The Pro-tumorigenic Role Of Cellular Senescence In Melanoma:Tumor Invasion And Vascularization

Part 1 Hypoxia-induced senescence in melanomas[Purpose]Senescence associated β-galactosidase(SA-β-gal)staining was used to detect whether the C57 mouse melanoma cells could develop senescence and to investigate the specific site where cellular senescence occurs and to speculate the potential causes of senescence in melanomas.In addition,the senescence model was made by hypoxia in vitro which lays research foundation for further understanding the characteristics of tumor cell in special microenvironment.[Methods]C57BL/6J mice were inoculated subcutaneously with B16 cells and tumors were obtained for preparing paraffin-embedded or frozen sections.SA-β-gal staining of tumor frozen sections was used to detect cellular senescence.Expression of endomucin and hypoxia probe(pimonidazole)was determined through immunohistochemical staining for analysis of the distribution of blood vessels and tissue hypoxia in tumors.Hypoxia-induced senescence model in vitro was established and Western Blot was used to analyze the relationship of HIF-1α expression and cellular senescence.[Results]1)By detecting senescence-associated β-galactosidase activity,we found that senescent cells(SA-β-gal+)were mainly located around the necrotic(hypoxic)areas of melanoma from C57BL/6J mice.2)Hypoxia probe and vascular endothelial staining indicated that no SA-β-gal positive cells were detected in areas with sufficient blood supply and SA-β-gal positive cells were in a hypoxic condition.3)Cellular senescence induced by hypoxic culture(CoCl2 and GasPak)was not temporary,but permanent.4)Expression of HIF-la was confirmed to be back to the low level when senescent cells got rid of CoCl2.[Conclusions]1)Tumor cell developed senescence in hypoxic microenvironment;2)Hypoxic model in vitro induced irreversible senescence;3)Expression of HIF-1α decreased when senescent cells got rid of hypoxic culture.Part 2 The mechanism of microenvironment modulation by senescent cells[Purpose]Effects of senescent cells on the ability of cell invasion and plasticity were analyzed to confirm that senescent cells were able to regulate the tumor microenvironment due to the specific secretion phenotype.Clinical specimens of human melanoma of 70 patients with complete follow-up data were studied in this part to evaluate the relationship of necrosis/ulceration with clinical prognosis[Methods]CoCl2 was used to simulate hypoxia to induce cellular senescence;MMPs activity of senescent cells was analyzed by western blot and gelatin zymography;Transwell assay was used to determine the effects of senescent cells on invasive ability and three-dimensional culture to evaluate cellular plasticity.Expression of MMP-2 was assessed by immunohistochemical staining of mouse and human melanoma tissue.Resected specimens of 70 patients identified as melanoma by pathologists with complete follow-up data were obtained Cancer Institute and Hospital of Tianjin Medical University from January 1996 to October 2007 to analyze the medical records and clinical follow-up data;HE staining was used to identify the association of existence of necrosis/ulceration and clinical diagnosis with statistical method.[Results]1)Expression and activity of MMP-2 of senescent cells increased;ability of cell invasion and plasticity was promoted in conditioned medium from senescent cells.2)Necrosis/ulceration existed in 32 of 70 malignant melanoma clinical cases;Survival analysis results showed that survival time of cases with presence of necrosis/ulceration was shorter;Cox regression analysis demonstrated that when the necrosis and ulceration were regarded as an indicator,it can be used as independent prognostic factors.[Conclusions]1)Senescent cells may regulate the biologic behaviors of neighboring cells through secreting factors such as MMP-2.2)The presence of necrosis/ulceration in melanomas had an inverse correlation with patient survival and the adverse effects of necrosis/ulceration in tumor may be attributed to the adjacent senescent cells that regulate tumor microenvironment.Part 3 Role of senescent fibroblasts in melanoma development and progression[Purpose]Mixed inoculation of mouse embryonic fibroblasts(MEF)and B16 was used to investigate the role of MEF in tumor formation.The presence of senescent MEF was examined in vivo and after several times of passages in vitro.Influence of senescent MEF on the proliferation and associated maker expression of endothelial cell was performed.Expression of inflammatory cytokines IL-6 was detected to investigate its role in senescent MEF and prognosis of patients with melanomas.The study on interaction of tumor stroma and parenchyma provided a basis for further understanding of senescence and its impact on tumor progression.[Methods]1)Primary cultured mouse embryonic fibroblasts were identified by immunofluorescence for vimentin expression.Senescent MEF was detected aftermultiple passages by SA-β-gal staining.Cultured in conditioned medium of senescent MEF was established to detect the effects of MEF on vascular endothelial cell proliferation and protein expression.Enzyme-linked immunosorbent assay(ELASA)was used to detect secretion of IL-6 by senescent MEF.2)Detection of cellular senescence by SA-β-gal staining and IL-6 expression by immunohistochemical staining in tumors from co-inoculation of MEF and B16 mixed at a ratio of 1:2 in C57 mice subcutaneously.3)The relationship of IL-6 expression with clinical characteristics of human melanoma was analyzed.[Results]1)After multiple passages,the major of MEF developed senescence.The proliferation capacity and expression of VEGFR2 of endothelial cells were increased when cultured in conditioned medium of senescent MEF.Secretion IL-6 of senescent MEF was increased compared to early passage MEF.2)The organizational structure of melanomas derived from co-inoculation of MEF and B16 changed:the tumor interstitial component increased and surrounding the tumor parenchyma cells;necrotic areas decreased;number of microvessels increased;MEF developed senescence and secreted IL-6.3)The statistical analysis showed that expression of IL-6 was associated with old patients and microvessel density in human melanoma.[Conclusions]1)Fibroblasts promote tumor growth and develop senescence.2)Senescent fibroblasts promote tumor growth by affecting the vascularazation.3)Inflammatory cytokine IL-6 may be involved in cellular senescence and its impact on tumors.Conclusions1.Senescent melanoma cells mainly distributed in the hypoxic region.Hypoxic environment simulated by cobalt chloride in vitro effectively induce senescence of melanoma cell and those cells which can not tolerate hypoxia cell died.Accordingly,hypoxia can induce senescence in vivo and in vitro.2.Although in an irreversible growth arrest,hypoxia-induced senescent cells remain metabolically activity and high expression of matrix metalloproteinase MMP-2 was detected.It was concluded that senescent cells could affect the ability of tumor cell invasion and migration of endothelial cells through its special secretary phenotype.3.Necrosis/ulcers in human melanoma tissue was associated with poor prognosis,which may due to the occurrence of senescent melanoma cells in hypoxic/necrotic region by modeling the tumor microenvironment.4.Embryonic mouse fibroblasts(MEF)developed senescence and IL-6 secretion was increased,which may contribute to tumor growth by affecting angiogenesis.