Carbon ion irradiation induces DNA damage in melanoma and optimizes the tumor microenvironment based on the cGAS-STING pathway.

Increased number of studies reveal the crucial role of the Cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway in anti-tumor immunity. In this study, we aim to explore the effect of cGAS/STING on tumor immune microenvironment of melanoma after carbon ion radiotherapy (CIRT) and the underlying mechanism. C57BL/6 mouse tumor models were used to evaluate the efficacy of different treatments (X-ray, carbon ion, PD-L1 inhibitor and combination therapies) on tumor growth and process. Mass cytometry was performed to assess tumor-infiltrating lymphocytes (TILs). DNA damage response (DDR) and cGAS/STING pathway were investigated by immunofluorescence-co-localization assays, γ-H2AX, P53-binding protein 1 (53BP1), Breast Cancer 1 (BRCA1), and cGAS measurements. Carbon ion irradiation caused more DNA damages and cGAS-STING pathway activation compared with X-ray irradiation, and the former slowed the melanoma growth in syngeneic model. Although X-ray irradiation is not sensitive for melanoma treatment, carbon ion irradiation showed a significant anti-tumor effect for melanoma treatment. TILs analysis revealed that CIRT boosted the infiltration of natural killer (NK), CD4+, and CD8+ T cells, meanwhile increased the number of immune checkpoint (programmed death-1, PD-1, lymphocyte activation gene 3, LAG-3 and T-cell immunoglobulin and mucin domain-containing protein 3, TIM-3). Moreover, CIRT increased PD-L1 exposure on cell surface compared with X-ray group. Furthermore, CIRT combined with PD-L1 inhibitor therapy increased the number of T cells and NK cells in melanoma, and slowed the growth of melanoma compared with other therapies. Our findings showed that CIRT displayed biological effects by increasing DNA damages of tumor cells and improving immunity in melanoma, which indicated that CIRT might be a potential synergetic treatment for radiotherapy and radioimmunotherapy in melanoma patients. Our works put forward a new insight to provide an effective strategy for melanoma therapy. These findings may help in the design of strategies on melanoma in clinical studies.

Guo Y ，Shen R ，Wang F ，Wang Y ，Xia P ，Wu R ，Liu X ，Ye W ，Tian Y ，Wang D ... -  《-》

ATR inhibitor AZD6738 enhances the antitumor activity of radiotherapy and immune checkpoint inhibitors by potentiating the tumor immune microenvironment in hepatocellular carcinoma.

Radioimmunotherapy has a promising antitumor effect in hepatocellular carcinoma (HCC), depending on the regulatory effect of radiotherapy on tumor immune microenvironment. Ionizing radiation (IR)-induced DNA damage repair (DDR) pathway activation leads to the inhibition of immune microenvironment, thus impairing the antitumor effect of radioimmunotherapy. However, it is unclear whether inhibition of the DDR pathway can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of DDR inhibitor AZD6738 on the combination of radiotherapy and immune checkpoint inhibitors (ICIs) in HCC. C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control and tumor recurrence inhibition. Effects of each treatment regimen on the alterations of immunophenotypes including the quantification, activation, proliferating ability, exhaustion marker expression, and memory status were assessed by flow cytometry. AZD6738 further increased radiotherapy-stimulated CD8+ T cell infiltration and activation and reverted the immunosuppressive effect of radiation on the number of Tregs in mice xenografts. Moreover, compared with radioimmunotherapy (radiotherapy plus anti-PD-L1 (Programmed death ligand 1)), the addition of AZD6738 boosted the infiltration, increased cell proliferation, enhanced interferon (IFN)-γ production ability of TIL (tumor-infiltrating lymphocyte) CD8+ T cells, and caused a decreasing trend in the number of TIL Tregs and exhausted T cells in mice xenografts. Thus, the tumor immune microenvironment was significantly improved. Meanwhile, triple therapy (AZD6738 plus radiotherapy plus anti-PD-L1) also induced a better immunophenotype than radioimmunotherapy in mice spleens. As a consequence, triple therapy displayed greater benefit in antitumor efficacy and mice survival than radioimmunotherapy. Mechanism study revealed that the synergistic antitumor effect of AZD6738 with radioimmunotherapy relied on the activation of cyclic GMP-AMP synthase /stimulator of interferon genes (cGAS/STING) signaling pathway. Furthermore, triple therapy led to stronger immunologic memory and lasting antitumor immunity than radioimmunotherapy, thus preventing tumor recurrence in mouse models. Our findings indicate that AZD6738 might be a potential synergistic treatment for radioimmunotherapy to control the proliferation of HCC cells, prolong survival, and prevent tumor recurrence in patients with HCC by improving the immune microenvironment.

Sheng H ，Huang Y ，Xiao Y ，Zhu Z ，Shen M ，Zhou P ，Guo Z ，Wang J ，Wang H ，Dai W ，Zhang W ，Sun J ，Cao C ... -  《Journal for ImmunoTherapy of Cancer》

Carbon ion irradiation exerts antitumor activity by inducing cGAS-STING activation and immune response in prostate cancer-bearing mice.

As an advanced radiotherapy technique, carbon ion radiotherapy has demonstrated good efficacy and low toxicity for prostate cancer patients, but the radiobiological mechanism of killing tumor cells has not been fully elucidated. This study aims to explore the antitumor effects of carbon ion irradiation (CIR) through investigating the immune response induced by CIR in prostate cancer-bearing mice and the underlying molecular mechanism. We established subcutaneous transplantation tumor models of prostate cancer to evaluate the tumor inhibition effect of CIR. Investigation of immunophenotype alterations were assessed by flow cytometry. Immunofluorescence, western blot, and real-time quantitative PCR was employed to analyze the activation of cGAS-STING pathway. CIR showed more powerful tumor growth control than photon irradiation in immunocompetent syngeneic C57BL/6 mice. CIR exerts antitumor effect by triggering immune response, characterized by increased CD4+ T cells and macrophages in tumor, enhanced CD8+ T cells and T effector memory cells in spleen, improved IFN-γ production of CD8+ tumor-infiltrating lymphocytes, and reduced exhausted T cells in tumor and spleen. Additionally, production of cytoplasmic double-stranded DNA, protein levels of p-TBK1 and p-IRF3 in the cGAS-STING pathway, and gene expression levels of downstream interferon-stimulated genes were significantly increased after CIR in a dose-dependent manner. Treatment of RM1 tumor-bearing mice with the STING inhibitor C-176 impaired the antitumor effect of CIR. The excellent antitumor activity of CIR in immunocompetent prostate cancer-bearing C57BL/6 mice may be attributed to stronger induction of antitumor immune response and higher activation of cGAS-STING pathway.

Hu W ，Zhang Z ，Xue Y ，Ning R ，Guo X ，Sun Y ，Zhang Q ... -  《Cancer Medicine》

Radiation Therapy Promotes Hepatocellular Carcinoma Immune Cloaking via PD-L1 Upregulation Induced by cGAS-STING Activation.

Radiation therapy (RT) is one of the main treatments for patients with unresectable hepatocellular carcinoma (HCC). Emerging evidence indicates that the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) stimulator of interferon gene (STING) pathway is crucial in RT-induced antitumor immune responses. Here, we discovered that activation of the cancer cell-intrinsic cGAS-STING pathway mediated immune cloaking after RT-induced DNA damage. Key regulatory proteins in the cGAS-STING signaling pathway in human and murine HCC cell lines were knocked out or down using CRISPR and CRISPR-associated protein 9 or small interfering RNA. The underlying mechanism of immune cloaking and clinical significance of cGAS-STING-induced programmed cell death ligand 1 (PD-L1) expression were studied with both ex vivo analyses and in vitro experiments. RT upregulated PD-L1 in patients with HCC, which correlated with poor survival. RT activated cGAS-STING, increasing immune-checkpoint PD-L1 expression in human and mouse liver cancer cells. Ionizing radiation activated the STING-TANK-binding kinase 1 (TBK1)-interferon regulatory factor 3 (IRF3) innate immune pathway, leading to PD-L1 upregulation in HCC cells and inhibiting cytotoxic T-lymphocyte activity and protecting tumor cells from immune-mediated eradication. Knockdown of cGAS, STING, TBK1, and IRF3 reversed the antitumor effect of cytotoxic T-lymphocyte-mediated cytotoxicity after ionizing radiation in vitro or in vivo. RT potentiated the antitumor effect of programmed cell death protein 1 and PD-L1 axis blockade and augmented cytotoxic T-cell (CTL) infiltration in HCC tumors in immunocompetent mice. CD8 depletion compromised the synergetic antitumor effect of combined RT and anti-PD-L1 blockade, demonstrating that CD8+ CTLs are required for antitumor immunity induced by combination therapy. Our results identified an immune-cloaking mechanism for RT-activated, innate immune cGAS-STING and suggested that RT enhances HCC immunotherapy.

Du SS ，Chen GW ，Yang P ，Chen YX ，Hu Y ，Zhao QQ ，Zhang Y ，Liu R ，Zheng DX ，Zhou J ，Fan J ，Zeng ZC ... -  《-》

Wnt/β-catenin inhibitor ICG-001 enhances the antitumor efficacy of radiotherapy by increasing radiation-induced DNA damage and improving tumor immune microenvironment in hepatocellular carcinoma.

Radiotherapy (RT) has a promising anti-tumor effect depending on its effects on both cancer cells and tumor immune microenvironment (TIME). As one of the most common alterations in hepatocellular carcinoma (HCC), wnt/β-catenin pathway activation, has been reported to induce radioresistance and suppressive TIME. In this study, we aim to explore the effect of wnt/β-catenin inhibitor ICG-001 on radiosensitivity and RT-related TIME of HCC and the underlying mechanism. C57BL/6 and nude mouse tumor models were used to evaluate the efficacy of different treatments on tumor growth, recurrence and mice survival. Flow cytometry was performed to assess tumor infiltrating lymphocytes (TILs). DNA damage response (DDR) and radioresistance was investigated by colony formation assays, γ-H2AX and micronuclei measurements. The addition of ICG-001 to RT exhibited better anti-tumor and survival-prolong efficacy in C57BL/6 than nude mice. TILs analysis revealed that ICG-001 plus RT boosted the infiltration and IFN-γ production of TIL CD8+ T cells, meanwhile reduced the number of Tregs. Moreover, mechanistic study demonstrated that ICG-001 increased the radiation-induced DDR of HCC cells by suppressing p53, thus leading to stronger activation of cGAS/STING pathway. Utilization of cGAS/STING pathway inhibitors impaired the therapeutic effect of combination therapy. Furthermore, combination therapy led to stronger immunologic memory and tumor relapse prevention. Our findings showed that ICG-001 displayed both local and systematic effects by increasing radiosensitivity and improving immunity in HCC, which indicated that ICG-001 might be a potential synergetic treatment for radiotherapy and radioimmunotherapy in HCC patients.

Huang Y ，Sheng H ，Xiao Y ，Hu W ，Zhang Z ，Chen Y ，Zhu Z ，Wu D ，Cao C ，Sun J ... -  《-》