Molecular Mechanism Of Melanoma Small Extracellular Vesicles Regulating Proangiogenic Phenotype Of Cancer-associated Fibroblasts & Case Report

Malignant melanoma is a malignant tumor characterized by strong invasiveness,metastasis and resistance.The high level of vascularization contributes to the progression of melanoma and is also one of the important factors for the poor prognosis of melanoma treated.So far,the efficacy of anti-angiogenesis medicine for melanoma is undesirable,which attributed to the mechanisms of angiogenesis and resistance.Exploring the mechanisms of melanoma angiogenesis is beneficial to provide better treatment strategies,which is of great significance for treating melanoma and improving prognosis.Cancer-associated fibroblasts(CAFs)are essential components of the tumor microenvironment(TME).Its switch of proangiogenic phenotype plays a great role in promoting tumorigenesis,invasion and metastasis.Studying how tumor affects the switch of proangiogenic phenotype of CAFs is helpful to comprehensively understand the process of tumor angiogenesis and improve the effect of anti-angiogenesis medicines.Small extracellular vesicles(sEVs)are significant mediators of communication between tumor and stromal cells,regulating the switch of phenotype of CAFs.Our previous studies show that melanoma-derived sEVs mediate the switch of proangiogenic phenotype of CAFs.Furthermore,hypoxia is one of the typical features of solid tumors and researches show that hypoxia can promote tumor cells to secrete sEVs and change the contents of sEVs,and such sEVs may play a greater part in intercellular communication.The mechanism of hypoxic melanoma-derived sEVs regulating proangiogenic CAFs is still unknown.Demystifying how melanoma-derived sEVs in hypoxia regulate proangiogenic CAFs can provide new strategies for melanoma treatment.In this thesis,we will discuss from following three parts.Part 1 Melanoma-derived sEVs regulate the proangiogenic phenotype of CAFs.Objective: Examine the regulation of normoxic and hypoxic melanoma sEVs on the proangiogenic CAFs.Methods: Used differential centrifugation,and TEM,DLS and Western blot to isolate and identify melanoma sEVs.NF were co-cultured with melanoma sEVs and the uptake of sEVs was proved by IF assay.The expression of markers of CAFs was detected by Western blot.Used CCK-8 assay,migration assay and in vitro tube formation assay to detect the proangiogenic phenotype of CAFs.Results: The isolated particles were possessed of biconcave disc like structures and the average diameter of them were about 150 nm.The particles contained sEVs protein markers TSG101,CD81 and CD9,but did not contain Calnexin.IF results verified the uptake of PKH26-labled sEVs by NF.After co-culture with melanoma sEVs,the markers of CAFs,α-SMA,FAP were significantly upregulated.The capability of sEVsinduced CAFs to facilitate proliferation,migration,and tube formation of ECs was markedly upregulated,and compared to normoxic sEVs,hypoxic sEVs performed a more significant effect.Conclusion: Melanoma sEVs can induce NF transforming into proangiogenic CAFs and hypoxic sEVs play a more significant role in promoting the transformation of proangiogenic CAFs than normoxic sEVs.Part 2 Melanoma-derived sEVs regulate the proangiogenic CAFs through NF-κB/CXCL1 axis.Objective: Explore the mechanism of normoxic and hypoxic melanoma-derived sEVs regulating the proangiogenic CAFs.Methods: Used RNA-seq analysis to compare the transcriptome of HFF-1 cells and hypoxic or normoxic sEVs-treated HFF-1 cells and then filtrated the target gene and verified the expression of it.Detected the expression of NF-κB/CXCL1 axis by Western blot.Measured the concentration of CXCL1 in the CM of CAFs by ELISA.CXCL1 Ab was applied to block CXCL1 in the CM,then the proangiogenic capability of CAFs was detected by CCK-8 assay,migration assay and tube formation assay.NF-κB inhibitor APDC was applied to block NF-κB signaling pathway,then the expression of CXCL1 was detected by Western blot and ELISA,and the proangiogenic capability of CAFs was detected by CCK-8 assay,migration assay and tube formation assay.Results: RNA-seq,Western blot and ELISA results showed that the transcription,translation and secretion of CXCL1 in the sEVs-induced CAFs were significantly upregulated,and hypoxic sEVs had a more significant effect.After blocked active CXCL1 in CM,the proangiogenic capability of the sEVs-induced CAFs was impaired.After APDC blocked NF-κB signaling pathway,the expression of CXCL1 in the sEVsinduced CAFs and the proangiogenic capability of CAFs were inhibited.Conclusion: Melanoma-derived sEVs regulate the proangiogenic phenotype of CAFs through NF-κB/CXCL1 axis.Hypoxic sEVs promote more efficiently the expression of NF-κB/CXCL1 axis in CAFs and the proangiogenic capability of CAFs than normoxic sEVs.Part 3 Melanoma-derived sEVs regulate the proangiogenic CAFs by transferring HSP90/p-IKKα/β complex to activate NF-κB/CXCL1 axis in CAFs.Objective: Explore the mechanism of melanoma sEVs regulating the NF-κB/CXCL1 axis and the proangiogenic CAFs.Methods: Used Proteomic analysis to compare the protein of hypoxic and normoxic melanoma-derived sEVs and filtrated the target protein by bioinformatic analysis.The expression of HSP90 and p-IKKα/β in normoxic and hypoxic sEVs was detected by Western blot.The interaction of HSP90 with p-IKKα/β in sEVs was verified by Co-IP.After HSP90 inhibitor 17-AAG blocked HSP90 in sEVs,NF were co-cultured with such sEVs and the uptake of sEVs was verified by IF assay.The expression of HSP90/NF-κB/CXCL1 axis in sEVs-treated HFF-1 cells was detected by Western blot and the concentration of CXCL1 in the CM was measured by ELISA.Used HPLCMS/MS analysis and Western blot to exclude the influence of 17-AAG residues on NF-κB/CXCL1 axis.After 17-AAG inhibit HSP90 in sEVs,the proangiogenic capability of sEVs-induced CAFs was detected by CCK-8 assay,migration assay and tube formation assay.Established xenograft tumor model and then used IHC and IF assay to investigate the effect of blocking HSP90 by 17-AAG on melanoma angiogenesis and development.Results: Proteomic analysis and Western blot results showed that HSP90 exhibited higher levels in hypoxic melanoma-derived sEVs and bioinformatic analysis showed that HSP90 had a close association with IKK/IκB/NF-κB/CXCL1 axis.As a client protein of HSP90,p-IKKα/β was enriched in hypoxic sEVs.IF results verified the uptake of 17-AAG-pretreated sEVs by NF.The influence of 17-AAG residues on NF-κB/CXCL1 axis in fibroblasts was excluded by HPLC-MS/MS analysis and Western blot.The interaction of HSP90AA1 and HSP90AB1 with p-IKKα/β in sEVs was verified by Co-IP.Blocking HSP90 with HSP90 inhibitor 17-AAG in sEVs could decreased p-IKKα/β in sEVs and downregulated the expression of IKK/IκB/NF-κB/CXCL1 axis,which impaired the proangiogenic capability of sEVs-induced CAFs.Xenograft tumor model,and IHC and IF assay verified that blocking HSP90 with 17-AAG could impair the angiogenesis and tumorigenesis in melanoma-CAFs xenograft tumor model.Conclusion: Melanoma-derived sEVs,especially hypoxic sEVs,transfer HSP90/pIKKα/β complex to activate NF-κB/CXCL1 axis in CAFs and then upregulate the proangiogenic capability of CAFs and promote tumor angiogenesis.