该文主要探讨端粒酶抑制剂6-硫代-2'-脱氧鸟苷(6-Thio-2'-deoxyguanosine,6-Thio-dG)是否诱导肿瘤细胞免疫原性死亡(immunogenic cell death,ICD),并揭示所产生的ICD的免疫应答特征,为利用6-Thio-dG诱导肿瘤细胞ICD开展免疫治疗提供基础.首先,用不同浓度的6-Thio-dG处理小鼠黑色素瘤B16-F10、结肠癌CT26、宫颈癌相关肿瘤TC-1和乳腺癌4T1细胞,在不同时间点用显微镜观察细胞死亡情况及形态特征;利用乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒定量分析细胞死亡情况;以检测试剂盒以及酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)分析细胞死亡过程中免疫介质释放情况;免疫荧光染色实验考察钙网蛋白(calreticulin,CALR)向细胞膜的迁移定位.进一步,建立皮下移植TC-1小鼠肿瘤模型,当肿瘤长至4～5 mm时,注射6-Thio-dG对荷瘤小鼠进行治疗性干预,通过酶联免疫斑点技术(enzyme-linked immunospot assay,EL1SPOT)与流式细胞术分析小鼠的细胞免疫应答情况;免疫组化检测肿瘤组织中高迁移率组蛋白B1(high mobil-ity group box protein B1,HMGB1)的释放情况.研究结果显示,6-Thio-dG可诱导4种肿瘤细胞死亡,死亡特征与细胞类型、药物剂量和处理时间有关;肿瘤细胞经6-Thio-dG诱导后促进了发挥免疫刺激作用的"发现我"信号分子三磷酸腺苷(adenosine triphosphate,ATP)和HMGB1、炎症细胞因子白细胞介素-1β(interleukin-1β,IL-1β)的释放,以及"吃了我"信号分子CALR在细胞膜的聚集.在肿瘤模型中,6-Thio-dG显著抑制了小鼠肿瘤生长,提高了肿瘤组织中HMGB1水平,增强了表达γ-干扰素(interferon-y,IFN-γ)的肿瘤抗原特异性的脾细胞应答,降低了脾细胞中髓源性抑制细胞(myeloid de-rived suppressor cell,MDSC)的水平.该研究揭示了 6-Thio-dG能够诱导肿瘤细胞ICD,在杀伤肿瘤细胞的同时,增强了 T细胞的抗肿瘤免疫应答能力,为肿瘤免疫治疗提供了新思路.
The Telomerase Inhibitor 6-Thio-2'-Deoxyguanosine Induced Immunogenic Death of Tumor Cells
This study focused on whether telomerase inhibitor 6-Thio-dG(6-Thio-2'-deoxyguanosine)induces ICD(immunogenic cell death)in tumor cells.The immune response characteristics of the generated ICD were revealed,which provided a basis for immunotherapy using 6-Thio-dG to induce ICD in tumor cells.Firstly,mouse melanoma B16-F10,colon cancer CT26,cervical cancer related tumor TC-1 and breast cancer 4T1 cells were treated with different concentrations of 6-Thio-dG,and the cell death and morphological characteristics were observed by microscope at different time points.The cell death was quantitatively analyzed by LDH(lactate dehy-drogenase)kits.Assay kit and ELISA(enzyme-linked immunosorbent assay)were used to analyze the release of immune mediators during cell death.The migration and localization of CALR(calreticulin)to the cell membrane were investigated by immunofluorescence staining.Further,the tumor model of subcutaneously transplanted TC-1 mice was established.When the tumor grew to 4-5 mm,6-Thio-dG was injected into the tumorbearing mice for therapeutic intervention.ELISPOT(enzyme-linked immunospot assay)and flow cytometry were used to analyze the immune response of mice cells.The release of HMGB1(high mobility group box protein B1)in tumor tissues was detected by immunohistochemistry.The results showed that 6-Thio-dG induced the death of four kinds of tu-mor cells,and the death characteristics were related to cell type,drug dose and treatment time.Tumor cells induced by 6-Thio-dG promote the release of the immune-stimulating"find me"signaling molecules ATP(adenosine tri-phosphate)and HMGB1,as well as the inflammatory cytokine IL-1β(interleukin-1β),and the aggregation of the"eat me"signaling molecule CALR in the cell membrane.In the tumor model,6-Thio-dG significantly inhibited tumor growth,increased the level of HMGB1 in tumor tissues,and enhanced tumor antigen-specific spleen cell re-sponse expressing IFN-y(interferon-γ).The levels of MDSC(myeloid derived suppressor cell)in spleen cells were decreased.This study revealed that 6-Thio-dG can induce ICD of tumor cells,and enhance the anti-tumor immune response ability of T cells while killing tumor cells,providing a new idea for tumor immunotherapy.
6-Thio-dGimmunogenic cell deathmelanomacervical cancercolon cancerbreast cancer
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