A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level
A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level
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DOI：
10.15252/embj.201592775
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发表时间：
2016-01-01
期刊：
影响因子：
11.4
通讯作者：
Kiefer, Friedemann
中科院分区：
文献类型：
--
作者：
Erapaneedi, Raghu;Belousov, Vsevolod V.;Kiefer, Friedemann
低氧是一种被广泛研究的疾病，在发育和疾病过程中对细胞的新陈代谢、迁移和血管生成有深远的影响。从生理上讲，低氧与组织的动态平衡和维持多能性有关。缺氧还会导致包括心血管疾病和癌症在内的病理变化。尽管它很重要，但缺氧的显微可视化在很大程度上仅限于通过免疫染色检测还原激活的探针。在这里，我们描述了一种新的基因编码的荧光传感器家族，它可以检测氧非依赖性荧光蛋白UnaG报告的HIF转录因子的激活。它包括具有不同开关和记忆行为的传感器，以及允许区分缺氧和复氧细胞的组合传感器。我们在原位移植的胶质瘤细胞系上测试了这些传感器。使用颅窗，我们可以在细胞分辨率下可视化体内的缺氧。在组织样本中，传感器的活性被检测到，这些区域基本上没有血管，与HIF-1α的稳定性相关，并且在细胞水平上具有高度的异质性。我们经常在血管附近的缺氧区外检测到最近复氧的细胞，这提示低氧促进了细胞迁移。
Hypoxia is an intensively investigated condition with profound effects on cell metabolism, migration, and angiogenesis during development and disease. Physiologically, hypoxia is linked to tissue homeostasis and maintenance of pluripotency. Hypoxia also contributes to pathologies including cardiovascular diseases and cancer. Despite its importance, microscopic visualization of hypoxia is largely restricted to the detection of reductively activated probes by immunostaining. Here, we describe a novel family of genetically encoded fluorescent sensors that detect the activation of HIF transcription factors reported by the oxygen-independent fluorescent protein UnaG. It comprises sensors with different switching and memory behavior and combination sensors that allow the distinction of hypoxic and reoxygenated cells. We tested these sensors on orthotopically transplanted glioma cell lines. Using a cranial window, we could visualize hypoxia intravitally at cellular resolution. In tissue samples, sensor activity was detected in regions, which were largely devoid of blood vessels, correlated with HIF-1 alpha stabilization, and were highly heterogeneous at a cellular level. Frequently, we detected recently reoxygenated cells outside hypoxic areas in the proximity of blood vessels, suggestive of hypoxia-promoted cell migration.