Abstract 174: Collagen fiber orientation influences tumor cell growth and Hedgehog signaling in the breast tumor microenvironment
Authors: Reyes Ana M. , Almodovar Jorge , Domenech Maribella 
Published: August 2018

Collagen matrix architecture is an important player in tumor transition to therapeutic resistance. Several studies have shown that high level of fiber orientation and density of collagen type I matrix enhances tumor cell invasion, decrease drug response and influence tumor metabolism, but the mechanisms of action that support transition to therapeutic resistance are not fully understood. We developed a 3D culture platform to evaluate the effects of collagen matrix architecture in the sensitivity of tumor and stromal cells to tumor promoting ligands and pharmacological inhibitors. Estrogen (E2) and Hedgehog (Hh) signaling pathways were selected for evaluation due to their relevance in breast cancer prognosis and tumor progression. In this 3D culture platform, electrospun collagen fiber sheets of random or aligned orientation are bound to double-sided medical grade tape to generate collagen fiber stickers. Automated cutting plotter allows razor-printing for precisely cutting shapes from collagen fiber sticker sheets. Round shapes were applied like a sticker to the bottom of culture well plates prior to cell seeding. The percentage of matrix porosity fiber orientation and diameter was quantified by Scanning Electron Microscopy (SEM) and Image J analysis. For estrogen signaling studies, MCF-7 cells were seeded in collagen fibers of random or aligned orientation using hormone free-culture conditions. Cells were treated +/- E2 (10nM) ligand and +/- estrogen receptor signaling inhibitors (Fulvestrant and Tamoxifen). Tumor cell proliferation was quantified at 48hrs via Image Cytometry. For Hh signaling studies, expression levels of GLI1, PTCH1 and SMO Hh target genes were monitored in triple negative breast cancer (MDA-MB-231 and MDA-MB-468) and mesenchymal cells. Cells were seeded on collagen fibers and treated with sonic hedgehog ligand for 24hrs. Tumor and mesenchymal cells were harvest for viability and qRT-PCR analysis respectively. Evaluation of cell growth in MCF-7 cultures shows that a fibrillar structure of collagen matrix in combination with high degree of fiber orientation can significantly increase cell proliferation as compared to gelatin or tissue culture plastic substrates. Collagen fibers significantly supported hormone-independent tumor growth at similar levels to those observed in E2 treatment. Increased proliferation levels were maintained in ER inhibition groups particularly when cultured in aligned collagen fibers. In Hh signaling studies, overexpression of GLI1 and SMO genes were observed for collagen fibers matrixes as compared to gelatin and tissue culture plastic. Overall our results show for the first time that collagen fiber architecture can promote hormone-independent growth dependence in breast cancer cells and activation of hedgehog signaling in tumor and mesenchymal cells.

Citation Format: Ana M. Reyes, Jorge Almodovar, Maribella Domenech. Collagen fiber orientation influences tumor cell growth and Hedgehog signaling in the breast tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 174.