Effect Of Gemcitabine On Tumor Microenvironment Enhancement Of PD-1 Antibody Against Hepatocellular Carcinoma In Mice

Hepatocellular carcinoma(HCC)is the most common type of primary liver cancer(90%)and the third leading cause of cancer-related death worldwide.At present,surgery,radiotherapy and chemotherapy are the main means of HCC treatment,but the overall therapeutic effect is poor due to the difficulty of early diagnosis and high recurrence rate of HCC.Heterogeneous etiology,complex tumor microenvironment,and immune regulation of HCC require specific therapeutic strategies with novel targeted therapies.There are a number of ongoing phase III trials of HCC therapy exploring combinations of tyrosine kinase inhibitors(TKIs)and immune checkpoint inhibitors(ICI),and cytotoxic T-lymphocyte associated antigen 4(CTLA-4)inhibitors with other ICIs.The next big challenge in this area is to identify new combination treatment regimens that can consistently improve overall survival for front-line patients.Recent studies have shown that gemcitabine can modulate the tumor immune microenvironment,particularly to clear myeloid derived suppressor cells(MDSCs)to activate endogenous antitumor immune responses.All these suggest that chemoimmunotherapy may trigger robust antitumor immunity.Based on some of the pitfalls of gemcitabine for cancer chemotherapy,such as enzymatic deamination,rapid systemic clearance,and chemoresistance through downregulation of cellular uptake,we investigated the therapeutic efficacy of gemcitabine combination with PD-1/PD-L1antibody in HCC.Objective:1.To investigate the alterations of the immunogenic microenvironment of HCC by gemcitabine.2.To investigate the therapeutic effects of combination therapy of gemcitabine plus PD-1 antibody in different mouse models of HCC.Methods:1.A subcutaneous H22 green fluorescent protein(GFP)cell inoculation model was constructed,it was randomly divided into 2 groups,control group and gemcitabine administration group(50 mg/kg)were administered once every 4 days for a total of 3times.Tumor tissues were collected and digested to examine subsets of immune cells,to quantify CD45~+tumor infiltrating immune cells by flow cytometry,and to further analyze the proportion of myeloid derived suppressor cells(MDSCs)and tumor associated macrophages(TAMs),CD4~+T,and CD8~+T cells.The expression levels of PD-1 and PD-L1 in tumor tissues were quantified by protein immunoblotting;As well as the quantitative determination of PD-L1 expression on H22-GFP cells by flow cytometry,and PD-1 expression on CD3~+tumor infiltrating T cells.2.A subcutaneous H22-GFP cell inoculation model was constructed,the mice were randomly divided into 4 groups,a control group,gemcitabine alone group(50 mg/kg),αPD-1 alone group(2.5 mg/kg),and gemcitabine combined withαPD-1 were administered once every 4 days for a total of 3 times.Tumor growth curves of tumorbearing mice were measured.The proportion of interleukin(IL)-2~+CD8~+T cells in mouse HCC tumor tissue was measured by flow cytometry.Enzyme linked immunosorbent assay(ELISA)assays were performed to detect the expression of immune cytokines including transforming growth factor(TGF)-βin tumor cells、Interleukin(IL)-12p70 and interferon(IFN)-γof the variation in content.3.4 days after final treatment,the primary tumor tissue of H22 tumor bearing mice was removed and separated,and 2×10~6H22 cells were injected subcutaneously on the opposite side to establish tumor recurrence model,and 4 days after the final treatment,the proportions of CD8~+and CD4~+central memory T cells(TCMs)in the spleen were examined by flow cytometry.On days 12-39 after the first treatment,the tumor volume change in mice with tumor recurrence in each group was evaluated,the tumor growth curve was plotted,and the number of tumor free mice in each group was counted.4.Three kinds of human hepatoma cells Hep G2,Hep3B and Huh7 were selected and treated with 10μM gemcitabine for 24 h,the expression of PD-L1 was detected by Western blot.The tumor samples obtained from liver cancer patients were digested into single cells and treated with 10μM gemcitabine for 24 h,and the expression of PD-1 on T cells was detected by flow cytometry.5.The Huh7 humanized mouse tumor transplantation model was constructed,and the mice were randomly divided into 4 groups:control group,gemcitabine alone group(50mg/kg),αPD-1 alone group(2.5 mg/kg),and gemcitabine combined withαPD-1 group(once every 4 days)for a total of 4 administration times.The tumor growth of mice was monitored.Results:1.Gemcitabine treatment can effectively increase the proportion of invasive immune cells in tumor tissue,reduce the proportion of MDSCs and TAMs in CD45~+cells,and increase the proportion of CD4~+T and CD8~+T cells.2.Gemcitabine treatment induces PD-L1 and PD-1 overexpression:gemcitabine treatment can increase the relative PD-L1 and PD-1 expression levels in tumor tissues,and PD-L1 expression on H22-GFP cells is upregulated compared with untreated control cells;PD-1 expression on CD3~+tumor infiltrating T cells was upregulated.3.Gemcitabine combinationαPD-1 can upregulate IL-2~+CD8~+T to inhibit the growth of H22 liver cancer:compared with gemcitabine orαPD-1 monotherapy,the subcutaneous tumor volume was significantly reduced,the proportion of IL-2~+CD8~+T cells was increased,the secretion of TGF-βwas down-regulated and the secretion of IL12p70 and IFN-γwas up-regulated after gemcitabine combined withαPD-1 treatment.4.Gemcitabine plusαPD-1 blockade can effectively suppress HCC recurrence:compared with gemcitabine orαPD-1,the proportions of CD8~+TCM and CD4~+TCM in the spleen of mice treated with gemcitabine andαPD-1 were significantly increased.Gemcitabine combined withαPD-1 significantly improved the proportion of tumor-free patients in the recurrence model from 1/6 to 4/6,and the volume of recurrent tumors decreased significantly.5.Gemcitabine treatment induces high expression of PD-L1 in human HCC cells:in the three groups of Hep G2,Hep3B and Huh7 cells,the expression level of PD-L1 in gemcitabine treated cells was higher than that in the control group.Gemcitabine induced high expression of PD-1 in T cells of hepatocellular carcinoma treated with single-cell suspension.6.Gemcitabine combined withαPD-1 effectively inhibited the growth of Huh7 liver cancer:compared with gemcitabine alone andαPD-1 alone,the size and mass of HCC tumors were significantly reduced in the gemcitabine plusαPD-1 group.Conclusions:1.Gemcitabine increased the frequency of CD8~+T and CD4~+T cell components,and decreased intracellular MDSCs(CD11b~+Gr-1~+)and TAMs(F4/80~+).2.The combination therapy of gemcitabine and PD-1 blockade can further inhibit HCC growth and recurrence.