Zhang Lichen, Feng Xinyu, Shen Yingzhuo, Wang Yingbin, Liu Zhuangzhuang, Ma Yuang, Gu Yanrong, Guo Guo, Duan Liangwei, Lu Liaoxun, Liang Yinming, Lawrence Toby, Huang Rong
Laboratory of Genetic Regulators in the Immune System, Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China.
Henan Key Laboratory of Immunology and Targeted Therapy, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, China.
Immunol Cell Biol. 2022 Oct;100(9):691-704. doi: 10.1111/imcb.12575. Epub 2022 Aug 5.
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is an efficient tool for establishing genetic models including cellular models, and has facilitated unprecedented advancements in biomedical research. In both patients and cancer animal models, immune cells infiltrate the tumor microenvironment and some of them migrate to draining lymph nodes to exert antitumor effects. Among these immune cells, phagocytes such as macrophages and dendritic cells engulf tumor antigens prior to their crosstalk with T cells and elicit adaptive immune response against tumors. Melanoma cells are frequently used as a tumor model because of their relatively high level of somatic mutations and antigenicity. However, few genetic models have been developed using melanoma cell lines to track tumor cell phagocytosis, which is essential for understanding protective immune response in vivo. In this study, we used CRISPR/Cas9-mediated DNA cleavage and homologous recombination to develop a novel knock-in tool which expresses the ultra-bright fluorescent probe ZsGreen in YUMM1.7 melanoma cells. Using this novel tool, we measured the macrophagic engulfment of melanoma cells inside the tumor microenvironment. We also found that in tumor-grafted mice, a subset of dendritic cells efficiently engulfed YUMM1.7 cells and was preferentially trafficking tumor antigens to draining lymph nodes. In addition, we used this knock-in tool to assess the impact of a point mutation of CD11b on phagocytosis in the tumor microenvironment. Our results demonstrate that the ZsGreen-expressing YUMM1.7 melanoma model provides a valuable tool for the study of phagocytosis in vivo.
成簇规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）是建立包括细胞模型在内的遗传模型的有效工具，推动了生物医学研究取得前所未有的进展。在患者和癌症动物模型中，免疫细胞浸润肿瘤微环境，其中一些迁移至引流淋巴结发挥抗肿瘤作用。在这些免疫细胞中，巨噬细胞和树突状细胞等吞噬细胞在与T细胞相互作用之前吞噬肿瘤抗原，并引发针对肿瘤的适应性免疫反应。黑色素瘤细胞因其相对较高的体细胞突变水平和抗原性，常被用作肿瘤模型。然而，利用黑色素瘤细胞系开发用于追踪肿瘤细胞吞噬作用的遗传模型较少，而这对于理解体内保护性免疫反应至关重要。在本研究中，我们利用CRISPR/Cas9介导的DNA切割和同源重组开发了一种新型敲入工具，该工具可在YUMM1.7黑色素瘤细胞中表达超亮荧光探针ZsGreen。利用这一新型工具，我们测量了肿瘤微环境中巨噬细胞对黑色素瘤细胞的吞噬作用。我们还发现，在接种肿瘤的小鼠中，一部分树突状细胞能有效吞噬YUMM1.7细胞，并优先将肿瘤抗原转运至引流淋巴结。此外，我们利用这一敲入工具评估了CD11b点突变对肿瘤微环境中吞噬作用的影响。我们的结果表明，表达ZsGreen的YUMM1.7黑色素瘤模型为体内吞噬作用的研究提供了一个有价值的工具。
