Hypoxic gradients direct the spatial organization of epithelial-to-mesenchymal transition in an in-vitro breast cancer tumor microenvironment

Authors

• MG Hirsch
• S Pal
• FR Mehrabadi
• S Malikic
• E Perez-Guijarro
• G Merlino
• SC Sahinalp
• EK Molloy
• CP Day
• TM Przytycka

Abstract

Poor prognosis in ER- breast cancer is predicted by high cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (NOS2) expression. In hypoxic gradients of the tumor microenvironment (TME) these proteins promote epithelial-to-mesenchymal transition (EMT), initiating metastasis. We define epithelial (E) and mesenchymal (M) phenotypes by E-cadherin (ECAD) and vimentin (VIM) expression, respectively. We hypothesize that hybrid (E/M) phenotypes, which are implicated in metastatic disease, (1) organize along hypoxic gradients and (2) correlate spatially COX2 and NOS2 expression.

Restricted exchange environment chambers (REECs) mimic the cell-generated hypoxic gradients of solid tumors in 2D live cell culture. We cultured 4T1 cells – a murine model of human triple-negative breast cancer – in REECs, and at 0, 48, 96, and 168 h, fixed and imaged them with multiplexed immunofluorescent microscopy.

The proportion of VIM+ cells in the REECs increased over 7 days, with a maximum in normoxic regions, as observed in 4T1 mouse tumors. Across all oxygen concentrations in the REEC, VIM+ expression was greater than in a monolayer of cells cultured under hypoxic conditions. E/M cells increased in all regions over time. In hypoxic regions, discrete VIM+ clusters emerged in spoke-like patterns aligned along the gradient. COX2 and NOS2 expression also increased significantly in response to hypoxic gradients.

Neighborhoods around clustered VIM+ cells are dominated by VIM+/ECAD+/COX2+ cells, but solitary VIM+ cells inhabit primarily VIM-/ECAD+/COX2- neighborhoods. These results support the hypothesis that COX2 and NOS2 contribute to the organization of E/M phenotypes in a hypoxic gradient.

Supported in part by NCI contract:75N91019D00024.

Scientific Focus Area: Cancer Biology

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