【作者】 刘凯；

【导师】 王建宁；

【作者基本信息】 山东大学 ， 外科学（泌尿外科）（专业学位）， 2022， 硕士

【摘要】 目的免疫治疗由于其副作用小、靶向精准,被认为是肾脏肿瘤治疗的新思路,例如酪氨酸激酶抑制剂联合免疫抑制药物和抗PD-L1抗体在临床被广泛使用1,其机制主要是通过重新激活被抑制的CD8+T细胞的功能,从而使CD8+T细胞达到杀伤肿瘤细胞的作用。然而并不是所有的肾肿瘤患者都对该疗法都有良好的应答,CD8+T细胞功能的衰竭是免疫治疗耐药的主要机制2,因此迫切需要找到一种在肾肿瘤状态下CD8+T细胞功能衰竭的新机制。铁死亡作为一种新的细胞死亡方式3,涉及细胞代谢的多个方面。目前关于CD8+T细胞的铁死亡状态的研究仅限于对黑色素瘤和骨肉瘤原位浸润CD8+T细胞的研究,对肾癌的CD8+T细胞铁死亡状态鲜有研究4,因此研究在肾肿瘤态下铁死亡在CD8+T细胞发生衰竭中的作用具有重要临床意义。同时,由于CD8+T细胞在浸润肿瘤组织前需要在胸腺和脾脏成熟,因此,探究肿瘤状态下CD8+T细胞在成熟过程中是否已经发生了耗竭,以及该状态下的铁死亡也具有重要意义。方法收集肾透明细胞癌患者和健康对照的血清样本,利用ICP-MS检测两组样本铁含量;收集肾透明细胞癌患者手术样本,利用单细胞转录组测序技术检测肿瘤组织和癌旁组织中的CD8+T细胞铁死亡相关基因转录水平的差异;利用免疫荧光检测肿瘤组织及其癌旁组织中CD8+T细胞的脂质过氧化产物4HNE含量。构建小鼠肾细胞癌模型,收集血液、肿瘤组织、胸腺和脾脏样本,首先利用免疫荧光检测荷瘤小鼠的癌旁组织和肿瘤组织中CD8+T细胞的4HNE含量;进一步探究CD8+T细胞成熟过程中铁死亡状态的改变:利用流式细胞术、免疫荧光、分光光度法检测脾脏、胸腺中CD8+T细胞的脂质过氧化产物的变化情况。为了探究铁代谢紊乱在CD8+T细胞发生铁死亡中的作用以及涉及的通路,首先利用分光光度法、普鲁士蓝染色检测血清、脾脏铁含量的改变;利用Western Blot、qPCR、免疫荧光检测肾细胞癌荷瘤小鼠与正常小鼠脾脏中CD8+T细胞的铁死亡相关基因尤其是与铁代谢相关基因的改变。最后,利用流式细胞术比较肾细胞癌荷瘤小鼠与正常小鼠外周血、脾脏、胸腺中CD8+T细胞凋亡-坏死指标的变化,以排除凋亡的影响,进一步确定铁死亡对荷瘤状态下CD8+T细胞成熟过程的影响。结果肾透明细胞癌患者的血清铁水平较健康组呈下降趋势;单细胞测序结果显示肿瘤组织CD8+T细胞促进铁死亡的基因转录增加、抑制铁死亡的基因转录降低,尤其是与铁代谢相关基因变化显著;与单细胞测序对应的是,肿瘤组织中CD8+T细胞脂质过氧化产物4HNE的水平较癌旁组织中更高。与临床样本结果一致的是,肾细胞癌荷瘤小鼠肿瘤原位CD8+T细胞脂质过氧化产物4HNE的水平较癌旁组织中更高。荷瘤小鼠的CD8+T细胞铁死亡水平在成熟过程中也升高:肾细胞癌荷瘤小鼠脾脏中CD8+T细胞脂质过氧化产物4HNE、MDA、BODIPY-C11的水平较正常小鼠的更高;虽然CD8+T细胞的BODIPY-C11水平较正常小鼠没有明显差别,但是肾细胞癌荷瘤小鼠较正常小鼠胸腺明显萎缩。与患者出现铁代谢紊乱的结果一致的是,肾细胞癌荷瘤小鼠血清铁降低,脾脏组织铁含量和铁沉积增加,提示荷瘤小鼠CD8+T细胞铁死亡的发生与铁代谢紊乱相关。与铁代谢结果一致的是,肿瘤原位、脾脏CD8+T细胞铁蛋白轻链表达水平较正常小鼠降低。此外,CD8+T细胞的凋亡-坏死水平在肾细胞癌荷瘤小鼠和正常小鼠中没有明显差别,进一步证明铁死亡是CD8+T细胞衰竭的主要机制。结论我们的研究揭示了肾透明细胞癌中CD8+T细胞衰竭与细胞凋亡无关。肾癌肿瘤原位CD8+T细胞发生了铁死亡;而且,CD8+T细胞在向肿瘤组织浸润前的脾脏成熟阶段和在外周血中的循环阶段已经发生了衰竭,铁死亡是主要机制。荷瘤小鼠整个机体的铁代谢发生了显著改变,铁向CD8+T细胞成熟的重要器官脾脏聚积。铁代谢的障碍尤其是铁蛋白的降低是CD8+T细胞发生铁死亡的重要机制。更多还原

【Abstract】 PurposeDue to the small side effect and targeted precision,immune therapy is regarded as a new treatment for renal tumors,such as tyrosine kinase inhibitor combined with immunosuppressive drugs and PD-L1 antibodies.The mechanism of immune therapy is mainly through the reactivation of CD8+T cells,which functions as a tumor killer.However,not all patients with renal tumor respond well to this therapy.Depletion of CD8+T cell function is the main mechanism of immunotherapy resistance.Therefore,it is urgent to find a new mechanism of CD8+T cell failure in renal tumor state.As a new way of cell death,ferroptosis involves various aspects of cell metabolism.Studies on the ferroptosis of CD8+T cells focused only on the in situ of melanoma and osteosarcoma.However,there are few studies on the state of ferroptosis of CD8+T cells in renal tumor.Therefore,it is of great clinical significance to study the role of ferroptosis in the failure of CD8+T cells in renal tumor state.At the same time,since CD8+T cells need to mature in thymus and spleen before infiltrating tumor tissue,it is also of great significance to explore whether depletion of CD8+T cells in tumor state has occurred during maturation and the ferroptosis in this state.MethodsSerum samples from patients with renal clear cell carcinoma and healthy controls were collected for the detection of the iron content of the two groups by ICP-MS.The tumor and paracancer tissue samples of patients with renal clear cell carcinoma were collected during operations.Further,the differences of ferroptosis-related genes of CD8+T cells in tumor and paracancer tissues were detected by single-cell transcriptome sequencing in the transcription levels.The 4HNE content of CD8+T cells in tumor and adjacent tissues was detected by immunofluorescence.Mouse renal cell carcinoma model was established for the collection of blood,tumor tissue,thymus and spleen samples,and the 4HNE content of CD8+T cells in paracancer tissues and tumor tissues of tumor bearing mice was detected by immunofluorescence.The changes of ferroptosis state during CD8+T cell maturation were further investigated.The changes of lipid peroxidation products of CD8+T cells in spleen and thymus were detected by flow cytometry,immunofluorescence and spectrophotometry.In order to explore the role of iron metabolism disorder in ferroptosis of CD8+T cells,spectrophotometry was used to detect iron content change in serum and Prussian blue staining were used to reflect the iron deposition state in spleen.Western Blot,qPCR and immunofluorescence were used to detect the changes of genes related to ferroptosis,especially iron metabolism,of CD8+T cells in spleen of tumor-bearing mice and normal mice.Finally,flow cytometry was used to compare the changes of apoptosis-necrosis indexes of CD8+T cells in peripheral blood,spleen and thymus of tumor-bearing mice and normal mice,so as to exclude the influence of apoptosis and further determine the effect of ferroptosis on the maturation of CD8+T cells in tumor bearing state.ResultsThe serum iron level decreased in patients with renal clear cell carcinoma compared with healthy controls.Single-cell sequencing results showed that the transcription of genes promoting ferroptosis increased in CD8+T cells from tumor tissues,especially those related to iron metabolism.Corresponding to single-cell sequencing,levels of 4HNE in CD8+T cells were higher in tumor tissues than in paracancer tissues.Consistent with the clinical sample results,the level of the lipid peroxidation product 4HNE was higher in the tumor in-situ CD8+T cells in the tumor bearing mice than in the adjacent tissues.The level of ferroptosis of CD8+T cells in tumor-bearing mice also increased during maturation:the level of 4HNE,MDA and Bodipy-C11 of CD8+T cells in spleen of tumor-bearing mice was higher than those of normal mice.Although the thymus of tumor-bearing mice shrank,there was no significant difference in the level of Bodipy-C11 in CD8+T cells between the two groups.Consistent with the results of iron metabolism disruption in ccRCC patients,the serum iron content of RCC mice was low,and the iron content and iron deposition in spleen tissues were increased,suggesting that the iron metabolism disorder of RCC mice may be involved in the occurrence of CD8+T cell ferroptosis.Consistent with the results of iron metabolism,ferritin light chain level decreased in tumor situ and spleen CD8+T cells compared with normal mice.In addition,there was no significant difference in the apoptosis-necrosis level of CD8+T cells between RCC and normal mice,further confirming that ferroptosis was the main mechanism of CD8+T cell failure.ConclusionOur study revealed that CD8+T cell failure was not associated with apoptosis in renal clear cell carcinoma.Ferroptosis occurred in CD8+T cells in situ in renal carcinoma;Moreover,ferroptosis occurred in CD8+T cells during maturation and in peripheral blood circulation before infiltration into tumor tissue.In tumor-bearing mice,iron metabolism was significantly changed,and iron accumulated in spleen,an important organ of CD8+T cell maturation.The disturbance of iron metabolism,especially the decrease of ferritin,was an important mechanism of iron death in CD8+T cells.更多还原