Cancer Cell-derived Immunoglobulin G Activates Platelets By Binding To Platelet FcγRⅡa And The Mechanism Of Platelet Infiltration In HCC Tissue

Part I Cancer cell-derived immunoglobulin G activates platelets by binding to platelet FcγRⅡaAims:Tumor-associated thrombosis is the second leading risk factor for cancer patient death,and platelets activity is abnormal in cancer patients.Discovering the mechanism of platelet activation and providing effective targets for therapy are urgently needed Cancer cell derived IgG has been reported to regulate development of tumors.However,studies on the functions of cancer cell derived IgG are quite limited.Here we investigated the potential role of cancer cell derived IgG in platelet activationMethods:We detected the expression of CD62P on platelets by Flow Cytometry and analyzed platelet function by platelets aggregation and ATP release.The content of IgG in cancer cell supernatants was detected by Enzyme-linked immune sorbent assay Western blot was performed to quantify the relative expression of FcγRⅡa,Syk,PLC y2 The interaction between cancer-derived IgG and platelet FcγRⅡa was analyzed by Co-immunoprecipitationResults:Higher levels of p-selectin were observed in cancer patients’ platelets compared with that of healthy volunteers.Cancer cell culture supernatants increased platelet p-selectin and PAC-1 expression,sensitive platelet aggregation and ATP release in response to agonists,while blocking FcγRⅡa or knocking down IgG reduced the activation of platelets.Coimmunoprecipitation results showed that cancer cell-derived IgG interacted directly with platelet FcγRⅡa.In addition,platelet FcγRⅡa was highly expressed in liver cancer patientsConclusions:cancer cell-derived IgG interacted directly with FcγRⅡa and activated platelets;targeting this interaction may be an approach to prevent and treat tumor-associated thrombosisPart II The mechanism of platelet infiltration in HCC tissueAims:Platelets has been shown to have the ability to actively migrate.Here we provide evidence that platelets migrate out of blood vessels in human and mouse hepatocellular carcinoma(HCC)tissues.However,the mechanism of platelet migration and the biological function of migrated platelets remains largely unknown,we investigated the mechanism of platelet migration and the role of migrated plateletsMethod:Immunofluorescence was used to analyze platelet infiltration in hepatocarcinoma tissues.Transwell was used to detect the effect of hepatoma cell culture supernatant on platelet migration.Flow cytometry was used to analyze the number of platelets migrated and analyze platelet intracellular calcium signal changes SiRNA and HCC orthotopic model were used to analyze of the role of CX3CL1 in platelet migration.Mitochondrial membrane potential and cell cycle assay to analyze the effect of migrated platelets on hepatoma cells.Western blot was used to analyze the expression of CX3CL1,p-Syk and p-Akt.Immunohistochemical analysis of the expression of CX3CL1,BAX,Bcl-2 and Ki67 in hepatocarcinoma tissues Subcutaneous xenografts to detect the effect of migrated platelets on the growth of HCCResults:There were extravascular infiltration of platelets in human and mouse HCC tissues,and no infiltration of platelets was detected in adjacent tissues.Hepatoma cell culture supernatants could directly induced platelet migration.Knockdown of CX3CL1 expression in hepatoma cells significantly decreased platelet migration and extravascular infiltration.Human CX3CL1 recombinant protein induced platelet migration in a concentration-dependent manner.Hypoxia promotes platelet migration by up-regulating CX3CL1 expression.Blocking platelet CX3CR1,Syk and PI3K inhibited platelet migration.Blocking CX3CR1 attenuates the phosphorylation of Syk and PI3K induced by CX3CL1.Platelet infiltration was associated with growth inhibition of HCC in orthotopic model.Migrated platelets induced mitochondrial membrane potential decline in hepatoma cells.Migrated platelets inhibited HCC growth in subcutaneous xenografts model.Conclusion:HCC cells derived CX3CL1 induced platelet migration through platelet CX3CR1/Syk/PI3K.Infiltrated platelets induced the apoptosis of HCC cells and inhibited the growth of HCC.