Experimental Study Of Sorafenib Combined With Anti-PD-L1 Antibody In The Treatment Of Hepatocellular Carcinoma And Multimodal Imaging Evaluation Of Its Efficacy

PART Ⅰ:Effect of hypoxia on immune microenvironment and resistance to sorafenib in hepatoma cells in vitroObjectives:The effect of hypoxia on the expression of hypoxia-inducible factor 1α(HIF-1α)and programmed death ligand 1(PD-L1)in hepatoma cell line Hepa 1-6 was investigated in vitro.Furthermore,the expression of multidrug resistance protein 1(MRP1)in Hepa 1-6 cells after different degrees of hypoxia and sorafenib intervention was investigated.Methods:The in vitro cell experiment of this study was mainly divided into two parts.In the first part,Hepa 1-6 cells were cultured at 21%O2 and 1%O2 for 12 hours.The expression of HIF-1α and PD-L1 protein were detected by western blot and immunofluorescence.In the second part,Hepa 1-6 cells were cultured under 21%O2,10%O2,5%O2 and 1%O2.Meanwhile,cells in the experimental group were treated with 10μM sorafenib for 12 hours.Western blot and real-time quantitative PCR were used to detect the expression of MRP1 protein and mRNA.Results:Western blot showed that compared with normoxic conditions,HIF-1α and PD-L1 were considerably increased in Hepa 1-6 cells under hypoxia(P<0.001).So it is with immunofluorescence.Meanwhile,western blot and PCR showed that with the aggravation of hypoxia,the expression levels of MRP1 protein and mRNA were gradually increased,and were further increased after the addition of 10 μM sorafenib.Conclusion:The up-regulated expressions of HIF-1α,PD-L1 and MRP1 in Hepa 1-6 hepatoma cells under hypoxia may increase the resistance of hepatoma cells to sorafenib.PART Ⅱ:Efficacy evaluation and mechanism exploration of sorafenib combined with anti-PD-L1 antibody in the treatment of hepatocellular carcinomaObjectives:To evaluate the efficacy of sorafenib combined with anti-PD-L1 antibody in the treatment of Hepa 1-6 hepatocellular carcinoma models in mice,and to investigate the synergistic mechanism of combination therapy.Methods:Mouse syngeneic Hepa 1-6 hepatoma cells were subcutaneously injected into the right flank near the hindlimbs of C57BL/6 mice(male,6-8 weeks old).About 5-6 days after inoculation,when the tumor volume reached 80-120 mm3,the mice were stochastically divided into four groups:(A)Control group:mice were intraperitoneally injected with isotype-matched IgG antibody(10 mg/kg)(n=6);(B)Sorafenib gavage(50 mg/kg)(n=6);(C)Intraperitoneal injection of anti-PD-L1 antibody(10 mg/kg)(n=6);(D)Combination therapy group:sorafenib gavage(50 mg/kg)combined with anti-PD-L1 antibody intraperitoneal injection(10 mg/kg)(n=6).On day 21 after intervention,the four groups of mice were euthanized and the tumor tissues were completely exfoliated from the skin.H&E staining,immunohistochemistry staining,immunofluorescence staining and flow cytometry were applied to detect the changes of subcutaneous tumor microenvironment of mice in the four groups.Meanwhile,ELISA kit and biochemical automatic analyzer were used to detect serum cytokines and liver and kidney functions of mice in the four groups.In addition,survival analysis was performed to evaluate the survival time of mice in each group.Survival analysis was conducted as an independent experiment with 10 mice in each group.Results:(1)On day 21 after treatment,subcutaneous tumor volume in the four groups was 1563.87±243.84 mm3 in group A,1100.68±198.81 mm3 in group B,491.79±92.44 mm3 in group C,and 120.51±15.97 mm3 in group D,respectively(P<0.001).Survival analysis showed that the median survival time of mice in the four groups(A-D)was 35,50,70 and 95 days,respectively(P<0.001).(2)The combination therapy group had the highest rate of tumor necrosis and apoptosis,and the lowest rate of tumor cell proliferation(P<0.001).(3)CD31 immunofluorescence showed that the tumor microvascular density of group B was appreciably lower than that of group A(P<0.001)and C(P<0.01),but there was no statistical difference with group D(P=0.064).a-SMA immunofluorescence showed that the positive rate of tumor perivascular cell in group D and C markedly exceeded that in groups A and B(P<0.001).Hypoxia staining demonstrated that hypoxia area of subcutaneous tumor in group B was the highest(P<0.001).(4)Flow cytometry showed that the number of CD8+T lymphocytes and M1 tumor-associated macrophages(P<0.001)in group D notably exceeded those in the other three groups,while the number of M2 tumor-associated macrophages and regulatory T cells(P<0.001)in group D was below those in the other three groups.The serum levels of IL-12,IFN-γ,granzyme B and TNF-α in groups C and D were appreciably greater than those in groups A and B(P<0.001),while the serum levels of IL-4 and IL-10 in groups C and D were not as much as those in group B(P<0.001).Conclusion:Sorafenib combined with anti-PD-L1 antibody could significantly inhibited tumor growth and prolonged survival time in mice compared with sorafenib or anti-PD-L1 antibody monotherapy.According to the experimental results:anti-PD-L1 antibody except by increasing the CD8+T lymphocytes and M1 tumor-associated macrophages infiltration to improve immunosuppression microenvironment,could increase by tumor vascular smooth muscle layer to reshape the tumor microvascular structure to improve the hypoxic microenvironment,which can reduce tumor sorafenib resistance in combination therapy.PART Ⅲ:Role of multimodal imaging in evaluating the early efficacy of sorafenib combined with anti-PD-L1 antibody in hepatocellular carcinomaObjectives:To investigate the role of magnetic resonance(MR)and 18F-FDG PET/CT multimodal imaging in the early efficacy evaluation of sorafenib combined with anti-PD-L1 antibody in the treatment of hepatocellular carcinoma.Methods:The four groups of mice were grouped and treated as in the part II.T2-weighted MR(T2WI),T1mapping,diffuse-weighted MR(DWI),dynamic enhanced MR(DCE-MR),MR spectroscopy(MRS)and 18F-FDG PET/CT scans were performed 1 day before and 7,14,and 21 days after intervention,respectively.T2WI appearance,T1 value,apparent diffusion coefficient(ADC)value,volume transfer constant(Ktrans)value,lactate/choline(Lac/Cho)ratio,and tumor/muscle standardized uptake ratio(SUVtumor/SUVmuscle)in each group at 4 time points were observed and quantitatively analyzed.Correlation analysis was conducted between the above imaging quantitative results and the results of tumor tissue H&E staining,immunofluorescence and flow cytometry in part Ⅱ.Results:(1)T1mapping showed that no remarkable difference of subcutaneous tumor T1 value was found among the four groups before and after treatment.(2)DWI showed that there was no extraordinary difference in ADC values of subcutaneous tumors in the four groups before treatment(P=0.646).On the 7th day after treatment,ADC value in group D appreciably exceeded that in group A(P=0.017).ADC value was positively correlated with tumor necrosis rate(r=0.810,P<0.001)and tumor cell apoptosis(r=0.710,P<0.001),and negatively correlated with tumor cell proliferation(r=0.655,P<0.001).(3)Before treatment,DCE showed that there was no obvious difference in Ktrans value of subcutaneous tumors in the four groups(P=0.487).On the 7th day after treatment,Ktrans value in group B was greatly lower than that in group A(P=0.03).The Ktrans value in groups C and D was markedly lower than that in group A on day 14 and 21 after treatment(P<0.01).Ktrans value was positively correlated with tumor microvascular density(r=0.766,P<0.01).(4)Before treatment,MRS showed that there was no statistically distinct difference in the Lac/Cho ratio of subcutaneous tumors in the four groups(P=0.969).After intervention,the Lac/Cho ratio of subcutaneous tumors in group B was greatly over and above that in groups A and C(P<0.01),but there was no remarkable difference compared with group D.Lac/Cho ratio was positively correlated with tumor hypoxia area(r=0.898,P<0.001).(5)Before treatment,18F-FDG PET/CT showed no marked difference in SUVtumor/SUVmuscle ratio among the four groups(P=0.978).On day 7 after treatment,the SUVtumor/SUVmuscle ratio was increased in all four groups,but there was no prominent difference among all groups(P=0.499).On day 21 after treatment,FDG uptake in both groups decreased due to significant reduction in tumor diameter in group C and group D.Correlation analysis showed that SUVtumor/S UVmuscle ratio was negatively correlated with the number of CD3+T lymphocytes(r=-0.792,P<0.01).Conclusion:On day 7 after combination therapy,increased ADC and high uptake of FDG in the tumor reflected decreased tumor cell density and increased inflammatory cell infiltration.Meanwhile,DCE quantitative parameters Krans and Lac/Cho ratio of MRS revealed early dynamic changes in tumor vascular and hypoxia microenvironment after sorafenib and anti-PD-L1 antibody treatment.The indirect detection results of image evaluation in this study have a good correlation with the direct detection results of animal model in the second part,which provides a theoretical and practical basis for the clinical application of multimodal imaging to evaluate the efficacy of anti-angiogenic drugs together with immune checkpoint inhibitors in the treatment of hepatocellular carcinoma.