肿瘤缺氧是抗癌治疗效果的主要障碍。由于技术上无法随着时间的推移识别和追踪这些细胞，因此理解单个缺氧细胞在治疗前、治疗期间和治疗后的细胞动态受到了阻碍。在这里，我们提出了一种基于缺氧细胞的条件表达的新型谱系追踪报告基因HIF1a-CreERT2-UnaG 生物传感器，可以以时间依赖性方式可视化缺氧细胞，并随着时间的推移追踪缺氧细胞的命运。我们将该系统与多光子显微镜、流式细胞术和免疫荧光相结合，以表征缺氧细胞在 H1299 肿瘤球体和体内异种移植物照射后肿瘤复发中的作用。我们在单层培养物中验证了报告基因，并表明标记的细胞在球体中共定位以及具有缺氧标记哌莫硝唑的人类肿瘤异种移植物。我们发现 H1299-HIFcreUnaG 球体的照射导致细胞优先从缺氧核心生长。类似地，在异种移植肿瘤中，虽然最初 UnaG 阳性细胞与哌莫硝唑阳性肿瘤区域一致并且它们只是静止的，但在照射后 UnaG 阳性细胞在再生肿瘤中富集并且主要是增殖性的。总的来说，我们的数据提供了明确的证据，表明缺氧细胞导致辐射后肿瘤复发。版权所有 © 2024。由 Elsevier B.V. 出版。

Tumor hypoxia imposes a main obstacle to the efficacy of anti-cancer therapy. Understanding the cellular dynamics of individual hypoxic cells before, during and post-treatment has been hampered by the technical inability to identify and trace these cells over time.Here, we present a novel lineage-tracing reporter for hypoxic cells based on the conditional expression of a HIF1a-CreERT2-UnaG biosensor that can visualize hypoxic cells in a time-dependent manner and trace the fate of hypoxic cells over time. We combine this system with multiphoton microscopy, flow cytometry, and immunofluorescence to characterize the role of hypoxic cells in tumor relapse after irradiation in H1299 tumor spheroids and in vivo xenografts.We validate the reporter in monolayer cultures and we show that tagged cells colocalize in spheroids and human tumor xenografts with the hypoxic marker pimonidazole. We found that irradiation of H1299-HIFcreUnaG spheroids leads to preferential outgrowth of cells from the hypoxic core. Similarly, in xenografts tumors, although initially UnaG-positive-cells coincide with pimonidazole-positive tumor areas and they are merely quiescent, upon Irradiation UnaG-positive cells enrich in regrowing tumors and are mainly proliferative.Collectively, our data provide clear evidence that the hypoxic cells drive tumor relapse after irradiation.Copyright © 2024. Published by Elsevier B.V.