Abstract
Hypoxia is known to be detrimental in cancer and contributes to its development. In this work, we present an approach to fate-map hypoxic cells in vivo in order to determine their cellular response to physiological O2 gradients as well as to quantify their contribution to metastatic spread. We demonstrate the ability of the system to fate-map hypoxic cells in 2D, and in 3D spheroids and organoids. We identify distinct gene expression patterns in cells that experienced intratumoral hypoxia in vivo compared to cells exposed to hypoxia in vitro. The intratumoral hypoxia gene-signature is a better prognostic indicator for distant metastasis-free survival. Post-hypoxic tumor cells have an ROS-resistant phenotype that provides a survival advantage in the bloodstream and promotes their ability to establish overt metastasis. Post-hypoxic cells retain an increase in the expression of a subset of hypoxia-inducible genes at the metastatic site, suggesting the possibility of a ‘hypoxic memory.’
Highlights
Hypoxia is known to be detrimental in cancer and contributes to its development
By mechanistically evaluating the potential of post-hypoxic tumor cells to accomplish each individual step in the metastatic cascade separately, we demonstrate that hypoxia-exposed cells have an enhanced ability to invade/intravasate and to survive in the bloodstream, but they are not more efficient at extravasating or proliferating at the metastatic site compared with their oxygenated counterparts
The findings demonstrate that (1) a portion of hypoxic cells in regions adjacent to the perinecrotic region of the tumor can overcome O2 deprivation and metastasize; (2) cells that leave the hypoxic region of the primary tumor and enter the bloodstream will be exposed to higher O2 levels; (3) green fluorescent protein (GFP)+ cells detected in the blood and lung became hypoxic in the primary tumor; (4) post-hypoxic cells have 4–5× higher chance at being found in the blood and lung
Summary
We present an approach to fate-map hypoxic cells in vivo in order to determine their cellular response to physiological O2 gradients as well as to quantify their contribution to metastatic spread. We identify distinct gene expression patterns in cells that experienced intratumoral hypoxia in vivo compared to cells exposed to hypoxia in vitro. The vast majority of studies on hypoxia-regulated gene expression have been conducted in an in vitro setting by exposing cells to 1% O2 for short periods of time. Both direct and indirect methods have previously been used to identify hypoxic tissue regions in vivo. O2 in human tumor tissue has been performed by using O2 electrodes[17] or fiber-optic probes[18], but these methods lack the ability to visualize, isolate, or separate individual hypoxic cells
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