1. 2036 Extracellular matrix as a novel approach to glioma therapy
- Author
-
Mark H. Murdock, Jordan T. Chang, George S. Hussey, Nduka M. Amankulor, Johnathan A. Engh, and Stephen F. Badylak
- Subjects
Medicine - Abstract
OBJECTIVES/SPECIFIC AIMS: Gliomas are the most lethal and common primary tumor type in the central nervous system across all age groups; affected adults have a life expectancy of just 14 months. As glioma cells invade the surrounding normal parenchyma they remodel the composition and ultrastructure of the surrounding extracellular matrix (ECM), suggesting that the native (i.e., “normal”) microenvironment is not ideal for their survival and proliferation. Recent reports describe suppressive and/or lethal effects of mammalian ECM hydrogels derived from normal (nonneoplastic) sources upon various cancer types. ECM-based bioscaffolds placed at sites of neoplastic tissue resection in humans have never been reported to facilitate cancer recurrence. The objective of the present research is to evaluate mammalian ECM as a novel approach to glioma therapy. METHODS/STUDY POPULATION: ECM hydrogels from porcine dermis, small intestine, and urinary bladder were produced as described previously. Primary glioma cells were graciously supplied by Drs. Nduka Amankulor and Johnathan Engh, and U-87 MG were ordered through ATCC. Cells were plated onto tissue culture plastic at ~60% confluence and allowed to attach for 24 hours before treatment. The saline-soluble fraction (SSF) of ECM was obtained by mixing lyophilized, comminuted ECM with 0.9% saline for 24 hours then filtering the resulting mixture through a 10 kDa molecular weight cutoff column. All assays and kits were followed according to the manufacturer’s instructions. Cell viability was measured via MTT assay (Vybrant® MTT Cell Proliferation Assay, Invitrogen) and by live/dead staining (LIVE/DEAD® Cell Imaging Kit, Invitrogen). Time lapse videos were created by taking images every 20 minutes for 18 hours (phase-contrast) or every 10 minutes for 12 hours (darkfield). NucView reagent was ordered from Biotium. Temozolomide was ordered through Abmole. All in vivo work was conducted according to protocols approved by the University of Pittsburgh’s IACUC office. RESULTS/ANTICIPATED RESULTS: ECM hydrogels derived from porcine dermis, small intestine, or urinary bladder all decreased the viability of primary glioma cells in vitro, with urinary bladder extracellular matrix (UBM) having the most dramatic effects. The SSF of UBM (UBM-SSF), devoid of the fibrillar, macromolecular components of ECM, was sufficient to recapitulate this detrimental effect upon neoplastic cells in vitro and was used for the remainder of the experiments described herein. In a cell viability assay normalized to the media treatment, non-neoplastic CHME5 and N1E-115 cells scored 103% and 114% after 48 hours when treated with UBM-SSF and 2 primary high-grade glioma cell types scored 17% and 30.5% with UBM-SSF (n=2). Phase-contrast time-lapse video showed CHME5 and HFF thriving in the presence of UBM-SSF for 18 hours while most primary glioma cells shriveled and died within this time. Darkfield time-lapse video of wells containing Nucview dye, fluorescent upon cleavage by active caspase-3, confirmed that within 12 hours most primary glioma cells underwent apoptosis while CHME5 and HFF did not. In culture with primary astrocytes, high grade primary glioma cells, and U-87 MG glioma cells for 24 hours, UBM-SSF was found to significantly increase the population of primary astrocytes compared with media (p
- Published
- 2018
- Full Text
- View/download PDF