title: Hypoxia Affects the Structure of Breast Cancer Cell-Derived Matrix to Support Angiogenic Responses of Endothelial Cells.
abstract: Hypoxia, a common feature of the tumor environment and participant in tumor progression, is known to alter gene and protein expression of several Extracellular Matrix (ECM) proteins, many of which have roles in angiogenesis. Previously, we reported that ECM deposited from co-cultures of Neonatal Fibroblasts (NuFF) with breast cancer cells, supported 3-dimensional vascular morphogenesis. Here, we sought to characterize the hypoxic ECM and to identify whether the deposited ECM induce angiogenic responses in Endothelial Cells (ECs). NuFF and MDA-MB-231 breast cancer cells were co-cultured, subjected to alternating cycles of 24 hours of 1% (hypoxia) and 21% (atmospheric) oxygen and de-cellularized for analyses of deposited ECM. We report differences in mRNA expression profiles of matrix proteins and crosslinking enzymes relevant to angiogenesis in hypoxia-exposed co-cultures. Interestingly, overt differences in the expression of ECM proteins were not detected in the de-cellularized ECM; however, up-regulation of the cell-binding fragment of fibronecin was observed in the conditioned media of hypoxic co-cultures. Ultrastructure analyses of the de-cellularized ECM revealed differences in fiber morphology with hypoxic fibers more compact and aligned, occupying a greater percent area and having larger diameter fibers than atmospheric ECM. Examining the effect of hypoxic ECM on angiogenic responses of ECs, morphological differences in Capillary-Like Structures (CLS) formed atop de-cellularized hypoxic and atmospheric ECM were not evident. Interestingly, we found that hypoxic ECM regulated the expression of angiogenic factors and matrix metalloproteinases in CLS. Overall, we report thatin vitro, hypoxia does not alter the composition of the ECM deposited by co-cultures of NuFF/MDA-MB-231, but rather alters fiber morphology, and induces vascular expression of angiogenic growth factors and metalloproteinases. Taken together, these results have important implications for understanding how the hypoxic matrix may regulate angiogenesis in tumors.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA ; Johns Hopkins Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Abigail
authors.initials: A
authors.lastname: Hielscher
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Connie
authors.initials: C
authors.lastname: Qiu
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA ; Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Josh
authors.initials: J
authors.lastname: Porterfield
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA ; Johns Hopkins Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Quinton
authors.initials: Q
authors.lastname: Smith
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA ; Johns Hopkins Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, MD 21218, USA ; Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Sharon
authors.initials: S
authors.lastname: Gerecht
keywords: Angiogenesis; Extracellular matrix; Hypoxia