ABSTRACT
Increasingly more evidence shows that TSCs possess the characteristics of stem-like cells. However, a link between stem cells and TSCs remains to be shown. We have sorted SP cells and non-SP cells from the B16F10 cell lines by FACS, and then studied their cellular biological characteristics by using a SFCM culture method, proliferative assay in vitro, clone formation assays in soft agar and normal media, tumorigenic assays in C57BL/6 mice, and resistance to chemotherapy assay in vitro, the quantitative detecting expression of ABCG2 and their CD phenotype analysis by a FCM. We detected 0.96% SP cells in the B16F10 cells and found that they had obvious differences in characteristics from non-SP cells. They possessed a marked capacity for self-renewal in soft agar and culture medium, strong tumorigenic potential in C57BL/6 mice, apparent resistance to vinblastin in vitro, upregulated ABCG2 expression, and a high expression of CD44+CD133+CD24+ phenotypes. We conclude that there were a few of SP cells that had the characteristics of tumor stem-like cells which may provide a useful tool and a readily accessible source for further study when specific TSCs markers are unknown.
Subject(s)
Melanoma, Experimental/metabolism , Neoplastic Stem Cells/pathology , AC133 Antigen , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/metabolism , Animals , Antigens, CD/metabolism , CD24 Antigen/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Separation , Flow Cytometry , Glycoproteins/metabolism , Hyaluronan Receptors/metabolism , Mice , Mice, Inbred C57BL , Neoplastic Stem Cells/metabolism , Peptides/metabolism , Vinblastine/pharmacologyABSTRACT
The ovarian cancer cells (SKOV3) secreting IL-21 alone or combination with GM-CSF cytokines was developed and its antitumor effect was evaluated in the nude mice. The gene of IL-21 was amplified from plasmid pRSC-IL-21 by PCR, cloned into the plasmid pRSC-GM-CSF, and the plasmid pRSC-GM-CSF-IL21 was constructed. The plasmids of pRSC-GM-CSF, pRSC-IL21, pRSC-GM-CSF-IL21 and pRSC were respectively transfected into the SKOV3 cells and antitumor efficacy induced by the SKOV3 cells secreting IL-21 or combination with GM-CSF was evaluated by surveying the tumor growth and the nude mice's survival. The results indicated that the secreted IL-21 and GM-CSF were functional because the culture supernatant of SKOV3 cells transfected with the plasmid pRSC-GM-CSF-IL21 enhanced NK cytotoxicity in vitro. The expressions of MIC A/B, NKG2D and ICAM-1 molecules on the SKOV3 cells were up-regulated. The level of IFN-gamma and TNF-alpha, the NK cytotoxicity and the antitumor efficacy were significantly increased in the null mice inoculated with the SKOV3 cells secreting both IL-21 and GM-CSF in comparison with the nude mice inoculated with the other different SKOV3 cells. We concluded that the SKOV3 cells genetically engineered to secrete biologically active IL-21 and GM-CSF elicited antitumor immunity effectively through enhancing NK cytotoxicity, promoting the expressions of MIC A/B , ICAM-1 and NKG2D molecules as well as elevating level of IFN-gamma and TNF-alpha in the nude mice model.
Subject(s)
Cytotoxicity, Immunologic , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interleukins/metabolism , Killer Cells, Natural/metabolism , Ovarian Neoplasms/immunology , Recombinant Fusion Proteins/metabolism , Animals , Female , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/immunology , Intercellular Adhesion Molecule-1/metabolism , Interferon-gamma/metabolism , Interleukins/genetics , Interleukins/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/immunology , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Neoplasm Transplantation , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Ovarian Neoplasms/therapy , Protein Engineering , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
In current study, cancer stem-like cells in the murine melanoma B16F10 cells were investigated. CD phenotypes of the B16F10 cells were analyzed by flow cytometry, and the specific CD phenotype cells from the B16F10 cells were isolated by MACS. Then we used colony formation assay in soft agar media, the cell growth assay in serum-free culture media as well as the tumorigenicity investigation of the specific CD phenotype cells in C57BL/6 mice, respectively, to identify cancer stem-like cells in the B16F10 cells. The results showed that the B16F10 cells could form spherical clones in serum-free culture media, and the rate of clonegenesis of CD133+, CD44+ and CD44+CD133+ cells was higher than that of CD133-, CD44- and CD44+CD133- cells in soft agar media, respectively. The tumorigenic potential of CD133+, CD44+, CD44+CD133+ cells and CD44+CD133+CD24+ cells was stronger than that of CD133-, CD44-, CD44+CD133- cells and CD44+CD133+CD24- cells in mice, respectively. In conclusion, the CD44+CD133+CD24+ cells have some biological properties of cancer stem-like cells or are highly similar to the characteristics of cancer stem cells (CSC). These results provide an important method for identifying cancer stem-like cells in B16F10 cells and for further cancer target therapy.
