目的：探讨活化转录因子 6（ATF6）对骨肉瘤细胞 MCA205 免疫原性的影响，初步解析其调控机制。方法：利用CRISPR-Cas9技术敲除MCA205细胞的Atf6基因，借助CCK-8实验、细胞能量代谢检测、流式细胞术、ATP检测试剂盒、干扰素刺激响应元件（ISRE）-荧光素酶报告细胞实验和qPCR法分别检测野生型（WT）和Atf6-/- MCA205细胞在PBS或衣霉素（Tm）处理后的活力、线粒体耗氧速率（OCR）和胞外酸化速率（ECAR）、磷脂酰丝氨酸外翻和细胞膜通透性、胞内钙离子动员、胞内外ATP浓度、IFN-a/b分泌和干扰素刺激基因（ISG）的表达水平。在免疫系统健全的小鼠皮下接种WT或Atf6-/- MCA205细胞，比较两者的成瘤速度、肿瘤组织基因转录图谱、局部抗肿瘤效应T细胞活化有无差异。将WT和Atf6-/- MCA205细胞分别接种于nu/nu小鼠背部两侧皮下，或将Atf6-/- MCA205细胞分别接种于免疫系统健全小鼠和Ifnar-/-小鼠皮下，记录肿瘤生长曲线。分别用Tm预处理的WT和Atf6-/- MCA205细胞对na?ve小鼠（未经免疫刺激的小鼠）进行初次免疫，使肿瘤抗原特异性T细胞发生初次活化（prime），随后收集引流淋巴结细胞并进行体外再刺激（boost），分析特异性T细胞再次活化有无差异。按照不同的效靶比，将NK细胞与染料标记的WT和Atf6-/- MCA205细胞共培养，流式细胞术检测细胞杀伤情况。结果：PBS或Tm处理后，WT和Atf6-/-骨肉瘤细胞活力和增殖、氧化磷酸化和糖酵解、离子霉素触发的胞内钙离子动员、胞内外ATP和IFN-a/b分泌均无显著差异。Tm处理后，Atf6-/-细胞死亡比例低于WT细胞（P<0.01）。在免疫系统健全的小鼠体内，Atf6-/-肿瘤的生长速度明显低于WT肿瘤（P<0.05）。然而，在缺乏T细胞的nu/nu小鼠体内，两种肿瘤生长速度的差异显著缩小。与免疫系统健全的小鼠相比，Ifnar-/-小鼠体内Atf6-/-肿瘤的生长速度略快（P<0.05）。Atf6-/-肿瘤内免疫应答相关基因的表达、效应T细胞的活化均显著高于WT肿瘤（P<0.05）。与WT MCA205细胞相比，Atf6-/- MCA205细胞与NK细胞共培养后死亡比例更高（P<0.05）。在Tm刺激后，Atf6-/- MCA205细胞比WT细胞表达更多的Irf3和Irf7，前者在prime-boost实验中可刺激T细胞分泌更多的IFN-g。结论：阻断ATF6信号通路能显著增强MCA205细胞的免疫原性，促进免疫监视，阻碍肿瘤进展。

Objective: To investigate the impact of activating transcription factor 6 (ATF6) on the immunogenicity of osteosarcoma cells MCA205, and to preliminarily explore the underlying regulatory mechanisms. Methods: The CRISPR-Cas9 technology was utilized to knock out Atf6 gene in MCA205 cells. By using CCK-8 assays, cell energy metabolism assays, flow cytometry, ATP detection kits, interferon-sensitive response element (ISRE) -luciferase reporter cells and qPCR, we analyzed cell viability,mitochondrial oxygen consumption rate (OCR), extracellular acidification rate (ECAR), phosphatidylserine exposure and permeabilization of cell membranes, intracellular calcium mobilization, intracellular and extracellular ATP concentration, IFN-a/b secretion, and the expression of interferon-stimulated genes (ISG) in wild-type (WT) and Atf6-/- MCA205 cells after PBS or tunicamycin (Tm) treatment, respectively. WT or Atf6-/- MCA205 cells were subcutaneously inoculated in immunocompetent mice. Tumor growth kinetics, gene transcription profiles in tumor tissues and activation of local anti-tumor effector T cells of the two groups were compared.WT and Atf6-/- MCA205 cells were simultaneously inoculated on two sides of nu/nu mice. Alternatively, Atf6-/- MCA205 cells were inoculated subcutaneously in immunocompetent mice and Ifnar-/- mice. Tumor growth curves were recorded. Tm-pretreated WT and Atf6-/- MCA205 cells were used to stimulate na?ve mice (without any previous immunostimulation) and prime tumor antigen-specific T cells, respectively. Cells from draining lymph nodes were then collected and boosted in vitro. Activation of antigen-specific T cells of the two groups were compared. At different effector-target ratios, NK cells were mixed with fluorescent dye-prelabeled WT and Atf6-/-MCA205 cells. NK cell-based killing was detected by flow cytometry. Results: Upon PBS or Tm treatment, we haven’t observed any significant differences between WT and Atf6-/- tumor cells in their viability and proliferation, oxidative phosphorylation and glycolysis,ionomycin-triggered intracellular calcium mobilization, intracellular and extracellular ATP content and IFN-a/b secretion. Upon Tm treatment, the death ratio of Atf6-/- tumor cells was significantly lower than that of WT tumor cells (P<0.01). In immunocompetent mice,the growth of Atf6-/- tumors was significantly slower than that of WT tumors (P<0.05). However, their differences in growth kinetics were largely diminished in nu/nu mice. The growth of Atf6-/- tumors in Ifnar-/- mice was slightly faster than that in immunocompetent mice (P<0.05). The expression of immune response-related genes and the activation of effector T cells in Atf6-/- tumors were significantly higher than those in WT tumors (P<0.05). Compared with that of WT MCA205 cells, Atf6-/- MCA205 cells were more sensitive to NK cell-mediated cytolysis. Upon Tm preconditioning, Atf6-/- MCA205 cells expressed more Irf3 and Irf7 and can stimulate more IFN-g production from T cells in the prime-boost setting than WT cells. Conclusion: Blocking ATF6 signaling pathway can significantly enhance the immunogenicity of MCA205 cells, promote immune surveillance and delay tumor progression.

国家自然科学基金面上项目资助（No. 81972701）；科技部科技创新2030重大项目资助（No. 2022ZD0205700）；中国医学科学院医学 与健康科技创新工程资助项目（No. 2021-I2M-1-074、2022-I2M-2-004）