研究背景：虽然免疫治疗策略在肿瘤的临床治疗中取得了令人鼓舞的成绩，但肿瘤患者对免疫疗法的响应率仍较低，其中一个重要原因是免疫抑制性肿瘤微环境（immunosuppressive tumor microenvironment, ITME）的存在，它是导致当前肿瘤免疫治疗效果不佳的主要原因之一。肿瘤细胞通过募集各种免疫抑制性细胞建立促瘤的ITME，导致肿瘤组织内细胞毒性T淋巴细胞（cytotoxic T lymphocytes, CTLs）浸润程度低，无法激活有效的抗肿瘤免疫应答。此外，肿瘤的低免疫原性和高异质性也使其能逃避免疫系统的监视与攻击。因此，如何提高肿瘤的免疫原性和重塑ITME成为当前肿瘤免疫治疗亟待解决的关键问题。诱导肿瘤细胞发生免疫原性细胞死亡（immunogenic cell death, ICD）已成为改善ITME的有效策略。肿瘤细胞发生ICD在释放新抗原表位的同时，还会释放损伤相关分子模式（damage associated molecular patterns, DAMPs），如高迁移率族蛋白B1（high mobility group protein 1, HMGB1）、ATP和钙网蛋白（calreticulin, CRT），激活适应性免疫应答，产生长效的抗肿瘤免疫效应。因此诱导肿瘤细胞发生ICD被认为是一种非常有前途的肿瘤免疫治疗策略。然而目前临床上可用做ICD诱导剂的化疗药物比较少，且副作用较大、作用机制还有待进一步研究。因此，寻找新型有效的ICD诱导剂，阐明其作用机制并将其应用到肿瘤治疗中，对于激活机体抗肿瘤免疫，产生长远的抗肿瘤效果具有重要意义。竹节香附素A（Raddeanin A, RA）是从中药竹节香附中提取分离的一种天然三萜皂苷，其在诱导ICD方面的作用至今未见报道。本文以诱导ICD和激活T细胞为出发点，评价RA的体内外抗肿瘤活性，并对其诱导ICD的机制进行了深入研究。 方法：基于HMGB1-Gluc报告基因实验和LacZ报告基因实验从天然活性化合物库中筛选能诱导肿瘤发生ICD同时激活T细胞的化合物。通过酶联免疫吸附分析法（enzyme linked immunosorbent assay, ELISA）检测B16肿瘤细胞、骨髓来源树突状细胞（Bone marrow-derived dendritic cells, BMDCs）与B3Z/OT-Ⅰ T细胞共培养后，B3Z/OT-Ⅰ T细胞分泌的白介素-2（interleukin-2, IL-2）、干扰素γ（interferon gamma, IFN-γ）和肿瘤坏死因子α（tumor necrosis factor alpha, TNF-α）的蛋白水平；ELISA检测肿瘤细胞中干扰素β（interferon bata, IFN-β）、TNF-α和CXC趋化因子配体10（C-X-C motif chemokine ligand 10, CXCL10）水平的变化。RA作用MC38或B16细胞后，流式细胞术检测肿瘤细胞表面CRT的表达，B3Z/OT-Ⅰ T细胞中CD69、IFN-γ和颗粒酶B（granzyme B, GZMB）的表达以及BMDCs表面CD40、CD80、CD86、MHC-Ⅰ、MHC-Ⅱ和MHC-Ⅰ SIINFEKL的变化情况。转录组测序方法筛选DMSO或RA处理的肿瘤细胞中的差异表达基因。qPCR检测RA作用后的肿瘤细胞中Cxcl10、Isg15、Ifnb1、Irf9、Ifit1、Ccl5和Tnf的mRNA变化。Western Blot检测RA作用后肿瘤细胞中TBK1、IRF3和P65蛋白及其磷酸化水平。通过生物素-链霉亲和素结合系统和LC-MS/MS筛选RA在肿瘤细胞中的潜在结合蛋白，并结合siRNA基因沉默实验对RA的直接作用靶点进行验证。细胞热位移实验（cellular thermal shift assay, CETSA）验证RA与TAR DNA 结合蛋白43（TAR DNA binding protein-43, TDP-43）的直接互作。表面等离子共振（surface plasmon resonance, SPR）分析实验测定TDP-43与RA或齐墩果酸（oleanolic acid, OA）的解离常数。通过分子对接模型预测TDP-43与RA的互作氨基酸位点。通过亚细胞分级分离实验对B16和MC38细胞中的细胞核、细胞质和线粒体进行提取分离，Western Blot检测RA作用后TDP-43在细胞中的分布情况。流式细胞术检测RA作用后肿瘤细胞的凋亡情况及其线粒体中ROS水平和线粒体膜电位的变化。通过免疫荧光实验和激光共聚焦观察cGAS和线粒体DNA（mitochondrial DNA, mtDNA）的共定位情况。使用siRNA对Tdp43等基因进行沉默。利用CRISPR-Cas9基因编辑技术敲除肿瘤细胞中的Tdp43。通过B16和MC38小鼠体内移植瘤模型评价RA的体内抗肿瘤活性，同时测定小鼠体重和瘤体积、瘤重变化情况。ELISA检测小鼠血清中谷草转氨酶和谷丙转氨酶的变化。多色流式细胞术检测肿瘤微环境（tumor microenvironment, TME）中T细胞和树突状细胞（dendritic cells, DCs）的比例及其活性变化以及免疫抑制细胞如髓源性抑制细胞（myeloid-derived suppressor cells, MDSCs）和调节性T细胞（regulatory T cells, Tregs）的浸润情况。 结果：通过HMGB1-Gluc报告基因和LacZ报告基因实验从天然活性化合物库中筛选诱导ICD的化合物，发现RA可显著诱导肿瘤细胞中HMGB1的释放和共培养的B3Z T细胞的LacZ活性。小鼠双侧肿瘤模型实验表明RA在体内可诱导产生免疫原性和免疫记忆，提示RA是一个有效的ICD诱导剂。通过ELISA、流式细胞术进一步验证发现，RA可诱导肿瘤细胞释放ATP，并促进CRT在肿瘤细胞膜表面暴露；同时，RA可促进共培养的B3Z/OT-Ⅰ T细胞表达CD69并分泌IL-2、IFN-γ、TNF-α及GZMB，证实RA能提高肿瘤的免疫原性。流式实验结果进一步表明，RA可明显升高与肿瘤细胞共孵育的BMDCs表面CD40、CD80、CD86、MHC-Ⅰ、MHC-Ⅱ和MHC-Ⅰ SIINFEKL的表达，说明RA可促进DCs的成熟并增强其抗原呈递功能。MC38和B16小鼠移植瘤模型实验结果表明，RA在体内具有良好的抗肿瘤活性，能明显抑制移植瘤的生长，同时能够促进TME中的CD8+ T细胞和DCs的浸润和活化，但在裸鼠体内或与CD8中和抗体同时使用时均无明显的抑瘤作用，证实RA的抗肿瘤活性依赖于T细胞。此外，当使用细胞色素C清除小鼠体内DCs后，RA的抑瘤效果也被逆转，说明RA的抗肿瘤作用也需要DCs介导。钓靶结果揭示，RA可与肿瘤细胞中的TDP-43蛋白发生直接相互作用，其结合位点为Ala228 和 His256。进一步的机制研究发现，RA与肿瘤细胞中的TDP-43蛋白结合后促进其在线粒体富集，导致线粒体膜电位下降、ROS水平升高以及mtDNA释放，激活cGAS-STING-Ⅰ型IFN信号通路，从而诱导ICD并激活TME中的DCs和CD8+ T细胞。小鼠MC38移植瘤实验进一步证实，RA与PD-1单抗联用可协同增加肿瘤浸润的CD8+ T细胞和DCs，并减少Tregs和M-MDSCs的累积，由此增强PD-1单抗的体内肿瘤免疫治疗作用。 结论：本研究发现RA通过mtDNA-cGAS/STING途径诱导ICD，促进DCs依赖性的CD8+ T细胞的激活，重塑肿瘤免疫微环境；同时揭示了TDP-43在肿瘤免疫治疗中的重要作用。RA与PD-1单抗等免疫治疗联合应用具有极大的潜力和广阔的应用前景，对天然活性产物RA的研究具有深远意义。 

Background: Although immunotherapies have achieved encouraging success in the clinical treatment of tumors, the response rate among tumor patients remains low. One of the major reasons is the presence of immunosuppressive tumor microenvironment (ITME), which is responsible for the poor effectiveness of current tumor immunotherapy. Tumor cells establish pro-tumor ITME by recruiting various immunosuppressive cells, resulting in a low infiltration of cytotoxic T lymphocytes (CTLs) in tumor tissues and the failure of activating an effective anti-tumor immune response. In addition, the low immunogenicity and high heterogeneity of tumors allow them to evade surveillance and attack by the immune system. Therefore, how to improve the immunogenicity of tumors and remodel ITME has become a critical issue to be addressed in current tumor immunotherapy. Induction of immunogenic cell death (ICD) in tumor cells has become an effective strategy to improve ITME. In addition to releasing neoantigenic epitopes, tumor cells undergoing ICD also release damage associated molecular patterns (DAMPs) such as high mobility group protein 1 (HMGB1), ATP and calreticulin (CRT), which activate adaptive immune responses and produce long-lasting antitumor immune effects. Therefore, induction of ICD in tumor cells is considered to be a very promising strategy for tumor immunotherapy. However, there are few chemotherapeutic agents that can be used as ICD inducers in clinical practice due to their severe side effects and unclear mechanism. Therefore, finding novel effective ICD inducers, elucidating their mechanism and applying them to tumor therapy are of great significance to activate antitumor immunity and produce long-term effects. Raddeanin A (RA) is a natural triterpenoid saponin isolated from the Chinese Medicinal Herb Anemone Raddeana Regel. Its role in inducing ICD has not been reported so far. In this study, taking the induction of ICD and activation of T cells as the starting point, we evaluated the in vitro and in vivo antitumor activity of RA, and conducted an in-depth study on the mechanism of its induction of ICD.

Methods: Screening for compounds that induce tumor ICD while activating T cells from a library of natural active compounds was based on HMGB1-Gluc reporter gene assay and LacZ reporter gene assay. The protein levels of interleukin-2 (IL-2), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) secreted by B3Z/OT-Ⅰ T cells after coculturing with B16 tumor cells and bone marrow-derived dendritic cells (BMDCs) were measured by enzyme linked immunosorbent assay (ELISA). The changes in levels of interferon bata (IFN-β), TNF-α and C-X-C motif chemokine ligand 10 (CXCL10) in B16/MC38 tumor cells were also determined by ELISA. The expression of CRT on the surface of B16/MC38 tumor cells treated with RA, and levels of CD69, IFN-γ and granzyme B (GZMB) on B3Z/OT-Ⅰ T cells and CD40, CD80, CD86, MHC-Ⅰ, MHC-Ⅱ and MHC-Ⅰ SIINFEKL on the surface of BMDCs were detected by flow cytometry. RNA sequencing was used to screen for differentially expressed genes in tumor cells treated with DMSO or RA. qPCR detected mRNA changes of Cxcl10, Isg15, Ifnb1, Irf9, Ifit1, Ccl5 and Tnf in tumor cells treated with RA. Western Blot was carried out to detect TBK1, IRF3 and P65 proteins and their phosphorylation levels in tumor cells treated with RA. The potential binding proteins of RA in tumor cells were screened by biotin-streptavidin binding system and LC-MS/MS, and the direct targets of RA were validated in combination with siRNA gene silencing assays. Cellular thermal shift assay (CETSA) was performed to verify the direct interaction of RA with TAR DNA binding protein-43 (TDP-43). Surface plasmon resonance analysis experiments were performed to determine the dissociation constants of TDP-43 with RA or oleanolic acid (OA). Prediction of the reciprocal amino acid sites of TDP-43 and RA was realized by molecular docking model. The nucleus, cytoplasm and mitochondria in B16 and MC38 cells were extracted and separated by subcellular hierarchical separation assay, and the distribution of TDP-43 in tumor cells treated with RA was detected by Western Blot. Flow cytometry was used to detect the apoptosis of tumor cells treated with RA and the changes in ROS levels in mitochondria and mitochondrial membrane potential. Co-localization of cGAS and mitochondrial DNA (mtDNA) was observed by immunofluorescence experiments and laser confocalization. Genes such as Tdp43 were silenced by siRNA. Knockout of Tdp43 in tumor cells was carried out with CRISPR-Cas9 gene editing technology. The in vivo antitumor activity of RA was evaluated by B16 and MC38 transplantation tumor models in mice, and changes in body weight, tumor volume and tumor weight were measured. ELISA was performed to detect the changes of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice serum. Multicolor flow cytometry detects the changes in the ratios of T cells and dendritic cells (DCs) and their activity in the tumor microenvironment (TME), as well as the infiltration of immunosuppressive cell populations such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs).

Results: Screening of ICD-inducing compounds from a library of natural active compounds by HMGB1-Gluc reporter gene and LacZ reporter gene assays revealed that RA significantly induced HMGB1 release from tumor cells and LacZ activity in co-cultured B3Z T cells. Experiments in a mouse bilateral tumor model showed that RA induced immunogenicity and immune memory in vivo, suggesting that RA is an effective ICD inducer. Further validation by ELISA and flow cytometry revealed that RA induced ATP release from tumor cells and promoted CRT exposure on the surface of tumor cell; meanwhile, RA promoted co-cultured B3Z/OT-Ⅰ T cells to express CD69 and secrete IL-2, IFN-γ, TNF-α, and GZMB, which confirms that RA enhances the immunogenicity of tumors. Flow cytometry further showed that RA significantly increased the expression of CD40, CD80, CD86, MHC-Ⅰ, MHC-Ⅱ and MHC-Ⅰ SIINFEKL on the surface of BMDCs co-incubated with tumor cells, indicating that RA promotes the maturation of DCs and enhances their function of antigen presentation. The results of MC38 and B16 mouse transplantation tumor models showed that RA had good anti-tumor activity in vivo and significantly inhibited the growth of transplanted tumors, while promoting the infiltration and activation of CD8⁺ T cells and DCs in TME. However, RA showed no significant tumor suppressive effect in nude mice or when used together with CD8 neutralizing antibody, confirming that the anti-tumor activity of RA was dependent on T cells. Moreover, the tumor suppressive effect of RA was also reversed when DCs were eliminated from mice using cytochrome C, suggesting that the antitumor effect of RA also requires DCs to mediate. Targets draping results revealed that RA could interact directly with the TDP-43 protein in tumor cells, and the binding sites are Ala228 and His256. Further mechanistic studies revealed that RA binding to TDP-43 protein in tumor cells promotes its enrichment in mitochondria, leading to a decrease in mitochondrial membrane potential, and an increase in ROS levels and mtDNA release, activating the cGAS-STING-type I IFN signaling pathway, which induces ICD and activates DCs and CD8⁺ T cells in TME. The mouse MC38 transplantation experiment further confirmed that RA in combination with PD-1 monoclonal antibody synergistically increased tumor-infiltrating CD8⁺ T cells and DCs and reduced the accumulation of Tregs and M-MDSCs, thereby enhancing the in vivo tumor immunotherapeutic effects of PD-1 monoclonal antibody.

Conclusion: This study found that RA induced ICD through the mtDNA-cGAS/STING pathway, promoted DCs-dependent activation of CD8⁺ T cells and reshaped the tumor immune microenvironment; meanwhile, it revealed the important role of TDP-43 in tumor immunotherapy. Moreover, the combination of RA with immunotherapy such as PD-1 monoclonal antibody has great potential and broad application prospects, which has far-reaching implications for the research of natural active product RA.