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.’

The rapid proliferation of cancer cells combined with deficient vessels cause regions of nutrient and O₂ deprivation in solid tumors. Some cancer cells can adapt to these extreme hypoxic conditions and persist to promote cancer progression. Intratumoral hypoxia has been consistently associated with a worse patient prognosis. In vitro, 3D models of spheroids or organoids can recapitulate spontaneous O₂ gradients in solid tumors. Likewise, in vivo murine models of cancer reproduce the physiological levels of hypoxia that have been measured in human tumors. Given the potential clinical importance of hypoxia in cancer progression, there is an increasing need to design methods to measure O₂ concentrations. O₂ levels can be directly measured with needle-type probes, both optical and electrochemical. Alternatively, indirect, noninvasive approaches have been optimized, and include immunolabeling endogenous or exogenous markers. Fluorescent, phosphorescent, and luminescent reporters have also been employed experimentally to provide dynamic measurements of O₂ in live cells or tumors. In medical imaging, modalities such as MRI and PET are often the method of choice. This review provides a comparative overview of the main methods utilized to detect hypoxia in cell culture and preclinical models of cancer.

Significance: Hypoxia (reduced oxygen availability) is a common finding in the tumor microenvironment and plays a critical role in stimulating the metastasis of breast cancer cells from the primary tumor to distant organs, which is closely related to patient mortality. Critical transcriptional responses to reduced O₂availability are mediated by hypoxia-inducible factors (HIFs). In this study, we demonstrate that hypoxia induces HIF-dependent expression of a disintegrin and metalloproteinase 12 (ADAM12), which clips off the extracellular domain of the membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF). The liberated extracellular domain of HB-EGF binds to the epidermal growth factor receptor, triggering a signal transduction pathway that endows breast cancer cells with increased capability for cell migration and invasion, leading to distant metastasis.

Hypoxia occurs in 90% of solid tumors and is associated with metastasis and mortality. Breast cancer cells that experience intratumoral hypoxia are 5x more likely to develop lung metastasis in animal models. Using spatial transcriptomics, we determine that hypoxic cells localized in more oxygenated tumor regions (termed ‘post-hypoxic’) retain expression of hypoxia-inducible and NF-kB-regulated genes, even in the oxygen-rich bloodstream. This cellular response is reproduced in vitro under chronic hypoxic conditions followed by reoxygenation. A subset of genes remains increased in reoxygenated cells. MUC1/MUC1-C is upregulated by both HIF-1α and NF-kB-p65 during chronic hypoxia. Abrogating MUC1 decreases the expression of superoxide dismutase enzymes, causing reactive oxygen species (ROS) production and cell death. A hypoxia-dependent genetic deletion of MUC1, or MUC1-C inhibition by GO-203, increases ROS levels in circulating tumor cells (CTCs), reducing the extent of metastasis. High MUC1 expression in tumor biopsies is associated with recurrence, and MUC1+ CTCs have lower ROS levels than MUC1- CTCs in patient-derived xenograft models. This study demonstrates that therapeutically targeting MUC1-C reduces hypoxia-driven metastasis.

The extracellular matrix (ECM) is often unaccounted for in studies that consider the stromal contribution to cancer cell signaling and response to treatment. To investigate the influence of a fibrotic microenvironment, we use fibroblast-derived ECM scaffolds as a cell culture platform. We uncover that estrogen receptor–positive (ER+) breast cancer cells cultured within ECM-scaffolds have an increase in ER signaling that occurs via an MAPK-dependent, but estrogen-independent manner. The ECM acts as a reservoir by binding, enriching, and presenting growth factors to adjacent epithelial cells. We identified FGF2 as a specific ECM-bound factor that drives ER signaling. ER+ cells cultured on ECM matrices have reduced sensitivity to ER-targeted therapies. The sensitivity to ER-targeted therapy can be restored by inhibiting FGF2–FGFR1 binding. ECM–FGF2 complexes promote Cyclin D1 induction that prevents G1 arrest even in the presence of antiestrogens. This work demonstrates that the ECM can drive ER signaling and resistance to endocrine therapy, and suggests that patients with ER+ breast cancer that have high mammographic breast density may benefit from existing FGFR-targeted therapies.Implications: This work uncovers how the ECM may mediate signaling between growth factors and ER+ breast cancer cells to promote estrogen-independent ER signaling and resistance to endocrine therapy.

Hypoxia occurs in 90% of solid tumors and is associated with treatment failure, relapse, and mortality. HIF-1α signaling promotes resistance to chemotherapy in cancer cell lines and murine models via multiple mechanisms including the enrichment of breast cancer stem cells (BCSCs). In this work, we utilize a hypoxia fate-mapping system to determine whether triple-negative breast cancer (TNBC) cells that experience hypoxia in the primary tumor are resistant to chemotherapy at sites of metastasis. Using two orthotopic mouse models of TNBC, we demonstrate that cells that experience intratumoral hypoxia and metastasize to the lung and liver have decreased sensitivity to doxorubicin and paclitaxel but not cisplatin or 5-FU. Resistance to therapy leads to metastatic recurrence caused by post-hypoxic cells. We further determined that the post-hypoxic cells that metastasize are enriched in pathways related to cancer stem cell gene expression. Overall, our results show that even when hypoxic cancer cells are reoxygenated in the bloodstream they retain a hypoxia-induced cancer stem cell-like phenotype that persists and promotes resistance and eventually recurrence.