OBJECTIVES/SPECIFIC AIMS: Gliomas are the most lethal and commonprimary tumor type in the central nervous system across all age groups; affectedadults have a life expectancy of just 14 months. As glioma cells invade thesurrounding normal parenchyma they remodel the composition and ultrastructure ofthe surrounding extracellular matrix (ECM), suggesting that the native (i.e.,“normal”) microenvironment is not ideal for their survivaland proliferation. Recent reports describe suppressive and/or lethaleffects of mammalian ECM hydrogels derived from normal (nonneoplastic) sourcesupon various cancer types. ECM-based bioscaffolds placed at sites of neoplastictissue resection in humans have never been reported to facilitate cancerrecurrence. The objective of the present research is to evaluate mammalian ECMas a novel approach to glioma therapy. METHODS/STUDY POPULATION: ECMhydrogels from porcine dermis, small intestine, and urinary bladder wereproduced as described previously. Primary glioma cells were graciously suppliedby Drs. Nduka Amankulor and Johnathan Engh, and U-87 MG were ordered throughATCC. Cells were plated onto tissue culture plastic at~60% confluence and allowed to attach for 24 hours beforetreatment. The saline-soluble fraction (SSF) of ECM was obtained by mixinglyophilized, comminuted ECM with 0.9% saline for 24 hours thenfiltering the resulting mixture through a 10 kDa molecular weight cutoff column.All assays and kits were followed according to the manufacturer’sinstructions. Cell viability was measured via MTT assay(Vybrant® MTT Cell Proliferation Assay, Invitrogen)and by live/dead staining(LIVE/DEAD® Cell Imaging Kit, Invitrogen). Timelapse videos were created by taking images every 20 minutes for 18 hours(phase-contrast) or every 10 minutes for 12 hours (darkfield). NucView reagentwas ordered from Biotium. Temozolomide was ordered through Abmole. All in vivowork was conducted according to protocols approved by the University ofPittsburgh’s IACUC office. RESULTS/ANTICIPATED RESULTS:ECM hydrogels derived from porcine dermis, small intestine, or urinary bladderall decreased the viability of primary glioma cells in vitro, with urinarybladder extracellular matrix (UBM) having the most dramatic effects. The SSF ofUBM (UBM-SSF), devoid of the fibrillar, macromolecular components of ECM, wassufficient to recapitulate this detrimental effect upon neoplastic cells invitro and was used for the remainder of the experiments described herein. In acell viability assay normalized to the media treatment, non-neoplastic CHME5 andN1E-115 cells scored 103% and 114% after 48 hours whentreated with UBM-SSF and 2 primary high-grade glioma cell types scored17% and 30.5% with UBM-SSF (n=2).Phase-contrast time-lapse video showed CHME5 and HFF thriving in the presence ofUBM-SSF for 18 hours while most primary glioma cells shriveled and died withinthis time. Darkfield time-lapse video of wells containing Nucview dye,fluorescent upon cleavage by active caspase-3, confirmed that within 12 hoursmost primary glioma cells underwent apoptosis while CHME5 and HFF did not. Inculture with primary astrocytes, high grade primary glioma cells, and U-87 MGglioma cells for 24 hours, UBM-SSF was found to significantly increase thepopulation of primary astrocytes compared with media(p<0.05) while decreasing the 2 glioma cell types toapproximately one-third as many cells as the media control(p<0.0001). A dose-response of temozolomide from 0to 10,000 μM showed that when treating 2 non-neoplastic cell types(CHME5 and HFF) and 2 types of primary glioma cell there was no difference insurvivability at any concentration. Contrasted to this, a dose-response ofUBM-SSF from 350 to 7000 μg/mL showed that thenon-neoplastic cells survived significantly better than the glioma cells atconcentrations of 875 μg/mL and upward(p<0.05). In preliminary animal experiments, largeprimary glioma tumors in the flanks of athymic nude mice were resected andreplaced with either UBM SSF or Matrigel (an ECM product of neoplastic cellorigin). After 7 days the resection sites with UBM-SSF had little tumor regrowthif any compared with the dramatic recurrence seen in the Matrigel injectionsites (n=2). In a separate survival study comparing PBS to UBM-SSFinjections in the flank-resection model, all animals given PBS had to besacrificed at 9, 11, and 11 days (n=3) whereas animals given UBM-SSFwere sacrificed at 15, 24, and 39 days (n=3), indicating a moderateincrease in survival due to the UBM-SSF. DISCUSSION/SIGNIFICANCE OFIMPACT: Since the introduction of the pan-cytotoxic chemotherapeutic agent TMZin 2005, the standard of care for patients with glioblastoma multiforme has notimproved. These findings indicate that non-neoplastic ECM contains potentbioactive regulators capable of abrogating malignancy. Our in vitro data suggestthese molecules appear to have no deleterious effect on non-neoplastic cellswhile specifically inducing apoptosis in glioma cells. Our in vivo data suggestthat these molecules may be useful in delaying glioma recurrence, thus resultingin extended lifespan. Delivering soluble fractions of ECM to a tumor site mayrepresent a novel approach to glioma therapy, sidestepping traditional cytotoxictherapies in favor of utilizing putative endogenous anti-tumor pathways.