Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O 2 ) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O 2 depletion. Here, we report the impact of hypoxic O 2 gradients on sarcoma cell invasion and migration. O 2 gradient measurements showed that large sarcoma mouse tumors (>300 mm 3 ) contain a severely hypoxic core [â€0.1% partial pressure of O 2 (pO 2 )] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1-6% pO 2 ). To analyze tumor invasion, we used O 2 -controllable hydrogels to recreate the physiopathological O 2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O 2 gradient accurately, we examined individual sarcoma cells embedded in the O 2 -controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O 2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O 2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O 2 -controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets.hydrogel | sarcoma | hypoxia | gradients | migration S oft tissue sarcomas are a heterogeneous group of malignant cancers derived from transformed cells of mesenchymal origin (1, 2). Approximately 13,000 new cases per year are diagnosed in the United States alone, with 25-50% of patients developing recurrent and metastatic disease (3-5). Current clinical data suggest that undifferentiated pleomorphic sarcoma (UPS) is one of the most aggressive sarcoma subtypes, which frequently results in lethal pulmonary metastases that are insensitive to radio/chemotherapy. It has recently become apparent that sarcoma progression and metastasis are regulated by microenvironmental cues, such as extracellular matrix (ECM) remodeling, stiffness modulation, cellto-cell/matrix interactions, signaling factors, and spatial gradients (6-8). Of all these factors, low intratumoral oxygen (O 2 ; hypoxia) is most dramatically associated with pulmonary metastasis and poor clinical outcomes (9, 10).Intratumoral hypoxia occurs when the partial pressure of O 2 (pO 2 ) falls below 5%, and it is a commonly observed feature of many sarcomas. Regional hypoxia develops as rapidly growing tumors outstrip their blood supply and as a consequence of aberrant tumor angiogenesis. As a result, O 2 gradients develop throughout the growing tumor. Tumor hypoxia promotes c...