摘要
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/β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/β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
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/βsecretion,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 Atf6tumor cells in their viability and proliferation,oxidative phosphorylation and glycolysis,ionomycin-triggered intrace
作者
黄恩厚
李莫寒
李培培
夏琳
马瑜婷
HUANG Enhou;LI Mohan;LI Peipei;XIA Lin;MA Yuting(Collaborative Innovation Center for Cancer Personalized Medicine,Nanjing Medical University,Nanjing 211166,Jiangsu,China;Institute of Systems Medicine,Chinese Academy of Medical Sciences,Beijing 100005,China;Suzhou Institute of Systems Medicine,Suzhou 215123,Jiangsu,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2022年第8期714-723,共10页
Chinese Journal of Cancer Biotherapy
基金
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)。
