Ummarino Aldo, Anfray Clément, Maeda Akihiro, Andón Fernando Torres, Allavena Paola
Department of Immunology, Humanitas Clinical and Research Center-IRCCS, Rozzano, Italy.
Humanitas University, Rozzano-Milano, Italy.
Methods Mol Biol. 2023;2614:81-91. doi: 10.1007/978-1-0716-2914-7_6.
Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the anti-tumoral efficacy of most treatments currently applied in the clinic. However, a key feature of macrophages is their phenotypical and functional plasticity, which called their attention as promising targets for therapeutic intervention based on their elimination or reprogramming toward M1-like cytotoxic effector cells, with anti-tumor functions. This polarization status of macrophages can be studied in terms of molecular markers and functional activities, using an appropriate combination of experimental methodologies, both in vitro and in vivo. Here we focus on describing in vitro protocols to isolate primary monocytes from buffy coats and to study macrophage phenotype and function, after exposure to new therapies, by a combination of flow cytometry, RT-PCR, and ELISA analysis. We also provide the methodology to evaluate in vitro the cytotoxic activity of treated macrophages toward cancer cells.
肿瘤相关巨噬细胞（TAMs）发挥着关键的免疫抑制作用，限制了免疫系统对抗癌症的能力，并阻碍了目前临床上应用的大多数治疗方法的抗肿瘤疗效。然而，巨噬细胞的一个关键特征是其表型和功能的可塑性，这使其成为基于消除或重编程为具有抗肿瘤功能的M1样细胞毒性效应细胞的治疗干预的有前景的靶点而受到关注。巨噬细胞的这种极化状态可以通过在体外和体内使用适当的实验方法组合，根据分子标志物和功能活性进行研究。在这里，我们重点描述从血沉棕黄层中分离原代单核细胞的体外方案，以及在接触新疗法后，通过流式细胞术、逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）分析相结合的方法来研究巨噬细胞的表型和功能。我们还提供了在体外评估经处理的巨噬细胞对癌细胞的细胞毒性活性的方法。
