Poster S12 - Restricted Exchange Environment Chambers: An Engineered, In Vitro System for Modeling Cell-Generated Molecular Gradients in the Breast Cancer Tumor Microenvironment

Introduction: Triple negative breast cancer (TNBC) is an aggressive, metastatic, and recurrent breast cancer subtype with 5-year survival rates as low as 13%. TNBC has a complex tumor microenvironment characterized by spatial gradients formed by cellular consumption of soluble molecules diffusing from the vasculature. These gradients drive a variety of phenotypic changes that encourage cancer cell survival and metastasis. Such changes include epithelial-mesenchymal transition (EMT), conversion to glycolytic metabolism, and formation of cancer stem cell (CSC) niches. EMT is a reversible, hypoxia-driven phenotype change from stationary, epithelial (E) cells to migratory, mesenchymal (M) cells. Hybrid (E/M) cells can act as leader cells, guiding invasion and colony formation during metastasis. Mitochondrial metabolism is significantly altered by cancer and hypoxia, switching from oxidative phosphorylation (OXPHOS) to glycolysis. These metabolic changes may correspond with changes in mitochondrial morphology and can contribute to drug resistance, resistance to cell death, and cancer stemness. The CSC niche, as an origin site, is generally characterized by hypoxia, acidic pH, and lack of nutrients.
We developed a novel, engineered system, Restricted Exchange Environment Chambers (REECs), which recapitulate the cell-generated molecular gradients observed in solid tumors in vitro and enable live-cell and high-resolution fluorescence microscopy. Here, we used the REECs to investigate how hypoxic gradients spatially organize EMT and metabolism in TNBC.
We hypothesized that:
1. Hypoxia drives EMT, increasing vimentin (VIM) expression and promoting cell migration out of oxygen deficient zones over time.
2. Hypoxia drives changes in mitochondrial metabolism that correspond to changes in morphology.

Materials and

Methods: The REEC is a glass or PDMS disk with a 500 μm central hole raised on a 100 μm gasket. This forms a ~15 µl volume of media over 75% confluent cell culture, resulting in cell-generated molecular gradients within the volume. These gradients form within a few hours of REEC application; over several days, a compact cell disk forms by a combination of cell death and migration. Glass REECs enforce oxygen and nutrient gradients, and PDMS REECs enforce a nutrient gradient alone. 4T1 cells, a murine model of TNBC, are cultured in the REECs for the desired time. Either live-cell dyes are applied for live imaging, or cells are fixed in 4% paraformaldehyde and stained with fluorescent antibodies for targets of interest. Spatial fluorescence data is extracted from the images with Imaris or Stardist, an ImageJ nuclei segmentation plugin, in combination with RStudio. Cells positive for a phenotype are identified by mean fluorescent intensity (MFI) exceeding a threshold, typically 1-3 standard deviations above the control MFI.
In our EMT experiment, cells were fixed at 0, 6, 12, 24, 48, 96, and 120 hr and imaged with immunofluorescent microscopy. Spatial changes in VIM expression over time were extracted from the images. In our metabolism experiment, we live-imaged a 48 hr disk stained with TMRE, a live-cell dye sequestered by active mitochondria, on an Elyra Lattice SIM microscope to visualize individual mitochondria. TMRE fluorescence and morphological characteristics per mitochondrion were extracted from the image and compared along the oxygen gradient.

Results, Conclusions, and Discussions: REECs were used to experimentally validate our hypotheses that hypoxia initiates pro-cancer mechanisms such as EMT and conversion to glycolytic metabolism within TNBC and that these phenotypic changes are spatially organized along the oxygen gradient.
In our EMT experiment, VIM+ and E/M cells were enriched in the hypoxic regions of the cell disk as early as 12 hr after REEC placement. After 48 hr, VIM+ and E/M cells increased in proportion in the normoxic regions of the disk, suggesting migration up the oxygen gradient. We conclude that hypoxia initiates EMT by conferring cells with VIM+ phenotypes. The increase in E/M cells from hypoxic to normoxic zones recapitulates how leading, migratory cells develop within the tumor core before invading surrounding tissue. Targeting this cell type therapeutically could decrease metastasis.
In our metabolism experiment, TMRE intensity was significantly lower in the hypoxic regions of the disk, suggesting a metabolic shift from OXPHOS to glycolysis. Mitochondrial morphology also differed between normoxia and hypoxia. TMRE MFI, density, sphericity, and volume per mitochondria varied continuously from normoxia to hypoxia and were significantly different in the extremes. TMRE MFI positively correlated with density and volume, and negatively correlated with sphericity. We conclude that hypoxia drives increased glycolytic metabolism and decreased mitochondrial bioenergetic capacity. Furthermore, this change in metabolism correlates with changes in mitochondrial morphology, an effect which may be exploited in cancer diagnostics.
The REEC system has many potential applications in the elucidation of the role of molecular gradients in the spatial organization of the tumor microenvironment, including the cancer stem cell niche and the effects of various drugs and treatments on cellular phenotypes. In particular, we anticipate investigating the intersection of EMT and metabolism in the context of oxygen and nutrient gradients to identify potential therapeutic targets.

Acknowledgements (Optional): GV, SA, and SJS were supported by the NIH Post- Baccalaureate Intramural Research Training Award Program at the Center for Cancer Research, National Cancer Institute, National Institute of Health and Mariam Malik and Chris McGinity, OSTR, CCR, NCI. Funded in whole or in part by the NCI Contract No. 75N91019D00024.