Radiotherapy Enhance Combining PD-L1 Inhibitor And VEGF Antibody Therapeutic Efficacy In Hepatocellular Carcinoma

Background and objectiveHepatocellular carcinoma(HCC),a primary form of liver cancer,has emerged as a focal point of oncological research due to its high incidence and mortality rates.The advent of immunotherapy has introduced new prospects for patients with HCC.Despite the results of the IMbrave150 trial indicating that the combination therapy of atezolizumab,a Programmed Death-Ligand 1(PD-L1)inhibitor,with the anti-angiogenic drug bevacizumab(hereafter referred to as the T+A regimen)significantly improves prognosis compared to sorafenib,only 27.3%of HCC patients benefit from this treatment.Radiation therapy,a local treatment modality for malignant tumors that has been in use for over a century,plays a dual role.It not only directly eradicates tumor cells but also modulates the tumor immune microenvironment,gaining increasing attention in the era of immunotherapy.This study aims to investigate the therapeutic efficacy of the combined radiation therapy with the T+A regimen on advanced HCC.It employs mass cytometry and other experimental methodologies to analyze the impact of radiation therapy on the tumor microenvironment(TME)of HCC.Furthermore,it seeks to elucidate the synergistic mechanisms of action of combining radiation therapy with the T+A regimen in the treatment of HCC.Methods(1)Efficacy of radiotherapy and T+AWe collected clinical and pathological data from a prospective single-arm clinical trial conducted at three hospitals including Eastern Hepatobiliary Surgery Hospital,to assess treatment response,overall survival time,and adverse reactions in patients receiving combined radiotherapy and T+A therapy for HCC.(2)Effect of radiotherapy on tumor microenvironmentWe utilized three immunocompetent mouse models of HCC to simulate clinical treatment regimens and evaluate their efficacy and safety.Mass cytometry was employed to explore the impact of different treatment regimens on the tumor microenvironment,and the results were validated using flow cytometry,immunohistochemistry,and multiplex immunohistochemistry.Immunocytes co-culture and in vivo macrophage depletion experiments were conducted to assess the role of PD-L1+macrophages in the radiotherapy combined with T+A treatment regimen.(3)Mechanism of PD-L1 ExpressionUtilizing RNA-seq to analyze the changes in macrophage m RNA expression under different irradiation doses,we concurrently conducted RNA-seq analysis on tumor tissues from various treatment groups to screen for potential effector molecules and signaling pathways.Real-time quantitative reverse transcription PCR(q PCR)and Western blot were employed to validate the role of the c GAS-STING signaling pathway in PD-L1 expression.Cell lines with Tmtc3 knockdown or overexpression were constructed to investigate the impact of TMTC3 on STING and PD-L1.Experimental analyses including liquid chromatography-mass spectrometry,immunofluorescence,immunoprecipitation,GST pull-down,and Western blot were conducted to explore the interaction between TMTC3 and STING.Molecular interaction binding sites were predicted using the Alphafold model.ATAC-seq,dual-luciferase reporter gene assays,and chromatin immunoprecipitation assays were employed to investigate the regulation of Tmtc3 by radiotherapy transcription factors.Results(1)Efficacy of radiotherapy and T+AThirty patients met the inclusion criteria and underwent radiotherapy combined with T+A therapy.The median age of the patients was 56.5 years(range,37-83 years),with 25patients(83.3%)being male,26 patients(86.7%)being carriers of the hepatitis B virus,and25 patients(83.3%)having cirrhosis.Eighteen patients(60.0%)had portal vein extrahepatic tumor thrombosis,16 patients(53.3%)had extrahepatic metastatic lesions,and 9 patients(30.0%)had concomitant hepatic vein tumor thrombosis.According to RECIST1.1 criteria,the overall best objective response rate was 76.6%,and the disease control rate was 96.7%.The median overall survival time for patients was 9.8 months.Treatment-related adverse events were assessed using CTCAE 5.0.The most common treatment-related adverse events of any grade included neutropenia(46.7%),fatigue(40.0%),and hypertension(33.3%),while the most common grade 3/4 treatment-related adverse events were hypertension(10.0%)and elevated aspartate aminotransferase(10.0%).(2)Effect of radiotherapy on tumor microenvironmentRadiotherapy enhances the therapeutic efficacy of T+A in Hepa1-6-C57BL/6subcutaneous and orthotopic tumor models,as well as in the H22-Balb/c orthotopic tumor model,while reducing the incidence of lung metastasis.Immunohistochemistry results suggest that radiotherapy combined with T+A significantly inhibits Ki-67 expression and increases the proportion of CD8+cells.Mass cytometry results show that radiotherapy alone increases the expression of chemokine receptors CXCR2,CX3CR1,and CXCR4,as well as the expression of exhaustion markers PD-L1 and Tim-3.Radiotherapy significantly increases the proportion of PD-L1-positive tumor-associated macrophages(TAMs),with the highest proportion of CD8~+T cells and the lowest proportion of PD-L1~+TAMs observed in the combined T+A group(hereafter referred to as the combination group).Flow cytometry results are consistent with mass cytometry findings,with an increase in the proportion of PD-L1~+TAMs observed after radiotherapy alone,and a decrease in the proportions observed in the T+A group and combination group,with the most significant decrease observed in the combination group.Compared to other treatment groups,the combination group exhibits the highest proportions of Gzmb~+CD8~+T cells,Ifn-γ~+CD8~+T cells,and Tnf-α~+CD8+T cells,with Ifn-γ~+CD8~+T cells being the highest among the three cell types.Clearance of tumor microenvironment macrophages with CSF1R inhibitor reveals that combination therapy enhances the efficacy of T+A by reducing TAM proportions.Co-culture of immune cells demonstrates that PD-L1~+TAMs inhibit the proliferation and cytotoxicity of CD8~+T cells.In patients receiving radiotherapy combined with T+A treatment,those who responded well to the treatment had a lower proportion of PD-L1+TAMs.(3)Mechanism of PD-L1 ExpressionFlow cytometry experiments revealed that the expression level of PD-L1 increased over time post-irradiation,with no significant differences observed between 24 and 48 hours post-irradiation.The impact of irradiation doses of 12Gy and 8Gy on PD-L1 expression was not significant.RNA sequencing results of macrophages subjected to various irradiation doses indicated that Tmtc3 was a significantly altered gene,ranking first in the list of upregulated genes according to the q-value.The expression of Tmtc3 exhibited an increasing trend with escalating irradiation doses(0Gy,2Gy,4Gy,8Gy).Gene Set Enrichment Analysis(GSEA)and immunoblotting results suggested that the c GAS-STING signaling pathway regulates PD-L1 expression.In RAW264.7 and THP-1 cells,the expression levels of TMTC3,STING,and PD-L1 proteins were higher post-irradiation compared to non-irradiated cells.Knockdown of TMTC3 followed by re-irradiation significantly reduced the expression of TMTC3,as well as STING and PD-L1.Knockdown of STING followed by re-irradiation markedly decreased the expression of STING and PD-L1,with no significant change in TMTC3 expression.TMTC3 regulates PD-L1 expression by directly interacting with STING at the Y104 site,enhancing STING glycosylation levels and reducing its ubiquitination levels.Radiation therapy regulates the transcription level of TMTC3 through the transcription factor Etv5.ConclusionsThis study found that the combination of radiotherapy with T+A significantly enhances the best objective response rate in patients with advanced HCC,while maintaining a favorable safety profile.The synergistic effect of radiotherapy and T+A is attributed to the reduction in the proportion of PD-L1~+TAM within the tumor microenvironment,which concurrently improves the proliferation and cytolytic activity of CD8~+T cells,thereby elevating the therapeutic efficacy.Mechanistically,TMTC3 directly interacts with the Y104site of STING,augmenting its glycosylation levels while reducing its ubiquitination,which in turn modulates the expression of PD-L1 on macrophages.Additionally,radiotherapy regulates the expression of TMTC3 via the transcription factor Etv5.