Champalimaud Foundation, Champalimaud Research, Lisbon, Portugal.
Methods Mol Biol. 2024;2811:155-164. doi: 10.1007/978-1-0716-3882-8_11.
The high prevalence of dormant disseminated tumor cells (DTCs) persisting systemically in patients with metastatic cancer is a major threat to long-lasting cure (Aguirre-Ghiso, Nat Rev Cancer 7:834-846, 2007; Klein, Nat Rev Cancer 20(11):681-694, 2020; Lyden et al. Cancer Cell 40:787-791, 2022). Despite its clinical significance, the study of what drives DTCs in and out of dormancy while they linger in distant sites has been challenged by the lack of tools to find and follow dormant DTCs inside a living organism. Here, leveraging the fact that dormant DTCs are mostly quiescent, we describe a live cell reporter to distinguish dormant from cycling DTCs (Correia, Nat Rev Cancer 22(7):379, 2022; Correia et al. Nature 594(7864):566-571, 2021). Cancer cell lines are engineered to coexpress a luciferase-tdTomato reporter and a fluorescent fusion protein of mVenus with a mutant form of the cell cycle inhibitor p27 (mVenus-p27K) that identifies quiescent cells. When implanted in animal models or assembled in cocultures in vitro, labeled cells can be imaged longitudinally over time or retrieved alive alongside their surrounding microenvironment for downstream gene, protein, and metabolite profiling, allowing the mapping of tissue-specific determinants of cancer dormancy and metastasis.
休眠性播散肿瘤细胞（DTCs）在转移性癌症患者体内系统地持续存在，其高发生率对长期治愈是一个主要威胁（Aguirre-Ghiso, Nat Rev Cancer 7:834-846, 2007; Klein, Nat Rev Cancer 20(11):681-694, 2020; Lyden et al. Cancer Cell 40:787-791, 2022）。尽管其具有临床意义，但由于缺乏在活体中寻找和跟踪休眠 DTC 的工具，研究导致 DTC 进入休眠和脱离休眠状态的因素以及它们在远处部位潜伏的机制一直受到挑战。在这里，我们利用休眠性 DTC 大多处于静止状态的事实，描述了一种活细胞报告基因，以区分休眠性和循环性 DTC（Correia, Nat Rev Cancer 22(7):379, 2022; Correia et al. Nature 594(7864):566-571, 2021）。将癌细胞系工程改造为共表达荧光素酶-tdTomato 报告基因和荧光融合蛋白 mVenus-p27K，该融合蛋白由细胞周期抑制剂 p27 的突变体组成，可识别静止细胞。当将标记的细胞植入动物模型中或在体外共培养中组装时，可以随时间对其进行纵向成像，或与周围微环境一起活体回收，用于下游基因、蛋白质和代谢物分析，从而可以绘制出癌症休眠和转移的组织特异性决定因素图谱。
