局部放射治疗（RT）可以对未经照射的肿瘤病变产生T细胞介导的异位效应，尤其是与免疫检查点阻断联合使用。然而，这种效应在临床上仍然很少见，需要进行改进。我们研究了临床批准的低剂量脂质体乐铂霉素（Doxil）与RT / αPD-1 和 Doxil / αPD-1 相比，能否增强异位效应。我们还研究了增强的异位效应是否依赖于线粒体DNA（mtDNA）/环磷酸磷酸酰化酶（cGAS）/干扰素基因（STING）/干扰素I（IFN-I）途径。我们在两个肿瘤模型（B16-CD133黑色素瘤和MC38结肠癌）中使用Doxil与RT和 αPD-1 相结合，这些模型为携带两个肿瘤的小鼠，其中一个肿瘤被照射。使用抑制剂和体外敲除细胞以及小鼠对mtDNA / cGAS / STING / IFN-I通路的机制研究。在两种模型中，添加低剂量的Doxil到RT和αPD-1中能够强化RT / αPD-1引起的异位效应。三重治疗主要在接受三重治疗的小鼠中观察到非照射肿瘤的完全治愈。与RT / αPD-1和Doxil / αPD-1相比，三重疗法在非照射肿瘤中诱导更多的交叉递呈树突状细胞（DC），以及更多的肿瘤特异性CD8 + T细胞。Doxil处理和/或RT处理的肿瘤细胞与DC联合培养可以增强DC抗原交叉递呈，这对于诱导CD8 + T细胞至关重要。CD8 + T细胞耗竭或植入cGAS缺陷或STING缺陷的肿瘤细胞会消除异位效应。多柔比星诱导的/Doxil诱导的IFNβ1明显依赖于cGAS / STING通路。缺乏mtDNA的Doxorubicin处理/ Doxil处理的肿瘤细胞分泌的IFNβ1较少，相关的T细胞招募趋化因子CXCL10和ATP。与缺乏mtDNA的肿瘤细胞共培养会显著减少DC分泌的IFNβ1。植入缺乏mtDNA的肿瘤细胞，尤其是在非照射的/异位部位，会显著减弱Doxil增强的异位效应和肿瘤特异性CD8 + T细胞的浸润。这些数据表明，单剂低剂量的Doxil可以显著增强RT / αPD-1引起的异位效应，尤其是在异位肿瘤中增加了交叉递呈的DC和肿瘤特异性CD8 + T细胞。此外，它们还表明mtDNA / cGAS / STING / IFN-I通路对多柔比星的免疫原性 / 免疫调节作用很重要。我们的发现可能有助于规划（少）转移患者临床放射化疗免疫治疗试验。© 作者（或其雇主）2023年。CC BY-NC 许可下的重复使用。不得进行商业再利用。由BMJ发布。

Localized radiotherapy (RT) can cause a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade. However, this effect is still clinically rare and improvements are highly desirable. We investigated whether triple combination with a low dose of clinically approved liposomal doxorubicin (Doxil) could augment abscopal responses compared with RT/αPD-1 and Doxil/αPD-1. We also investigated whether the enhanced abscopal responses depended on the mitochondrial DNA (mtDNA)/cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING)/IFN-I pathway.We used Doxil in combination with RT and αPD-1 in two tumor models (B16-CD133 melanoma and MC38 colon carcinoma) with mice bearing two tumors, only one of which was irradiated. Mechanistic studies on the role of the mtDNA/cGAS/STING/IFN-I axis were performed using inhibitors and knockout cells in vitro as well as in mice.Addition of a single low dose of Doxil to RT and αPD-1 strongly enhanced the RT/αPD-1-induced abscopal effect in both models. Complete cures of non-irradiated tumors were mainly observed in triple-treated mice. Triple therapy induced more cross-presenting dendritic cells (DCs) and more tumor-specific CD8+ T cells than RT/αPD-1 and Doxil/αPD-1, particularly in non-irradiated tumors. Coincubation of Doxil-treated and/or RT-treated tumor cells with DCs enhanced DC antigen cross-presentation which is crucial for inducing CD8+ T cells. CD8+ T cell depletion or implantation of cGAS-deficient or STING-deficient tumor cells abolished the abscopal effect. Doxorubicin-induced/Doxil-induced IFNβ1 markedly depended on the cGAS/STING pathway. Doxorubicin-treated/Doxil-treated tumor cells depleted of mtDNA secreted less IFNβ1, of the related T cell-recruiting chemokine CXCL10, and ATP; coincubation with mtDNA-depleted tumor cells strongly reduced IFNβ1 secretion by DCs. Implantation of mtDNA-depleted tumor cells, particularly at the non-irradiated/abscopal site, substantially diminished the Doxil-enhanced abscopal effect and tumor infiltration by tumor-specific CD8+ T cells.These data show that single low-dose Doxil can substantially enhance the RT/αPD-1-induced abscopal effect, with a strong increase in cross-presenting DCs and CD8+ tumor-specific T cells particularly in abscopal tumors compared with RT/αPD-1 and Doxil/αPD-1. Moreover, they indicate that the mtDNA/cGAS/STING/IFN-I axis is important for the immunogenic/immunomodulatory doxorubicin effects. Our findings may be helpful for the planning of clinical radiochemoimmunotherapy trials in (oligo)metastatic patients.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.