Platelets Reprogram The Immunosuppressive Microenvironment Of Ovarian Cancer Via Upregulating The Expression Of PD-L1 In Cancer-associated Fibroblasts

Background: Ovarian cancer is a gynecological malignancy with the highest mortality rates in the world.Current therapies cannot significantly improve the long-term prognosis of ovarian cancer patients.Although immune checkpoint blockade therapy targeting PD-1/PDL1 has achieved significant efficacy in melanoma,non-small cell lung cancer and other tumors,its response rate was less than 10% in clinical trials of ovarian cancer,suggesting a complicated immunosuppressive microenvironment.Both cancer-associated fibroblasts and platelets are abundant in the stroma and contribute to the immunosuppression of ovarian cancer.However,it's unknown if there is an interaction between them involving in the formation of an immunosuppressive tumor microenvironment.Object: To explore how platelets and CAFs in cooperatively reshape the immune suppression microenvironment of ovarian cancer and its possible new mechanism.Methods: Through the analysis of ovarian cancer dataset TCGA,GSE9891 and ovarian stroma dataset GSE40595,combining immunohistochemistry and immunofluorescence in clinical samples,the correlation between infiltrating platelet and stromal PD-L1 expression and its potential immunoregulatory effects were verified.Further in vitro co-culture assays and bioinformatics analysis confirmed the role of platelets in upregulation of PD-L1 expression in ovarian CAFs and the possible mechanism.Finally,the feasibility of downregulating the stromal PD-L1 expression by anti-platelet drugs thus reversing the immunosuppressive microenvironment was evaluated in the xenograft models.Results: 1)In ovarian cancer dataset TCGA and GSE9891,platelet-related signature score were significantly positively correlated with immunosuppression-related signature score,and the expression of platelet characteristic genes GP1 BA and SELP were significantly positively correlated with PD-L1.2)There was a significant positive correlation between platelet infiltration and stromal PD-L1 expression in clinical samples of ovarian cancer.In dataset GSE40595,there was a significant positive correlation between platelet activation-related signature and stromal PD-L1 expression,and a significant positive correlation between stromal PD-L1 expression and immunosuppression-related signature.3)Platelets upregulated PD-L1 expression in ovarian CAFs in both two-dimensional and three-dimensional co-cultures in vitro.4)Western blot and flow cytometry results showed that platelets might up-regulate PD-L1 expression in ovarian CAFs through NF?B and STAT3 pathways.5)Bioinformatics analysis and in vitro intervention of the possible pathways showed that platelets might up-regulate PD-L1 expression of ovarian CAFs through PDPN/NF?B/STAT3 signaling.6)Compared with the control group,the tumor volume of aspirin and ticagrelor groups was significantly reduced,the expression of PD-L1 in tumor stroma was significantly down-regulated,and the infiltration of CD8 positive T cells was increased.Conclusions: In ovarian cancer,platelets contribute to the reprogramming of the immunosuppressive microenvironment by upregulation of stromal PD-L1 expression.This finding complements the formation mechanism of the immunosuppressive microenvironment in ovarian cancer,and provides inspiration and theoretical basis for potential schemes of immunotherapy combined with anti-platelet drugs,which has been proved to be safe for clinical use.