Natural killer (NK) cells are cytotoxic innate lymphocytes that are specialized to kill tumor cells. NK cells are responsive to the primary cytokine IL-2 in the tumor microenvironment (TME), to activate its effector functions against tumors. Despite their inherent ability to kill tumor cells, dysfunctional NK cells observed within advanced solid tumors are associated with poor patient survival. Hypoxia in the TME is a major contributor to immune evasion in solid tumors that could contribute to impaired NK cell function. HIF-1α is a nodal regulator of hypoxia in driving the adaptive cellular responses to changes in oxygen concentrations. Whether HIF-1α is expressed in hypoxic NK cells in the context of IL-2 and whether its expression regulates NK cell effector function are unclear. Here, we report that freshly isolated NK cells from human peripheral blood in hypoxia could not stabilize HIF-1α protein coincident with impaired anti-tumor cytotoxicity. However, ex vivo expansion of these cells restored HIF-1α levels in hypoxia to promote antitumor cytotoxic functions. Similarly, the human NK cell line NKL expressed HIF-1α upon IL-2 stimulation in hypoxia and exhibited improved anti-tumor cytotoxicity and IFN-γ secretion. We found that ex vivo expanded human NK cells and NKL cells required the concerted activation of PI3K/mTOR pathway initiated by IL-2 signaling in combination with hypoxia for HIF-1α stabilization. These findings highlight that HIF-1α stabilization in hypoxia maximizes NK cell effector function and raises the prospect of NK cells as ideal therapeutic candidates for solid tumors.

Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein that selfassembles into FN fibrils, forming a FN matrix contributing to the stiffness of the ECM. Stromal FN stiffness in cancer has been shown to impact epithelial functions such as migration, cancer metastasis, and epithelial-to-mesenchymal transition. The role of the FN matrix of epithelial cells in driving such processes remains less well understood and is the focus of this study. Hypoxia, defined by low oxygen tension (<5%) is one of the hallmarks of tumor microenvironments impacting fibril reorganization in stromal and epithelial cells. Here, using the MCF10 breast epithelial progression series of cell lines encompassing normal, preinvasive, and invasive states, we show that FN fibril formation decreases during hypoxia, coinciding with a decrease in migratory potential of these cells. Conversely, we find that FN fibril disruption during three-dimensional acinar growth of normal breast cells resulted in acinar luminal filling. Our data also demonstrates that the luminal filling upon fibril disruption in untransformed MCF10A cells results in a loss of apicobasal polarity, characteristic of pre-invasive and invasive breast cell lines MCF10AT and MCF10 DCIS.com. Overall this is the first study that relates fibrilmediated changes in epithelial cells as critical players in lumen clearing of breast acini and maintenance of the untransformed growth characteristic.

Natural Killer cells (NK), the major innate effector cells, with their broad cytotoxicity against tumors are ideal candidates for immunotherapy. Once having entered the malignant site, NK cells encounter a complex environment composed of tumor cells, non-tumor cells along with the extra cellular matrix (ECM). As one of the integral components of the tumor microenvironment (TME), the normal regulation of the extra cellular matrix (ECM) is necessary to prevent tumor progression and metastasis. Nevertheless, the impact of the matrix architecture found in solid tumors on immune cells and especially NK cells is not well characterized. Here we investigated the role of Fibronectin, a fibrous ECM protein, and its assembly and localization on NK cell infiltration into clear cell renal cell carcinoma (ccRCC) spheroids. 3D tumor spheroids established from the ccRCC cell line 786-O (RCC-) that lacks the Von Hippel-Lindau (VHL) gene are incapable of fibronectin assembly in their ECM. However, addition of the VHL gene into the 786-O cells (RCC+) reversed this phenotype facilitating fibronectin assembly. Fibronectin in RCC-spheroids exhibited prominent peripheral localization in contrast to RCC+ spheroids where fibronectin was detected towards the center of the spheroid. Coincident with fibronectin localization, PKH26 labeled Natural killer cell line, NKL, showed significantly increased infiltration into RCC+ spheroids compared to RCC-spheroids that lacked the tumor suppressor VHL. Thus, assembly and localization of fibronectin modulates NK cell infiltration in RCC tumors. Supported by grants from NIH U54MD012388 (NR and AV) and NIH U54CA143925 (NACP-NAU) to NR.