The Role Of Type ? Interferons In The Combinational Treatment Of PIC And PDL1 Blockade In Colorectal Liver Metastases

Background:Despite the low incidence of colorectal cancer several decades ago,now it is the third most common cancer and the fourth most common cancer mortal factor globally,accounting for roughly 1·2 million new cases and 600 000 deaths per year.Due to the high metastatic activity of colorectal cancer,hepatic metastases became the primary fetal factor.Surgery is the main curative treatment non-metastasized colorectal cancer.The 5-year overall survival rate of patients with colorectal cancer who have isolated liver and/or lung metastases is about 20%.Recently,targeted agents are used for metastatic colorectal cancer treatment.PDL1 antibody specially blocks the PD-1-PD-L1 axis to save the exhausted T cells,making it better for the TILs proliferation,infiltration and memory T cells increase to finally enhance the adaptive immune response.TLR3 agonist poly I:C activates IRF3,thereby inducing type ? interferons(IFN)and IFN-inducible genes enhance antigen-specific CD8+T cell responses dependent upon host IFN-? signaling polyIC also could synergize with type ? inteferons to boost the tumor apoptosis.It was already reported that the mice HCC was dramatically suppressed by combinational treatment of polyIC and PDL1 antibody.Now we plan to explore the therapuetic effect of combinational treatment of polyIC and PDL1 antibody and the role of type ? inteferon in the combo therapy.Aims:To explore the role of of type ? interferons in the combinational treatment of pIC and PDL1 blockade in CRC liver metastases.Methods:For animal CRC metastasis model establishment,1×105MC38-GFP cells were cultured and 1x105 cells were injected into the spleen slowly.Mice were administered i.p.with drugs,such as polyIC 4mg/kg every other days(at day7,9,11,13,15),PDL1 antibody 0.2mg/mouse every other days(at day 10,12,14),PD1 antibody,CTLA4 antibody,sorafenib,accordingly.To block the type I interferon signaling,mice were injected intraperitoneally with the initial dose of 0.2mg IFNAR1 antibodies at day 6,followed by 0.lmg/mouse every other days(at day7,9,11,13,15)or IFNabR-/-mice were used.The survival times(day)for all mice were recorded.For combinational treatment of polyIC and PDL1 antibody,mice were euthanized at the endpoint to show the suviving time.After serums were collected,livers and spleens were dissected and weighed.The tissues from mice were frozen in Tissue-Tek O.C.T.compound or fixed in formalin.Liver and spleen tumors were observed generally and microscopically before treatment,in the middle of treatment,at the end of treatment and at the death point.We also examined the proliferation and apoptosis of hepatic metastasis tumor by immunofluorescence and immunohistochemistry.Furthermore,the immune cell subgroups and numbers were also explored by flow cytometry.The type ? interferon associated proteins were assessed to show the signaling pathway.The mice were also treated by sorafenib+PDL1 antibody,polyIC+PD1 antibody,polyIC+CTLA4 antibody to assess the efficacy of different combination therapies.Results:After the splenic transplantation,the mice developed hepatic metastases successfully We found the combination of polyIC and PD-L1 blockade dramatically suppressed CRC liver metastases.The median suvival time of control group is 18 days,while the mice from combo group all stayed alive.There were huge splenic and hepatic tumors in control mice,accompanied by massive bloody ascites,hemorrhage,necrosis and adhesion.There was no tumor in combo group whether by general observations or under the microscope Blocking the type ? interferons eliminated the effect of combination treatment,whether by IFNAR1 antibody or IFNabR-/-mice.However,IFNAR1 Ab alone neither enhance nor suppress tumor growth.IFN?+PDL1 had some therapeutic effect,but not as good as pIC+PDL1 antibody.The median survival time is 26.5 days,showing IFN?+PDL1 could somehow prolonged the surviving time.The ratio and activity of CD8+cells increased in the combo treatment group.STAT1 and pSTAT1,which is the downstream moleculars involved in type I interferon signaling pathway,were also upregulated in combo group As for other drug combinations,pIC+PD1 antibody and pIC+CTLA4 antibody had similar result with pIC+PDL1 antibody,whose mice stayed alive without tumor Sorafenib+PDL1 antibody had no effect,showing comparable outcome as controlConclusions:The combination of polyIC and PD-L1 blockade can suppress CRC liver metastases and prolonged the suvival time.Type ? interferon played a crucial role in the treatment for the blockade of type ? interferon could totally eliminated this treatment outcome There is still some therapuetic effect if polyIC was replaced by IFN?.Our observations suggest that polyIC and PDL1 antibody may be further developed into an effective combo treatment.