Carbon ion irradiation plus CTLA4 blockade elicits therapeutic immune responses in a murine tumor model

Radiotherapy can act as in situ vaccine activating preventive tumor-specific immune responses in treated patients. While carbon ion radiotherapy holds superior biophysical properties over conventional photon irradiation, little is known about the immunological effects induced by this radiation type. Multiple strategies combining radiotherapy with immune checkpoint inhibition (radioimmunotherapy) to enhance anti-tumor immunity have been described, however, results on the immune cell composition in tumors following radioimmunotherapy with carbon ions are lacking. Here, we present a bilateral tumor model based on time-shifted transplantation of murine, Her2+ EO771 tumor cells onto the flanks of immune competent mice followed by selective irradiation of the primal tumor. aCTLA4- but not aPD-L1-based radioimmunotherapy induced complete tumor rejection and mediated eradication of even non-irradiated, distant tumors. Cured mice were protected against EO771 rechallenge indicative of long lasting, tumor-specific immunological memory. Single cell RNA-sequencing and flow cytometric analyses of irradiated tumors revealed activation of NK cells and distinct tumor-associated macrophage clusters with upregulated expression of TNF and IL1 responsive genes. Distant tumors of irradiated mice showed higher frequencies of naïve T cells, which were activated upon combination with CTLA4 blockade. Thus, radioimmunotherapy with carbon ions plus CTLA4 inhibition reshapes the tumor-infiltrating immune cell composition and can induce complete rejection even of non-irradiated tumors. Our data suggest to combine radiotherapy approaches with CTLA4 blockade to achieve durable anti-tumor immunity. Furthermore, evaluation of future radioimmunotherapy approaches should not be restricted to immunological impacts at the irradiation site, but should also consider systemic immunological effects on non-irradiated tumors. Overall design: Library preparation: Single cells suspensions of eight digested tumors per group were pooled. Afterwards, cells were incubated with LIVE/DEAD® Fixable Dead Cell Stain Kit (Thermo Fisher Scientific) diluted 1:1000 in PBS for 30 min at 4 °C and stained with PE Rat Anti-Mouse CD45 antibody (BD) in FACS buffer for 30 min at 4 °C. Sorting for viable CD45+ cells was performed with a BD FACS Aria III. The cell suspension of CD45 positive cells was adjusted to a concentration of approximately 1000 cells/µl. The cells were then loaded onto the 10x Genomics Chromium controller for droplet-enabled single cell RNA sequencing according to the manufacturer's instruction manual. Library generation was performed following the Chromium Single Cell 3' Reagents Kit version 3.1 user guide. The libraries were sequenced on the Illumina NovaSeq 6000 platform.

放射治疗（radiotherapy）可作为原位疫苗（in situ vaccine），在接受治疗的患者中激活预防性肿瘤特异性免疫应答（tumor-specific immune responses）。尽管碳离子放射治疗（carbon ion radiotherapy）相较于常规光子照射（conventional photon irradiation）具有更优异的生物物理学特性，但目前对于该放射类型诱导的免疫学效应仍知之甚少。已有多项研究将放疗与免疫检查点抑制（immune checkpoint inhibition）相结合（即放射免疫治疗（radioimmunotherapy）），以增强抗肿瘤免疫，然而关于碳离子放射免疫治疗后肿瘤内免疫细胞组成的相关研究仍存在空白。

本研究构建了双侧肿瘤模型：将小鼠Her2阳性EO771肿瘤细胞按时间差接种于免疫健全小鼠（immune competent mice）的双侧背部侧翼，随后对原发肿瘤（primal tumor）进行选择性照射。

采用抗CTLA4（aCTLA4，anti-CTLA4）而非抗PD-L1（aPD-L1，anti-PD-L1）的放射免疫治疗方案可诱导肿瘤完全消退（complete tumor rejection），甚至可介导未照射的远处肿瘤被清除。经该方案治愈的小鼠可抵御EO771肿瘤再攻击（tumor rechallenge），表明其形成了持久的肿瘤特异性免疫记忆（immunological memory）。

对照射后的肿瘤进行单细胞RNA测序（single cell RNA-sequencing）与流式细胞术分析（flow cytometric analyses），结果显示自然杀伤（NK）细胞与特定肿瘤相关巨噬细胞（tumor-associated macrophage）亚群被激活，其肿瘤坏死因子（TNF）与白细胞介素1（IL1）应答基因的表达上调。未接受照射的远处肿瘤中，初始T细胞（naïve T cells）的比例更高，而联合CTLA4阻断（CTLA4 blockade）治疗可激活这些初始T细胞。

综上，碳离子放射免疫治疗联合CTLA4阻断可重塑肿瘤浸润免疫细胞（tumor-infiltrating immune cell）组成，甚至可诱导未照射肿瘤完全消退。本研究数据表明，应将放疗方案与CTLA4阻断相结合，以实现持久的抗肿瘤免疫。此外，未来评估放射免疫治疗方案时，不应仅局限于照射部位的免疫学影响，还应考虑其对未照射肿瘤的全身性免疫学效应。

【总体实验设计】
文库制备（library preparation）：
将每组8个酶解肿瘤组织（digested tumors）的单细胞悬液（single cells suspensions）混合。随后，将细胞置于用磷酸盐缓冲液（PBS）按1:1000稀释的LIVE/DEAD® 固定型死活细胞染色试剂盒（LIVE/DEAD® Fixable Dead Cell Stain Kit，赛默飞世尔科技Thermo Fisher Scientific）中，于4℃孵育30分钟；再用流式细胞术缓冲液（FACS buffer）稀释的PE标记大鼠抗小鼠CD45抗体（PE Rat Anti-Mouse CD45 antibody，碧迪医疗BD）于4℃染色30分钟。使用BD FACS Aria III流式分选仪对活的CD45阳性细胞进行分选。

将CD45阳性细胞悬液调整至约1000个细胞/微升的浓度，随后按照制造商操作手册，将细胞上样至10x Genomics Chromium控制器（10x Genomics Chromium controller）以进行微滴式单细胞RNA测序（droplet-enabled single cell RNA sequencing）。文库构建遵循Chromium单细胞3'端试剂试剂盒v3.1（Chromium Single Cell 3' Reagents Kit version 3.1）用户指南完成。最终文库在Illumina NovaSeq 6000测序平台（Illumina NovaSeq 6000）上进行测序。