<br>永生化骨髓源性巨噬细胞（iBMDM）的表型比较和潜在抗肿瘤功能,Frontiers in Immunology

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永生化骨髓源性巨噬细胞（iBMDM）的表型比较和潜在抗肿瘤功能
Frontiers in Immunology ( IF 5.7 ) Pub Date : 2024-04-08 , DOI: 10.3389/fimmu.2024.1379853
Dong-Kun Xie _{1,

2} , Jin Yao _{1,

2} , Peng-Hui Li _{2,

3} , Yan-Wen Zhu ₂ , Jia-Nuo Chen ₂ , Xiu-Li Cao ₂ , Shi-Lin Cheng ₂ , Ya-Miao Chen ₁ , Yi-Fei Huang ₂ , Liang Wang ₂ , Zan-Han Wang ₂ , Rong Qiao ₂ , Jia-Mei Ge ₂ , Huan Yue ₁ , Li Wei ₂ , Zhong-Yuan Liu ₂ , Hua Han _{2,

4} , Hong-Yan Qin ₂ , Jun-Long Zhao _{1,

2}

Affiliation

简介巨噬细胞是先天免疫的重要组成部分，参与多种疾病的免疫调节。功能的多样性和可塑性使得巨噬细胞在不同的刺激后表现出不同的极化表型。在肿瘤进展过程中，M2 样极化肿瘤相关巨噬细胞 (TAM) 通过协助免疫逃逸、促进肿瘤细胞转移和切换肿瘤血管生成来促进肿瘤进展。我们之前的研究表明，通过工程修饰或基因编辑对 TAM 进行功能重塑为肿瘤提供了潜在的免疫治疗。然而，输注巨噬细胞缺乏增殖能力和维持免疫记忆，限制了基于巨噬细胞的治疗策略在抑制性肿瘤免疫微环境（TIME）中的应用。尽管J2逆转录病毒感染能够实现骨髓源性巨噬细胞（iBMDMs）的永生化并促进其机制的探索和应用，但对于多种巨噬细胞之间的表型和功能差异知之甚少。采用实时定量PCR、免疫荧光染色和ELISA检测iBMDMs中的极化反应和趋化因子的表达。采用流式细胞术、划痕实验、实时定量PCR和结晶紫染色分析其吞噬功能以及对肿瘤细胞迁移、增殖和凋亡的影响。不仅如此，通过皮下肿瘤加载检测iBMDMs对肿瘤生长的抑制作用，同时对肿瘤组织进行石蜡切片并使用流式细胞术检测其对肿瘤微环境的影响。结果在本研究中，我们证明 iBMDM 具有快速增殖和长期存活的特点。我们还将 iBMDM 与 RAW264.7 细胞系以及小鼠原代 BMDM 与体外和体内实验表明，iBMDMs 可以经历与正常巨噬细胞相同的极化响应，极化后没有明显的细胞形态变化。此外，iBMDMs对肿瘤细胞具有更强的吞噬和促凋亡功能。此外，M1极化的iBMDMs可以维持抗肿瘤表型，并驯化受体小鼠募集的巨噬细胞，从而进一步提高TIME并抑制肿瘤生长。讨论iBMDMs可以作为巨噬细胞功能和机制研究的良好对象以及巨噬细胞免疫治疗的可选来源。

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Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs)

IntroductionMacrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages.MethodsHE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment.ResultsIn this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What’s more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth.DiscussioniBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

更新日期：2024-04-08

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