Study On The Molecular Mechanisms Of Exosome-mediated Proangiogenic Switch Of Melanoma-associated Fibroblasts

Malignant melanoma is a highly aggressive cancer.The incidence of melanoma in China tends to raise year by year,and the annual rate of increase is nearly 3-5%.Although several medicine of targeted therapy have been adopted into melanoma treatment and huge progress has been made in fighting against melanoma,the prognosis of melanoma is still undesirable.The vascularization level of malignant melanoma is quite high.Angiogenesis facilitates tumor cell proliferation and migration.But so far,the underlying mechanisms of melanoma angiogenesis haven't been fully understood.Therefore,thoroughly exploring the underlying mechanisms of melanoma progression and metastasis especially angiogenesis will be significant for the diagnosis and treatment of melanoma and the welfare of melanoma patients.Cancer-associated fibroblasts(CAFs)constitute a major part of tumor microenvironment.They could serve as tumor's accomplice and promote tumor proliferation,invasion,angiogenesis and metastasis.How normal cells transform into CAFs including the proanigenic switch of CAFs and how CAFs affect tumor progression,however,we haven't known much about these questions yet.Therefore,research on the transformation mechanisms of CAFs and their functions on tumor progression especially angiogenesis has high clinical values in searching new therapy against tumor.Exosome is an important vehicle of intercellular crosstalk.By transporting numerous bioactive molecules into stroma cells,tumor-derived exosomes could induce alteration of functions and phenotype of these recipient cells,and thus regulate tumor microenvironment and tame the stromal cells to support tumor progression.Therefore,exploring the mechanisms of how exosomes modulate the tumor microenvironment and trigger its proangiogenic switch will contribute to reveal the tumor invasion and metastasis mechanisms and provide new strategy against tumor.Part ? Melanoma exosomes induce reprogramming of normal fibroblasts into proangiogenic cancer-associated fibroblasts.Objective: Examine whether melanoma exosomes could transform normal fibroblasts into proangiogenic cancer-associated fibroblasts.Methods: Melanoma cell-derived exosomes was isolated by using differential centrifugation and verified by transmission electron microscopy,dynamic light scattering and Western blot.Fibroblasts were co-cultured with melanoma exosomes and the intake of exosomes was observed by immunofluorescence.The morphology of these fibroblasts was observed and expression of CAFs' markers was detected by qRTPCR and Western Blot.The expression of proangiogenic factors in these CAFs was detected by qRT-PCR,Western Blot,and ELISA.And the proangiogenic phenotype of CAFs was examined by CCK-8 proliferation assay,Transwell migration assay,in vitro tube formation assay,and in vivo Matrigel plug angiogenesis assay.Results: The results of transmission electron microscopy showed that the isolated particles remained cup-tray like structure.Dynamic light scattering showed that the diameter of melanoma exosomes fell between 30 nm to 150 nm.The presence of exosome protein markers Hsp90,Tsg101,and CD63 was testified by Western Blot.The results of immunofluorescence showed that exosome-carried red fluorescent signal appeared in the cytoplasm of fibroblasts.After co-culture with melanoma exosomes,fibroblasts obtained spindle-like shape,and the expressions of CAFs' markers ?-SMA,FAP were significantly upregulated.The expressions of proangiogenic factors FGF2,VEGFa,and MMP9 were also upregulated.The capability of CAFs to promote proliferation,migration,and tube formation of endothelial cells was remarkably enhanced.Conclusion: Melanoma exosomes could trigger the transformation of fibroblasts into proangiogenic CAFs.Part ? JAK2/STAT3 signaling pathway modulates the melanoma exosomeinduced proangiogenic switch of cancer-associated fibroblasts.Objective: Exploring the regulatory role of JAK2/STAT3 signaling pathway in proangiogenic switch of CAFs.Methods: Using Western blot to detect the phosphorylation levels of JAK2/STAT3 signaling pathway in fibroblasts after treatment with melanoma exosomes.Using immunofluorescence to detect the accumulation of phosphorylated STAT3 in fibroblast nuleus.The expression of SOCS1 was detected by qRT-PCR and Western blot.JAK inhibitor AG490 was applied to block JAK2/STAT3 signaling pathway,then the phosphorylation level of JAK2/STAT3 signaling and the expressions of downstream proangiogenic factors was investigated.CCK-8 proliferation assay,Transwell migration assay,and Matrigel tube formation assay was carried out to determine the alteration of proangiogenic capability of CAFs in promoting endothelial cell proliferation,migration and tube formation.Results: After incubation with melanoma exosomes,the phosphorylation level of JAK2/STAT3 signaling pathway in fibroblasts was significantly upregulated,the expression of SOCS1 was downregulated,nuclear accumulation of phosphorylated STAT3 was much more obvious.After JAK2/STAT3 signaling pathway was inhibited by AG490,the phosphorylation level of JAK2/STAT3 in CAFs was remarkably downregulated,and the expressions of VEGFa,FGF2,and MMP9 significantly decreased.And the capability of CAFs to promote endothelial cell proliferation,migration,and tube formation was obviously weakened.Conclusion: Melanoma exosomes could induce the proangiogenic switch of CAFs through activating JAK2/STAT3 signaling pathway.Part ? Melanoma exosomal miR-155-5p modulates the proangiogenic switch of CAFs via SOCS1/JAK2/STAT3 pathway.Objective: Investigating the regulatory roles of exosomal miR-155-5p on JAK2/STAT3 signaling pathway and proangiogenic switch of CAFs.Methods: Using bioinformatics analysis to predict the microRNAs which might target SOCS1.Applying qRT-PCR to detect the expressions of microRNA candidates in melanoma exosomes.When we ascertained the highest expressed microRNA(miR-155-5p),we detected its expression in fibroblasts after the stimulation with melanoma exosomes and compared the difference of miR-155-5p expression between B16,B16F10,and NIH/3T3 cells.We used miR-155-5p mimic and inhibitor to transfect B16 and B16F10 cells respectively and investigated the changes of miR-155-5p expression in both cell lines,their exosomes,and the CAFs.Transfected B16F10 cells with FITCmiR-155-5p,we used immunofluorescence to determine whether miR-155-5p was transferred by exosomes.We used dual-luciferase reporter assay to testify that miR-155-5p targets SOCS1.We isolated exosomes from miR-155-5p mimic-transfected B16 cells and miR-155-5p inhibitor-transfected B16F10 cells and co-cultured them with fibroblasts.Then the phosphorylation level of JAK2/STAT3 signaling pathway and the expressions of SOCS1,MMP9,VEGFa,and FGF2 in these CAFs were detected by Western blot.CCK-8 proliferation assay,Transwell migration assay,and tube formation assay were carried out to examine the influence of miR-155-5p on the capability of CAFs to promote endothelial cell proliferation,migration,and tube formation.We mixed fibroblasts stimulated with exosomes from miR-155-5p mimictransfected B16 cells and miR-155-5p inhibitor-transfected B16F10 cells with melanoma cells,and set up tumor bearing mouse models,and examined the microvessel density in xenografted tumors.Results: The results of qRT-PCR showed that miR-155-5p enriched in B16-and B16F10-derived exosomes,and the expression in B16F10 cells and exosomes was much higher than those of B16.The expression of miR-155-5p in NIH/3T3 cells was lower than those in melanoma cells,but significantly increased after stimulation with melanoma exosomes.The expression of miR-155-5p in CAFs increased when B16 cells were transfected with miR-155-5p mimic,and didn't change remarkably when B16F10 cells were transfected with miR-155-5p inhibitor.We used FITC to label miR-155-5p and PKH26 to label exosomes,then detected green and red fluorescence signal in the cytoplasm of NIH/3T3 cells.The results of dual-luciferase reporter assay showed that miR-155-5p mimic could weaken the activity of luciferase reporter containing the wildtype 3`UTR of SOCS1 and miR-155-5p inhibitor had the reverse effects.Transfection of miR-155-5p mimic could inhibit the expression of SOCS1.After stimulated with exosomes from miR-155-5p mimic-transfected B16 cells,the expression of SOCS1 was downregulated,the phosphorylation level of JAK2/STAT3 signaling pathway and the expressions of MMP9,VEGFa,FGF2 was upregulated.The exosomes from miR-155-5p inhibitor-transfected B16F10 cells made opposite effects.After transfection of miR-155-5p mimic,the capability of B16-associated fibroblasts to promote endothelial cell proliferation,migration,tube formation,and in vivo angiogenesis was significantly upregulated.And transfection of miR-155-5p inhibitor didn't change the proangiogenic capability of B16F10-associated fibroblasts remarkably.Conclusion: Melanoma exosomal miR-155-5p regulates the expressions of MMP9,VEGFa,and FGF2 via SOCS1/JAK2/STAT3 pathway and modulates the proangiogenic switch of CAFs.