RESUMO
Many tools from the field of tissue engineering can be used to develop novel model systems to study cancer. We have utilized biomimetic synthetic hydrogels, based on poly(ethylene glycol) (PEG) modified with cell adhesive peptides (RGDS) and peptides sensitive to degradation by matrix metalloproteinases 2 and 9 (GGGPQGIWGQGK), as highly controlled 3D substrates for cell culture. We have previously shown that this hydrogel can support growth of tumor cells and also growth and assembly of microvascular networks. Based on this technology, a 3D in vitro tumor angiogenesis model was developed using a dual layer PEG-based hydrogel comprised of vascular cells (endothelial cells, pericytes) and lung adenocarcinoma cells in separate layers to support recapitulation of the vessel recruitment process as it occurs in vivo. This model was previously used to study highly metastatic murine 344SQ cells and in this paper was used to investigate 2 additional types of lung adenocarcinoma cells: nonmetastatic murine 393P cells and somewhat metastatic human A549 cells. All three cell types readily formed spheroid structures in the 3D hydrogels. When cultured in the dual layer format, where tumor cell spheroids were adjacent to a hydrogel layer with microvascular tubule networks, all three tumor cell types recruited vascular cells into the cancer cell layer. Interactions between vessels invading the cancer layer and the cancer cell structures was nearly twice as high for the highly metastatic 344SQ cells as for the other two cell types. Secretion of angiogenic growth factors by the tumor cells was evaluated. 344SQ cells produced the greatest amount of VEGF and FGFb, which probably accounts for the greater degree of vessel recruitment observed. Upon interaction with vessel structures, the 344SQ spheroids underwent a dramatic change in morphology, increasing in size and adopting highly irregular shapes, suggestive of invasive phenotype. This behavior was observed to a much lesser degree for A549 cells and 393P cells.
RESUMO
Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. In vitro models that mimic in vivo tumor neovascularization facilitate exploration of this role. Here we investigated lung adenocarcinoma cancer cells (344SQ) and endothelial and pericyte vascular cells encapsulated in cell-adhesive, proteolytically-degradable poly(ethylene) glycol-based hydrogels. 344SQ in hydrogels formed spheroids and secreted proangiogenic growth factors that significantly increased with exposure to transforming growth factor beta 1 (TGF-ß1), a potent tumor progression-promoting factor. Vascular cells in hydrogels formed tubule networks with localized activated TGF-ß1. To study cancer cell-vascular cell interactions, we engineered a 2-layer hydrogel with 344SQ and vascular cell layers. Large, invasive 344SQ clusters (area > 5,000 µm(2), circularity < 0.25) developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed cluster morphological changes required close proximity to vascular cells. Additionally, TGF-ß1 inhibition blocked endothelial cell-driven 344SQ migration. Our findings suggest vascular cells contribute to tumor progression and establish this culture system as a platform for studying tumor vascularization.