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 that in 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.

Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis

“…NuFF (passages 18–28) were co-cultured with MDA breast cancer cells for establishment of cell-derived ECM. Both cell lines were seeded on the same day in a 1:1 ratio in 4 well Nunc LabTek II Chamberslides (Sigma) using methods previously described [ 11 , 14 ]. In brief, co-cultures were maintained in one-half NuFF media and one-half MDA media with a final concentration of 10% fetal bovine serum (FBS).…”

“…Isolation of cell-derived ECM and subsequent seeding of HUVECs for vascular morphogenesis were based off of methods described in prior published reports [ 11 , 14 ]. HUVECs were used at passages 4–5 for all analyses of vascular morphogenesis on de-cellularized ECM and were cultured in EGM media containing 2% serum.…”

“…Fibronectin is produced by several cell types including fibroblasts [ 11 – 13 ] and is a component of the ECM milieu of several organs. Here, we describe for the first time, the use of a completely biological 3D de-cellularized ECM [ 11 , 14 ] rich in fibronectin for analyses of vascular morphogenesis and matrix assembly. Using this 3D matrix as a culture platform, we tested the hypothesis that both fibronectin polymerization in the matrix and fibronectin deposition by ECs participate in vascular morphogenesis of ECs on the de-cellularized ECM.…”

Characterizing Human Pluripotent-Stem-Cell-Derived Vascular Cells for Tissue Engineering Applications

“…Vascular derivatives, including both EVCs and derived ECs, obtained under low-oxygen differentiation conditions demonstrated a greater propensity to produce ECM proteins collagen IV, fibronectin, and laminin. In our previous studies, we also document a correlation between hypoxic conditions and ECM production [26,30]; hypoxic exposure promoted ECM deposition from mature ECs and yielded a more fibrous ECM ultrastructure from cocultures of fibroblasts and breast cancer cells. In both our previous studies and current study, we could not observe collagen I deposition from any types of ECs tested in atmospheric conditions; however, 5% O 2 conditions stimulated collagen I production by ECs harvested from human umbilical vein or artery [26].…”

Transitions from mono- to co- to tri-culture uniquely affect gene expression in breast cancer, stromal, and immune compartments

“…Advances in modeling breast cancer in vitro using multi-culture techniques has recently been reviewed (Regier et al 2016). Though less common than mono- and co-culture models, heterotypic models comprised of breast cancer cells with two or more other cell types have successfully generated in vitro functional recapitulation of in vivo processes including migration (Torisawa et al 2010), intravasation (Zervantonakis et al 2012), and extravasation (Jeon et al 2015) as well as other critical functions such as angiogenesis induction (Hielscher et al 2012; Hielscher et al 2013), and micrometastasis formation (Bersini et al 2014). However, the role of the increase in heterotypic complexity in the success of these models is difficult to define for two primary reasons.…”