Wang Weinan, Chen Sufang, Duan Yubei, Jing Yuying, Huang Tianping, Chen Kai, Yang Kaige, Luo Chenghua, Zhou Wenhu, Hu Jianming
Department of Pathology, School of Medicine, Shihezi University/First Affiliated Hospital of Shihezi University, Shihezi 832002, China.
Department of Pharmacy, Xiangya College of Pharmacy, Central South University, Changsha 410013, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2024 Oct;40(10):887-893.
Objective To explore the impact of M2 macrophages on the malignant biological behavior of esophageal cancer by inhibiting the anti-tumor ability of CD8 T cells. Methods Using phorbol myristate acetate (PMA) combined with interleukin 4 (IL-4)/IL-13, we induced human monocytic leukemia cells (THP-1) to become M2 macrophages and the detected related inflammatory factors by real-time quantitative PCR. We used magnetic bead sorting to isolate CD8 T cells from healthy volunteers' peripheral blood, and verified the purity of the sorted cells by flow cytometry. We established a non-contact co-culture system between CD8 T cells and esophageal squamous carcinoma cells (CD8 T cell), and established a non-contact co-culture system between M2 macrophages, CD8 T cells, and esophageal squamous carcinoma cells (M2 macrophages combined with CD8 T cell). The plate clone formation assay and CCK-8 cell toxicity assay were used to detect the proliferation ability of tumor cells in each group. The Transwell assay was used to detect the invasive and migratory abilities of tumor cells in each group. Flow cytometry was used to detect and analyze the apoptosis of tumor cells in each group. GraphPadPrism9.5 software was used for statistical analysis and graphing. Results After induction, the expression of IL-10 and arginase 1 (Arg1) in macrophages was upregulated, while the expression of IL-12 and tumor necrosis factor-alpha (TNF-α) was downregulated, showing the characteristics of M2 macrophages. Peripheral blood CD8 T cells were successfully selected by magnetic bead sorting, with a positive rate of over 90%. The proliferation, invasion, migration and anti-apoptosis ability of esophageal squamous carcinoma cells co-cultured with CD8 T cells in the non-contact manner were significantly lower than those of the single cancer cell group; while the proliferation, invasion, migration and the anti-apoptosis ability of esophageal squamous carcinoma cells co-cultured with CD8 T cells pretreated with M2 macrophage conditioned medium were significantly enhanced compared with those of esophageal squamous carcinoma and CD8 T cells co-cultured group. Conclusion M2 macrophages promote the proliferation, invasion, migration, and anti-apoptosis of esophageal cancer cells by inhibiting the anti-tumor ability of CD8 T cells.
目的 通过抑制CD8 T细胞的抗肿瘤能力，探讨M2巨噬细胞对食管癌恶性生物学行为的影响。方法 采用佛波酯（PMA）联合白细胞介素4（IL-4）/IL-13诱导人单核细胞白血病细胞（THP-1）成为M2巨噬细胞，并通过实时定量PCR检测相关炎性因子。利用磁珠分选法从健康志愿者外周血中分离CD8 T细胞，并通过流式细胞术验证分选细胞的纯度。建立CD8 T细胞与食管鳞癌细胞的非接触共培养体系（CD8 T细胞组），以及M2巨噬细胞、CD8 T细胞与食管鳞癌细胞的非接触共培养体系（M2巨噬细胞联合CD8 T细胞组）。采用平板克隆形成实验和CCK-8细胞毒性实验检测各组肿瘤细胞的增殖能力。采用Transwell实验检测各组肿瘤细胞的侵袭和迁移能力。采用流式细胞术检测和分析各组肿瘤细胞的凋亡情况。运用GraphPadPrism9.5软件进行统计分析和绘图。结果 诱导后，巨噬细胞中IL-10和精氨酸酶1（Arg1）的表达上调，而IL-12和肿瘤坏死因子-α（TNF-α）的表达下调，呈现M2巨噬细胞的特征。通过磁珠分选成功筛选出外周血CD8 T细胞，阳性率超过90%。非接触方式下与CD8 T细胞共培养的食管鳞癌细胞的增殖、侵袭、迁移及抗凋亡能力明显低于单癌细胞组；而与经M2巨噬细胞条件培养基预处理的CD8 T细胞共培养的食管鳞癌细胞的增殖、侵袭、迁移及抗凋亡能力较食管鳞癌与CD8 T细胞共培养组明显增强。结论 M2巨噬细胞通过抑制CD8 T细胞的抗肿瘤能力促进食管癌细胞的增殖、侵袭、迁移及抗凋亡。
