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 [≤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...

Collagen is prevalent in the microenvironment of many cancer types and has been demonstrated to play an important role during disease progression. We previously showed the importance of hypoxic gradients in sarcoma cell migration. Here, we utilized an oxygen gradient collagen gel platform to determine the impact of collagen fiber density and hypoxic gradient on sarcoma cell migration. The oxygen gradient was created by regulating the oxygen diffusion coefficient along with the cellular oxygen consumption rate. Collagen fiber density in the hydrogels is modified by changing the preincubation period of the collagen gel solution at 4 °C, controlling fiber density independently of collagen concentration and oxygen tension. High fiber density gels have wider and longer fibers but a similar microscale pore size with a larger nanoscale pore size and quicker stress relaxation time, compared to the low fiber density gel. Both gels have the same Young's modulus. We analyzed responses of sarcoma cells encapsulated in the different hydrogels for 3 days. In the nonhypoxic low fiber density constructs, sarcoma cells exhibit a larger aspect ratio, and the matrix has less fiber alignment compared to the nonhypoxic high fiber density constructs. Interestingly, we found a minimal effect of fiber density on cell migration and the ability of the cells to degrade the matrix in nonhypoxic constructs. When compared with hypoxic constructs, we observed the opposite trend, where cells in low fiber density constructs exhibit a lower aspect ratio and the matrix has more aligned fibers compared to hypoxic high fiber density constructs. Sarcoma cells encapsulated in high fiber density hypoxic gels migrated faster and degraded the matrix more rapidly compared to the low fiber density hypoxic constructs. Overall, we show that hypoxic cell migration and matrix degradation are enhanced in high fiber density gels, while hypoxic matrix alignment is prominent in low fiber density gels. Our results suggest that the differences in cellular responses under hypoxic gradients are due to the hydrogel architecture including fiber density, size (length and width), and stress relaxation.

Background Dendritic cells (DCs) are potent antigen‐presenting cells critical for immunity. We previously demonstrated a significant association between pre‐transplant blood myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) deficiency and post‐transplant BK viremia in renal transplant recipients. In the current post‐hoc analysis, we studied the association of these same pre‐transplant DC levels with other post‐transplant outcomes. Methods Pre‐transplant peripheral blood mDC and pDC levels were quantified using flow cytometry in 78 patients undergoing kidney transplantation. Post‐transplant outcomes were analyzed, including infection, rejection, and patient death, with a median follow‐up of 5.3 years. Associations between DC levels and outcomes were assessed using logistic regression analysis and Cox proportional hazards models. Results An independent association of mDC levels with post‐transplant cytomegalovirus infection (adjusted odds ratio 7.0, P = 0.01) and patient death (adjusted hazard ratio 13.0, P = 0.015) was found. No associations were demonstrated between levels of either DC subtype and bacterial infections or rejection. Conclusions Pre‐transplant mDC deficiency is significantly associated with CMV infection and death after kidney transplantation.

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