Investigating The Role And Mechanism Of Immune Escape-Related Key Molecules In Melanoma

Background: Melanoma is the most malignant skin tumor,with a high mortality rate in the late stage and a lack of treatment strategies.Although the immune checkpoint blockades therapy has greatly benefited melanoma patients,its overall response rate is even low.One of the key factors leading to this phenomenon is that melanoma can affect the tumor immune microenvironment through a variety of ways,thereby promoting immune escape.And the specific mechanism of melanoma immune escape is still unclear.In this study,we found two key molecules,CCL8 and PEG10,which may affect the immune escape of melanoma.We then explored their correlation with the prognosis of melanoma and the response to immunotherapy.At the same time,we further clarified their functions in the immune escape of melanoma through in vivo and in vitro experiments,and revealed their molecular mechanisms which have not yet been reported.Finally,based on our results,it provides a new clinical treatment and combined treatment strategy with ICBs for melanoma,and provides a new biomarker for the prediction of ICBs in melanoma.Objective:(1)To determine the correlation between the expression of CCL8,PEG10 and ICBs response and prognosis in melanoma;(2)To reveal the molecular mechanism of CCL8 and PEG10 affecting the immune escape of melanoma;(3)To develop treatments and prediction schemes for melanoma based on CCL8 and PEG10.Methods:(1)Bioinformatics was used to analyze the correlation between target molecule expression,clinical features,and treatment prognosis in public databases and clinical samples from hospital;(2)ELISA was used to detect CCL8 expression in the plasma of melanoma patients;(3)Gene-knockdown stable cell line constructed by lentivirus;plasmid transient transfection to construct gene expression cell line;(4)Western blot to detect changes in protein levels,real-time quantitative PCR to detect changes in m RNA levels,flow cytometry to detect changes in membranous protein levels,immunofluorescence to detect changes in protein location and expression levels;(5)Construct C57BL/6 immunocompetent mice subcutaneously transplanted tumor models to study the effects of different interventions on the occurrence and development of melanoma;(6)Second-generation sequencing(NGS)technology to detect transcriptome changes in melanoma cells;(7)Luciferase reporter gene to detect the targeting relationship between transcription factors and target genes;(8)Extracting mouse BMDMs and primary peritoneal macrophages for macrophage polarization induction experiment;(9)Extracting volunteer peripheral blood PBMC and using magnetic beads to select CD8+ T cells for in vitro killing experiments to detect the effect of target molecules on T cell tumor killing function.Results:Part One:(1)The expression of CCL8 RNA and plasma protein are negatively correlated with the therapeutic effect and prognosis of melanoma patients with ICBs treatment;(2)CCL8 can promote macrophage polarization towards M2 type through the CCR5/SPHK1 axis,then inhibit the anti-tumor immunity of melanoma;(3)Bindarit can reduce the exhaustion of cytotoxic T cells by inhibiting M2 polarization of tumor related macrophages,and exerts anti-tumor effects on melanoma in vivo;(4)In the melanoma mouse model,the small molecule compound Bindarit,which is a drug with clinical application potential,can enhance ICBs effect.Part Two:(1)The expression of PEG10 is negatively correlated with the therapeutic effect and prognosis of melanoma patients undergoing ICBs therapy;(2)In animal models,knockdown PEG10 in melanoma cells can reduce the expression of membranous PD-L1,increase the infiltration and activation of CD8+ T cells and reduce the infiltration of Tregs cells,promote anti-tumor immunity,and then inhibit tumor progression;(3)In animal models,Knockdown PEG10 in melanoma cells can up regulate the expression of CD73 on tumor infiltrating Tregs cells;(4)PEG10 regulates the expression of PD-L1 in melanoma cells from the transcriptional level through the transcription factor ATF3;(5)Knockdown PEG10 can cooperate with CD73 monoclonal antibody in the treatment of melanoma.Thus,it can be used as a new predictor of melanoma ICBs treatment and a new target of single or combined treatment strategies.Conclusions:(1)CCL8 expression is associated with poor prognosis of ICBs therapy in melanoma,and can promote M2 polarization of macrophages through CCR5/SPHK1 axis,then inhibit anti-tumor immunity.CCL8 detection can be used as a non-invasive,efficient and economical method to predict the prognosis of ICBs therapy in melanoma.Bindarit can be used as a novel strategy alone or combine with ICBs therapy in melanoma;(2)PEG10 expression is related to the poor prognosis of ICBs therapy in melanoma,and can regulate the expression of PD-L1 in melanoma through ATF3 to inhibit anti-tumor immunity.PEG10 can be used as a novel biomarker to predict the prognosis of melanoma.PEG10 is a potential therapeutic target for melanoma,which can cooperate with CD73 monoclonal antibody to play an anti-tumor role.Figures: 42,Tables: 37,References: 98...