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Abstract：
OBJECTIVE: Immunotherapy, specifically immune checkpoint inhibitors (ICIs), has revolutionized cancer treatment. However, it can also cause immune-related adverse events (irAEs). This study aimed to develop a clinically practical animal model of irAEs using BALB/c mice.
METHODS: Subcutaneous tumors of mouse breast cancer 4T1 cells were generated in inbred BALB/c mice. The mice were treated with programmed death-1 (PD-1) and cytotoxic t-lymphocyte antigen 4 (CTLA-4) inhibitors once every 3 days for five consecutive administration cycles. Changes in tumor volume and body weight were recorded. Lung computed tomography (CT) scans were conducted. The liver, lungs, heart, and colon tissues of the mice were stained with hematoxylin-eosin (H&E) staining to observe inflammatory infiltration and were scored. Serum samples were collected, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ferritin, glutamic-pyruvic transaminase (ALT), tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and interleukin-6 (IL-6). Mouse liver and lung cell suspensions were prepared, and changes in macrophages, T cells, myeloid-derived suppressor cells (MDSCs), and regulatory (Treg) cells were detected by flow cytometry.
RESULTS: Mice treated with PD-1 and CTLA-4 inhibitors showed significant reductions in tumor volume and body weight. The tissue inflammatory scores in the experimental group were significantly higher than those in the control group. Lung CT scans of mice in the experimental group showed obvious inflammatory spots. Serum levels of ferritin, IL-6, TNF-α, IFN-γ, and ALT were significantly elevated in the experimental group. Flow cytometry analysis revealed a substantial increase in CD3+T cells, Treg cells, and macrophages in the liver and lung tissues of mice in the experimental group compared with the control group, and the change trend of MDSCs was opposite.
CONCLUSIONS: The irAE-related animal model was successfully established in BALB/c mice using a combination of PD-1 and CTLA-4 inhibitors through multiple administrations with clinical translational value and practical. This model offers valuable insights into irAE mechanisms for further investigation.
METHODS: Subcutaneous tumors of mouse breast cancer 4T1 cells were generated in inbred BALB/c mice. The mice were treated with programmed death-1 (PD-1) and cytotoxic t-lymphocyte antigen 4 (CTLA-4) inhibitors once every 3 days for five consecutive administration cycles. Changes in tumor volume and body weight were recorded. Lung computed tomography (CT) scans were conducted. The liver, lungs, heart, and colon tissues of the mice were stained with hematoxylin-eosin (H&E) staining to observe inflammatory infiltration and were scored. Serum samples were collected, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ferritin, glutamic-pyruvic transaminase (ALT), tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and interleukin-6 (IL-6). Mouse liver and lung cell suspensions were prepared, and changes in macrophages, T cells, myeloid-derived suppressor cells (MDSCs), and regulatory (Treg) cells were detected by flow cytometry.
RESULTS: Mice treated with PD-1 and CTLA-4 inhibitors showed significant reductions in tumor volume and body weight. The tissue inflammatory scores in the experimental group were significantly higher than those in the control group. Lung CT scans of mice in the experimental group showed obvious inflammatory spots. Serum levels of ferritin, IL-6, TNF-α, IFN-γ, and ALT were significantly elevated in the experimental group. Flow cytometry analysis revealed a substantial increase in CD3+T cells, Treg cells, and macrophages in the liver and lung tissues of mice in the experimental group compared with the control group, and the change trend of MDSCs was opposite.
CONCLUSIONS: The irAE-related animal model was successfully established in BALB/c mice using a combination of PD-1 and CTLA-4 inhibitors through multiple administrations with clinical translational value and practical. This model offers valuable insights into irAE mechanisms for further investigation.
摘要：
目的：免疫治疗，特别是免疫检查点抑制剂（ICIs），彻底改变了癌症治疗。然而,它也可以引起免疫相关的不良事件（irAEs）。本研究旨在使用BALB/c小鼠开发临床实用的irAE动物模型。
方法:在近交BALB/c小鼠中产生小鼠乳腺癌4T1细胞的皮下肿瘤。每3天一次用程序性死亡-1(PD-1)和细胞毒性t淋巴细胞抗原4(CTLA-4)抑制剂处理小鼠,连续5个给药周期。记录肿瘤体积和体重的变化。进行肺部计算机断层扫描（CT）扫描。肝脏，肺,心,小鼠结肠组织用苏木精-伊红(H&E)染色观察炎性浸润并评分。收集血清样本，酶联免疫吸附试验(ELISA)检测铁蛋白水平,谷丙转氨酶（ALT），肿瘤坏死因子-α(TNF-α),干扰素-γ（IFN-γ），和白细胞介素-6（IL-6）。制备小鼠肝脏和肺细胞悬液，和巨噬细胞的变化，T细胞,骨髓来源的抑制细胞（MDSCs），通过流式细胞术检测调节性(Treg)细胞。
结果:用PD-1和CTLA-4抑制剂处理的小鼠显示肿瘤体积和体重显著减少。实验组的组织炎症评分明显高于对照组。实验组小鼠肺部CT扫描显示明显的炎性斑点。血清铁蛋白水平,IL-6,TNF-α,IFN-γ，实验组ALT明显升高。流式细胞术分析显示CD3+T细胞大幅增加，Treg细胞,与对照组相比，实验组小鼠肝脏和肺组织中的巨噬细胞，MDSCs的变化趋势相反。
结论：PD-1和CTLA-4抑制剂联合应用于BALB/c小鼠多次给药成功建立了irAE相关动物模型，具有临床转化价值和实用性。该模型为进一步研究irAE机制提供了有价值的见解。
方法:在近交BALB/c小鼠中产生小鼠乳腺癌4T1细胞的皮下肿瘤。每3天一次用程序性死亡-1(PD-1)和细胞毒性t淋巴细胞抗原4(CTLA-4)抑制剂处理小鼠,连续5个给药周期。记录肿瘤体积和体重的变化。进行肺部计算机断层扫描（CT）扫描。肝脏，肺,心,小鼠结肠组织用苏木精-伊红(H&E)染色观察炎性浸润并评分。收集血清样本，酶联免疫吸附试验(ELISA)检测铁蛋白水平,谷丙转氨酶（ALT），肿瘤坏死因子-α(TNF-α),干扰素-γ（IFN-γ），和白细胞介素-6（IL-6）。制备小鼠肝脏和肺细胞悬液，和巨噬细胞的变化，T细胞,骨髓来源的抑制细胞（MDSCs），通过流式细胞术检测调节性(Treg)细胞。
结果:用PD-1和CTLA-4抑制剂处理的小鼠显示肿瘤体积和体重显著减少。实验组的组织炎症评分明显高于对照组。实验组小鼠肺部CT扫描显示明显的炎性斑点。血清铁蛋白水平,IL-6,TNF-α,IFN-γ，实验组ALT明显升高。流式细胞术分析显示CD3+T细胞大幅增加，Treg细胞,与对照组相比，实验组小鼠肝脏和肺组织中的巨噬细胞，MDSCs的变化趋势相反。
结论：PD-1和CTLA-4抑制剂联合应用于BALB/c小鼠多次给药成功建立了irAE相关动物模型，具有临床转化价值和实用性。该模型为进一步研究irAE机制提供了有价值的见解。
