Interestingly, Sox2 transcription factor is the pre dominant downstream target of EGFR signaling in these cells and plays a major role in self renewal growth and expansion of SP cells, independent of Oct4 and Nanog. Results SP cells are enriched with tumorigenic cells and the produce highly invasive tumors In an attempt to identify NSCLC stem like cells, SP ana lysis was conducted on four primary human NSCLC explants grown in athymic nude mice. SP cells appeared as a well separated population as described previously. As shown in Figure 1A, a specific inhibitor of ABCG2, Fumitremorgin C could block the ap pearance of SP phenotype. All the four tumor samples dis played the presence of SP cells with varying frequency ranging from 0. 6 3%, and could be significantly blocked by FTC.

Self renewing normal or cancer stem like cells can be expanded as Inhibitors,Modulators,Libraries non adherent spheres when cultured at low density in serum free, stem cell selective medium. differ entiated cells do not grow or form spheres under these conditions. The self renewal property of SP cells was examined by performing sphere formation assay on sorted SP and MP cells isolated from human tumor xenografts. While sorted SP cells were able to grow as spheres, MP cells had markedly Inhibitors,Modulators,Libraries less capacity to grow under identical conditions. Attempts were then made to assess the presence of SP cells in human NSCLC cell lines. As shown in later sections, A549, H1650 and H1975, contained SP cells with varying fre quency. Appearance of SP cells was completely blocked by FTC.

Sorted SP cells were able to grow Inhibitors,Modulators,Libraries as spheres whereas MP cells showed markedly reduced capability Inhibitors,Modulators,Libraries suggesting that NSCLC SP cells are enriched with CSCs. The stem cell like property of NSCLC SP cells was verified by evaluating its ability to form tumors in the lung microenvironment. Sorted SP and MP cells from A549 cells stably expressing the luciferase gene were orthotopically implanted into the left lung of SCID mice and tumor growth was monitored for 12 weeks. As shown in Figure 1E, SP cells generated primary tumors in the lung more efficiently than MP cells. At the end of the experiment, lungs, liver, kidney and Inhibitors,Modulators,Libraries brain were excised from each mouse and ex vivo images were examined for the presence of metastasized luciferase positive cells. Mice injected with SP cells demonstrated substantial tumor burden throughout the lungs and showed luminescent metastatic loci in liver, kidney and brain.

In contrast, MP cells formed only one luminescent focus in the lung of one mouse injected with 50 000 MP cells and there was no metastasis. These results were confirmed by H E staining. further, Sorafenib Tosylate tumors formed within the lung from SP cells, recapitulated the histo pathology of adenocarcinoma as confirmed by positive staining with pan keratin antibody as well as mucicar mine dye.