Background and goal: Carbon ion beam is radio-biologically more efficient than photons and is beneficial for treating radio-resistant tumors. Several animal experiments with tumor-bearing suggest that carbon ion beam irradiation in combination with immunotherapy yields better results, especially in controlling distant metastases. This implies that carbon ion induces a different anti-tumor immune response than photon beam. More complex molecular mechanisms need to be uncovered. This in vivo and in vitro experiment was carried out in order to examine the radio-immune effects and the mechanism of action of carbon ion beam versus X-ray in combination with PD-1 inhibitors./nMethods and Materials: Lewis lung adenocarcinoma cells and C57BL/6 mice were used to create a tumor-bearing mouse model, with the non-irradiated tumor growing on the right hind leg and the irradiated tumor on the left rear. 10Gy carbon ion beam or X-ray radiation, either alone or in combination with PD-1 inhibitor, were used to treat the left back tumor. The expression of molecules linked to immunogenicity and the infiltration of CD8+ T lymphocytes into tumor tissues were both identified using immunohistochemistry. IFN-β in mouse serum was measured using an ELISA, while CD8+ T cells in mouse peripheral blood were measured using flow cytometry. Lewis cells were exposed to different dose of X-ray and carbon ion. TREX1, PD-L1, and IFN-β alterations in mRNA and protein levels were identified using Western blot or RT-PCR, respectively. TREX1 knockdown was created by siRNA transfection and exposed to various radiations. Using the CCK8 test, EdU assay, and flow cytometry, changes in cell viability, proliferation, and apoptosis rate were discovered./nResults: Bilateral tumors were significantly inhibited by the use of carbon ion or X-ray in combination with PD-1, particularly to non-irradiated tumor(p<0.05). The percentage of infiltrating CD8+ T cells and the level of IFN-β expression were both raised by 10Gy carbon ion irradiation in the irradiated side tumor, although PD-L1 and TREX1 expression levels were also elevated. Lewis cell in vitro experiment further demonstrated that both X-ray and carbon ion irradiation can up-regulate the expression levels of PD-L1 and TREX1 with dose-dependent in tumors, particularly the trend of up-regulation TREX1 is more apparent at a higher dose in carbon ion, i.e. 8 or 10Gy, while the level of IFN-β is decreased. IFN-β levels were considerably raised under hypofractionated doses of carbon ion radiation by gene silencing TREX1./nConclusions: By enhancing tumor immunogenicity and increasing CD8+T infiltration in TME through a threshold dosage, X-ray or carbon ion radiation and PD-1 inhibitors improve anti-tumor activity and cause abscopal effect in Lewis lung adenocarcinoma-bearing mice. TREX1 is a possible therapeutic target and prognostic marker.

中文翻译：

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碳离子照射联合PD-1抑制剂通过阈剂量触发Lewis肺癌的远隔效应

背景和目标：碳离子束在放射生物学上比光子更有效，有利于治疗放射抗性肿瘤。几项荷瘤动物实验表明，碳离子束照射与免疫疗法相结合可产生更好的效果，特别是在控制远处转移方面。这意味着碳离子诱导与光子束不同的抗肿瘤免疫反应。需要揭示更复杂的分子机制。进行体内和体外实验的目的是检查碳离子束与 X 射线联合 PD-1 抑制剂的放射免疫效应和作用机制。/n方法和材料： Lewis 肺腺癌细胞采用C57BL/6小鼠制作荷瘤小鼠模型，未照射肿瘤生长在右后腿，照射肿瘤生长在左后腿。单独使用10Gy碳离子束或X射线辐射或与PD-1抑制剂联合治疗左背部肿瘤。与免疫原性相关的分子表达以及 CD8+ T 淋巴细胞向肿瘤组织的浸润均通过免疫组织化学进行鉴定。使用 ELISA 测定小鼠血清中的 IFN-β，使用流式细胞术测定小鼠外周血中的 CD8+ T 细胞。Lewis细胞暴露于不同剂量的X射线和碳离子。分别使用蛋白质印迹或 RT-PCR 鉴定了 TREX1、PD-L1 和 IFN-β mRNA 和蛋白质水平的变化。TREX1 敲低是通过 siRNA 转染并暴露于各种辐射而产生的。使用 CCK8 测试、EdU 测定和流式细胞术，发现细胞活力、增殖和凋亡率的变化。/n结果：使用碳离子或 X 射线联合 PD-1 显着抑制双侧肿瘤，特别是对于未照射的肿瘤（p<0.05）。10Gy碳离子照射使受照射侧肿瘤的浸润CD8+T细胞百分比和IFN-β表达水平均升高，但PD-L1和TREX1表达水平也升高。Lewis细胞体外实验进一步证明，X射线和碳离子照射均可上调肿瘤中PD-L1和TREX1的表达水平，且呈剂量依赖性，特别是在较高剂量下上调TREX1的趋势更为明显。碳离子剂量，即8或10Gy，同时IFN-β水平降低。通过基因沉默 TREX1，在大分割剂量的碳离子辐射下，IFN-β 水平显着升高。/n结论：通过阈值剂量、X 射线或碳离子辐射和 PD- 增强肿瘤免疫原性并增加 TME 中的 CD8+T 浸润。 1 抑制剂可提高 Lewis 肺腺癌小鼠的抗肿瘤活性并引起远隔效应。TREX1 是一个可能的治疗靶点和预后标志物。