative group (P<0.05). and the survival time of uPAR positive tumor group was shorter (P<0.05). Expression of CXCR4in SCLC has no relationship with tumor size, sexy, age and lymph metastasis.Conclusion:1. In the H446of SCLC cell line, uPAR+cells processed high tumorigenic and self-renewal capacity.2. uPAR+cells were capable of differentiation. uPAR+cells can differentiate into vascular endothelial cells.3. uPAR+cells showed high capacity of invasion. EMT is proposed to attribute to the invasive capacity of the uPAR+cells.4. uPAR+cells could be divided into uPAR+CXCR4+and uPAR+CXCR4+cells.5. uPAR+CXCR4+cells showed higher migration capacity than uPAR+CXCR4+cells in Transwell assay.6. The occurrence of lung metastasis was observed in the group injected with uPAR+CXCR4+cells, indicating that the subpopulation of uPAR+CXCR4+cells were associated with development of metastasis.7. The expression of uPAR had close relationship with tumor sizes and patient prognosis.