Mechanism Of Oncoprotein SND1 Inducing Melanoma Immune Escape Through Exosomes

Objectives:The tumor microenvironment is created by the tumor and dominated by tumor-induced interactions.Although various immune effector cells are recruited to the tumor site,immune escape of tumor cells from surveillance will occur when these immune-functioning cells in the microenvironment are unable to recognize and kill Tumor cells.Therefore,it is very important to explore the molecular mechanism underlying immune evasion of tumors so as to guide relevant strategies of tumor treatment.The reasons behind these are multifaceted,among which the tumor-derived Exosomes（TEX）secreted by tumor cells play an indispensable role.In recent years,numerous results have revealed TEX’s role in the immune system as follows.TEX is an important link in tumor-induced immunosuppression:they carry immunosuppressive molecules and specific factors known to interfere with immune cell function.TEX directly or indirectly affects the development,maturation,and antitumor activity of immune cells by delivering inhibitory.Tumor oncoprotein SND1was previously identified as a key factor promoting immune evasion via degradation of MHC-I molecules in tumor cells,according to one of our reports last year.However,the role of SND1 protein in the regulation of tumor metastasis by exosomes secreted by tumors remainselusive.This study aims to study the potential role and underlying mechanism of SND1 in tumor microenvironment promoting tumorigenesis via exosomes,and also seek to shed light on the scientific basis for the biological role and immune regulation mechanism of tumor exosomes.Method:The current study falls into three sectionSection 1:（1）Isolation and purification of exosomes from supernatant of B16F10WT/SND1-KO cells culture by ultra-centrifugation;（2）Establishing lung metastasis model with exosome intervention;（3）Co-culture of different exosomes with B16F10 WT cells in vitro.Flow cytometry was used to detect the cycle and apoptosis of B16F10 WT cells co-cultureed with Exo^WT and Exo^SND1-KO.The proliferation,migration and invasion of B16F10 WT cells after co-culture were detected by clone formation,CCK8 assay,and cell-migration assay.（4）The infiltration of immune cells in mice lung metastatic model was detected by flow cytometry.Section 2:（1）The proteome of B16F10 WT/SND1-KO cells and exosomes were detected and analyzed by isotope labelling relative and absolute quantitative technique（i TRAQ）.（2）The expression of CD47 was verified by western blot and q PCR,and the binding of SND1 to CD47 protein in vivo was verified by immunoprecipitation and immunofluorescence assay.（3）The co-localization of early endosome marker EEA1,recycling endosome marker HRS,and late endosome marker CD63 in B16F10 WT/SND1-KO cells was verified by immunofluorescence assay.（4）The binding of CD47 in B16F10 WT/SND1-KO cells to RAB family members（Rab5,Rab7,Rab11A,Rab35）and the exosomal secretion-related protein SNARE family member VAMP7 in cells was verified by immunoprecipitation and immunofluorescence assay.Section 3:（1）Constructing of Exo ^WT-mplum and Exo ^{SND1-KO-mplum} RAW264.7 cells in vitro with deep red fluorescent protein markers of tracer secrete m Plum.Immunofluorescence and flow cytometry analysis on efficiency of macrophage phagocytosis.western blot on verification of the phagocytosis by protein level of phosphorylated SHP1 level.Further validation by by q PCR analysis;（2）Different exosomes(Exo^WT and Exo^SND1-KO)were constructed in vitro,M1 macrophages selected from the spleen of mice and murine melanoma B16F10 co-culture model.PBS treatment was used as blank control to detect the killing effect of M1macrophages on tumor cells under different exosomes treatment.Result:Result:Section 1:（1）Isolation and purification of exosomes secreted by B16F10WT/SND1-KO cells;（2）In the mouse lung metastasis model with exosome intervention,the number of lung metastasis in the Exo^SND1-KO intervention group was significantly less than that in the PBS and Exo^WT intervention group;（3）PBS group,Exo^WT group and Exo^SND1-KO group had no significant effects on apoptosis,cycle,proliferation,migration and invasion of B16F10 WT cells in vitro.（4）More immune cells（CD4⁺T,CD8⁺T,DC,M1 macrophages）were infiltrated in lung metastases of mice in Exo^SND1-KO treatment group;Section 2:（1）The differential proteins of the cell group and the exosome group were analyzed,and CD47 was screened by GO and KEGG enrichment analysis.（2）The expression of CD47 did not change in the cell group,but the expression of Exo^SND1-KOdecreased significantly.The expression of CD47 m RNA did not change in the cell group,and SND1 did not bind to CD47.（3）CD47 did not co-locate with EEA1 and HRS in B16F10 WT,but did co-locate with CD63.CD47 did not co-locate with EEA1 and HRS in B16F10 SND1-KO cells,but the co-location with CD63 was significantly reduced.（4）Rab35 binds to SND1 in B16F10 WT,and CD47 binds to Rab35 in B16F10 WT,while CD47 binds to Rab35 in SND1-KO cells significantly decreased.Section 3:（1）The absorption efficiency of RAW264.7 WT and mouse spleen Macrophages to Exo^{SND1-KO-m Plum} was significantly higher than that of Exo^{WT-m Plum},and the phosphorylation level of SHP-1 was significantly higher than that of Exo^{WT-m Plum} and the activation of Macrophages was promoted after Exo^{WT-m Plum}treatment.（2）The effect of Macrophages killing B16F10WT in the Exo ^SND1-KOtreatment group was significantly stronger than that in the Exo^WT intervention group and PBS group.Conclusion:（1）Exo^SND1-KO inhibited the metastasis of B16F10 in the lung of mice and promoted the infiltration of more immune cells in the metastatic lesions.Exo^SND1-KO had no significant effect on the growth,migration and invasion of B16F10.（2）By binding to RAB35,SND1 promoted a large amount of CD47 aggregation in the late endosome,which was secreted by exosomes.（3）Exo^WT can inhibit the phagocytic ability of Macrophages,thus weakening the ability of Macrophages to kill tumor cells.On the contrary,Exo^SND1-KO can promote the phagocytic ability of Macrophages,thus enhancing the ability of Macrophages to kill tumor cells.