The Investigation Of Mechanism Of Targeting Cancer-associated Fibroblasts In Melanoma Therapy

BackgroundMelanoma is the most malignant skin cancer,and the incidence has been increasing in recent years.Moreover,the age of onset tends to be younger.For early-stage melanoma patients,the five-year survival rate is around 93%after timely surgery.However,the five-year survival rate is as low as 5%in advanced melanoma.The current treatment for advanced melanoma patients are mainly chemotherapy,radiotherapy and immunotherapy,etc.Although these approaches partially block the progression of cancer,the efficacy is still limited.For immunotherapy,the treatment responses vary a lot in different patients.Therefore,investigating novel strategies for melanoma therapy are of great importance.Several factors may affect the therapeutic efficiency.Besides tumor cells,the tumor microenvironment(TME)is an essential part affecting the treatment.Various components,including melanoma cells,fibroblasts,endothelial cells,immune cells,extracellular matrix,cytokines and chemokines constitute the TME of melanoma.Among them,cancer-associated fibroblasts(CAFs)are the most abundant cells.On the one hand,they participate in the remodelling of tumor stroma thus forming the physical barrier to prevent immune cells and drugs.On the other hand,CAFs could promote the growth of tumor cells,angiogenesis,and regulate other immune cells by secreting cytokines.Given the important role of CAFs in tumor development,targeting CAFs may be a novel strategy for the treatment of melanoma.Our previous studiy identified that tumor necrosis factor receptor-associated factor 6(TRAF6)was highly expressed in melanoma and promoted tumor progression.Another study suggested that TRAF6 might be involved in the activation of fibroblasts by secreting cytokines.However,the role of TRAF6 in the TME of melanoma remains unclear.Death receptor 5(DR5)is another protein that has been confirmed to be highly expressed in both melanoma cells and CAFs,indicating that DR5 could be a therapeutic target for melanoma TME.Anti-DR5 monoclonal antibodies have been developed for cancer therapy in recent years.However,the clinical efficacy of the antibodies was limited.Moreover,whether the anti-DR5 antibodies have therapeutic efficiency on CAFs is not yet clarified.Therefore,it is critical to explore new strategies to improve the effectiveness of anti-DR5 antibodies in cancer therapy.Based on these,we investigated the role of TRAF6 and DR5,which were highly expressed in both melanoma cells and CAFs,in melanoma TME,with the aim to explore novel strategies for melanoma therapy.Aims1.To study the role of TRAF6 in CAF and its impact on melanoma;2.To investigate the effect of TRAF6 inhibitor in melanoma treatment;3.To study the therapeutic effect of targeting DR5 in melanoma cells and tumor microenvironment;4.To explore new strategies for melanoma treatment using engineered exosomes.Method1.The expression of target proteins,including TRAF6,α-SMA,Vimentin,Desimin,PDPN,MMP-9,and DR5,were detected by western blot,immunohistochemistry or immunofluorescence;2.The knockdown or overexpression of target genes(TRAF6 or DR5)in cell lines were generated by lentiviral infection;3.The cell proliferation was detected through CCK-8 and xenograft mouse models;The malignant phenotype of tumor cells was tested through Transwell,Migration,and wound healing assays;4.The mechanisms of gene regulation were identified by gene luciferase reporter assay and chromatin immunoprecipitation;5.The killing efficiency was detected by LDH and luciferase killing assays;the apoptosis was tested by flow cytometry and confocal;6.The anti-tumor effects of TRAF6-and DR5-based therapy were detected by xenograft mouse model;7.The statistical analysis of the data was performed through Graphpad Prism.Results:Part One1.TRAF6 was highly expressed in cancer-associated fibroblasts in melanoma tissue;2.Overexpression of TRAF6 promoted the proliferation,migration and invasion of fibroblasts;increased the expression of a-SMA,Vimentin,Desmin,PDPN,MMP-9,which were markers of CAFs;3.The expression of TRAF6 in fibroblasts regulated the growth,migration and invasion of melanoma cells in vitro and in vivo;4.The expression of TRAF6 in melanoma regulated the expression and secretion of FGF19;FGF19 promotes the activation of fibroblasts to CAFs;5.Overexpression of TRAF6 up-regulated the nuclear NF-κB1 in melanoma cells;NF-κB1 regulated the expression of FGF19;6.The expression level of plasma FGF19 was elevated in melanoma patients compared to healthy donors;7.CAFs activated by FGF19 promoted the growth of melanoma cells in vivo;8.TRAF6 inhibitor suppressed the proliferation of melanoma cells and CAFs.Part Two1.DR5 was highly expressed in melanoma cells and CAF s;2.Anti-DR5-scFv-BBZ-NK92 and anti-DR5-scFv-PDGFR-NK92 cells were built;the expression of anti-DR5-scFv was elevated in PDGFR5-DR5 cells derived exosomes;3.Exosomes(sEVs)derived from anti-DR5-scFv NK92 cells killed DR5 highly expressed melanoma cells(DR5high)and induced apoptosis;the PDGFR-DR5-sEVs exhibited the highest cytotoxic efficiency;4.DR5-NK92-sEVs killed CAFs cytotoxic efficiency in melanoma tissue slices;the PDGFR-DR5-sEVs exhibited the highest cytotoxic efficiency;5.DR5-NK92-sEVs killed myeloid-derived suppressor cells(MDSCs)in melanoma tissue slices;the PDGFR-DR5-sEVs exhibited the highest cytotoxic efficiency;6.DR5-NK92-sEVs specifically target DR5high melanoma cells and inhibit tumor growth in vivo;7.γδ T cell-derived sEVs showed better cytotoxicity in melanoma cells than and NK92 cells;8.DR5-γδ T-sEVs significantly inhibited the growth of melanoma cells in vitro and in vivo.Conclusions1.TRAF6 played a dual role in the tumor microenvironment of melanoma.Besides regulating the activation of CAFs to affect the biological behaviour of melanoma cells,TRAF6 also promoted the secretion of FGF19 in melanoma cells through NF-κB1,thereby activating fibroblasts and promoting the progression of melanoma.TRAF6 inhibitors suppressed the proliferation of both melanoma cells and CAFs;2.The engineered sEVs derived from anti-DR5-scFv-NK92 cells could specifically kill DR5high melanoma cells,as well as CAFs and MDSCs in melanoma TME.The PDGFR transmembrane domain increased the expression of anti-DR5 scFv on exosome thus enhancing its anti-tumor efficiency.sEVs derived from γδ T cells expressed higher cytotoxic molecules.The killing efficiency of DR5-γδ T-sEVs was further enhanced,which provides a novel strategy for melanoma therapy.