A novel ZsGreen knock-in melanoma cell line reveals the function of CD11b in tumor phagocytosis
A novel ZsGreen knock-in melanoma cell line reveals the function of CD11b in tumor phagocytosis
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新型 ZsGreen 敲入黑色素瘤细胞系揭示 CD11b 在肿瘤吞噬作用中的功能
DOI：
10.1111/imcb.12575
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发表时间：
2022
影响因子：
4
通讯作者：
Rong Huang
中科院分区：
文献类型：
--
作者：
Lichen Zhang;Xinyu Feng;Yingzhuo Shen;Yingbin Wang;Zhuangzhuang Liu;Yuang Ma;Yanrong Gu;Guo Guo;Liangwei Duan;Liaoxun Lu;Yinming Liang;Toby Lawrence;Rong Huang
聚集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，促进了生物医学研究的前所未有的进步。在患者和肿瘤动物模型中，免疫细胞浸润肿瘤微环境，部分细胞迁移到引流淋巴结发挥抗肿瘤作用。在这些免疫细胞中，吞噬细胞如巨噬细胞和树突状细胞在与T细胞串音之前吞噬肿瘤抗原，并引发针对肿瘤的适应性免疫反应。黑色素瘤细胞经常被用作肿瘤模型，因为它们的体细胞突变和抗原性相对较高。然而，很少有利用黑色素瘤细胞系来追踪肿瘤细胞吞噬的遗传模型，这对于了解体内保护性免疫反应至关重要。在这项研究中，我们利用CRISPR/Cas9介导的DNA切割和同源重组，开发了一种新的敲入工具，在YUMM1.7黑色素瘤细胞中表达超亮荧光探针ZsGreen。使用这种新工具，我们测量了肿瘤微环境中黑色素瘤细胞的巨噬细胞吞噬。我们还发现，在肿瘤移植小鼠中，树突状细胞亚群有效地吞噬YUMM1.7细胞，并优先将肿瘤抗原运送到引流淋巴结。此外，我们使用这种敲入工具来评估CD11b点突变对肿瘤微环境中吞噬的影响。我们的研究结果表明，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。
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 responsein 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 phagocytosisin vivo.