Abstract 362: Fibronectin facilitates extracellular matrix assembly and vascular morphogenesis .

Abstract

Abstract Tumor growth is facilitated by oxygen and nutrients supplied by tumor blood vessels. Recently, we reported that de-cellularized extracellular matrix (ECM) deposited by co-cultures of fibroblasts with breast cancer cell lines supported vasculogenesis of endothelial cells (ECs). The most robust changes in vasculogenesis and phenotype were attributed to ECM deposited by co-cultures with metastatic MDA-MB-231 breast cancer cells. We have utilized this co-culture set up to investigate how fibronectin, a matrix protein involved in angiogenesis and breast tumor progression, influences vasculogenesis of ECs grown on de-cellularized ECM. Based on our previous study, MDA-MB-231 and human neonatal foreskin fibroblasts were grown at a 1:1 ratio for 9 days followed by de-cellularization using 50mM NH4OH and 0.4% triton-X. Following ECM de-cellularization, we found that ECM proteins collagens I and IV, laminin and tenascin-C were highly co-localized with a fibrillar fibronectin matrix and absent in regions where the fibrillar fibronectin matrix was lacking. Through use of a fibronectin inhibitor peptide, pUR4B (Dr. Jane Sottile, University of Rochester), reported to prevent fibronectin accumulation in the matrix, we were able to eliminate fibronectin assembly in the matrix. Interestingly, the deposition of collagen I and tenascin-C in the matrix could not be detected with the addition of the fibronectin inhibitor pUR4B. Examining vasculogenesis of ECs on de-cellularized ECM, we found that the presence of fibronectin was increased both intra-and extra-cellularly following vascular morphogenesis. Moreover, the vascular network appeared to degrade laminin and collagens I and IV in the de-cellularized ECM. Up-regulated expression of the gelatinases and collagenases MMPs 1,2 and 9 and MT1-MMP was observed in ECs which had formed a vascular network on ECM, a mechanism likely contributing to decreased laminin and collagens I and IV after vasculogenesis. Blocking fibronectin integrins α5β1 and αvβ3prevented vascular formation of ECs on intact, de-cellularized ECM, suggesting that fibronectin is important for EC attachment and subsequent vasculogenesis. Inhibition of fibronectin assembly into the matrix during vasculogenesis of ECs on intact, de-cellularized ECM reduced vascular formation, but did not abrogate EC attachment to the ECM, implicating a contribution from EC-derived fibronectin during vasculogenesis. In vivo experiments are being performed to elucidate the role of fibronectin in the assembly of the tumor ECM and in the growth of tumor vessels during the progression of human xenograft breast tumors. Together, these results suggest that fibronectin is a critical component of the ECM, facilitating the assembly of a number of matrix proteins and promoting vasculogenesis on de-cellularized ECM. Citation Format: Abigail Hielscher, Connie Qiu, Sharon Gerecht. Fibronectin facilitates extracellular matrix assembly and vascular morphogenesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 362. doi:10.1158/1538-7445.AM2013-362