The Experimental Study On Tumors Treated By Inhibiting Yap1-induced Immunogenic Cell Death Coordinated With Ionizing Radiation And PD-1 Blockade

Objective:1.To explore the effects of Yap1 on immunogenic cell death(ICD)and the expression of immunorecognition related molecules.2.To explore the mechanism of Yap1regulating immune response.3.To observe the effect of radiation on the expression of Yap1and the anti-tumor effect of inhibiting Yap1 combined with radiation.4.To observe the anti-tumor effect of inhibiting Yap1 on the radiotherapy and PD-1 blockade resistance model.Methods:1.Verteporfin,a small molecule inhibitor,or small interfering RNA(si RNA)were used to inhibit and silence Yap1.CCK8 experiments were conducted to determine the effects of different concentrations of Verteporfin on the proliferation and viability of CT26 and MC38 cell and to determine IC50 concentrations of both cell lines.Western Blot was used to verify the effective si RNA sequences.2.ICD of CT26 and MC38 cell after inhibiting Yap1 were observed.Specific indicators were the expression of damage-related molecular patterns(DAMPs),including apoptosis,calreticulin translocation,ATP release,and secretion of high mobility protein B1(HMGB1).And whether tumor cells pretreated with lethal doses of Verteporfin had the ability to"immunize"to verify ICD induction.In brief,CT26/MC38 tumor cells were treated with Verteporfin in vitro and the dead cells were injected into the left flank of immunocompetent BALB/c or C57BL/6 mice.The mice were then rechallenged with live CT26/MC38 cell inoculation into the right flank 8 days later and the tumor formation of the right flank was observed within 30 days.3.The changes of molecules involved in immune recognition after inhibiting Yap1 were observed,including the expression of MHC-I,MHC-II,ICAM-1 and PD-L1 detected by flow cytometry and the m RNA levels of?2M,TAPBP,TAP1 and TAP2,components of MHC,were detected by q PCR.4.After subcutaneous inoculation of MC38 cells in the flank of C57BL/6 mice and intraperitoneal injection of Verteporfin,T cell activation in spleen of tumor-bearing mice was detected by flow cytometry.5.The DNA damage of CT26 and MC38 cells after inhibiting Yap1 was observed by immunofluorescence.The changes of the innate immune pathway c GAS-STING and type I interferon pathway were detected by q PCR and Western Blot.In addition,lentiviral infection was used to silence c GAS or STING,so as to verify the regulation of ICD and immune response by inhibiting Yap1 through this signaling pathway.6.CT26 and MC38 cells were irradiated with different radiation doses(0Gy,2Gy,4Gy and 8Gy),and the effect of radiation on the expression of Yap1 was analyzed by Western Blot.7.MC38 colon cancer xenograft model was used to observe the anti-tumor effect of inhibiting Yap1 combined with radiation.Tumor volume was measured,survival time was observed,and the microenvironment of the transplanted tumor was detected and analyzed by flow cytometry.8.Pan02 pancreatic cancer model was used to observe the efficacy of a novel treatment method of inhibiting Yap1 combined with radiation and PD-1 bloackde in the treatment of radiation and PD-1 bloackde-resistant pancreatic cancer xenograft.Results:1.Inhibiting Yap1 can induce apoptosis of CT26 and MC38 cells,and lead to increased expression of calreticulin,ATP release and HMGB1 secretion.We observed 100%tumor-free survival among mice immunized with Verteporfin-treated dead tumor cells in the 30 days after challenge,while all mice that were vaccinated with freeze-thawed tumor cells developed tumors.2.The expression of MHC-I and MHC-II in CT26 and MC38 cells was up-regulated by inhibiting Yap1,and the m RNA levels of?2M,TAPBP,TAP1 and TAP2 related components of MHC molecules were also increased.The expression of ICAM-1,an adhesion molecule involved in T cell activation,was increased,while the expression of PD-L1,which is involved in immune escape,was down-regulated.After intraperitoneal injection of Verteporfin in MC38 colon cancer-bearing mice,the proportion of CD8~+T cells expressing CD69~+in the spleen increased,but there was no significant change in CD4~+T cells,suggesting that CD8~+T cell activation increased.3.Immunofluorescence detection of?-H2AX showed that inhibiting Yap1 induced increased DNA damage in CT26 and MC38 cells.Western Blot detected the up-regulation of c GAS and STING expression,increased IRF3 phosphorylation,and increased IFN?m RNA level after inhibiting Yap1.After silencing c GAS or STING,it was found that splenic T cell activation induced by intraperitoneal injection of Verteporfin was significantly reduced,and the“vaccine effect”induced by a dead dose of Verteporfin disappeared.4.Irradiation can up-regulate the expression of Yap1 in a time-dose-dependent manner,with the highest level of Yap1 expression at 48 hours after 8Gy irradiation.5.Radiation combined to inhibit Yap1 effectively reduced the size of MC38 transplanted tumors and significantly prolonged the survival time of tumor-bearing mice.Analysis of the microenvironmental analysis of transplanted tumors found that not only the number of CD8~+T cells increased,but also their activation degree and killing ability were also improved,which was manifested as an increase in the proportion of CD69~+,IFN-?+and TNF-?+.In addition,the combination therapy also reduced the infiltration of regulatory T cells(Tregs)and improved the tumor immune response.6.Inhibiting Yap1 and radiation combined with PD-1 blockade effectively inhibited the growth of Pan02 pancreatic cancer transplantation tumors,significantly improving the therapeutic effect of radiotherapy and PD-1 blockade resistant tumors.Concluisons:1.Inhibiting Yap1 can cause immunogenic cell death(ICD).2.Inhibiting Yap1 can strengthen the recognition of tumor cells,weaken immune escape,and enhance T cell activation.3.Inhibiting Yap1 enhanced the anti-tumor immune response through the c GAS-STING pathway.4.Radiation can up-regulate the expression of Yap1,and radiation combined to inhibiting Yap1 had a significant anti-tumor effect.5.Inhibiting Yap1 can effectively improve the therapeutic effect of pancreatic cancer that is resistant to both radiotherapy and anti-PD-1 therapy.