Abstract A70: Targeting pediatric bone sarcoma stem cell with metabolic inhibitors
Authors: Trucco Matteo , Rainsusso Nino , Techavichit Piti , Bernardi Ronald , Shuck Ryan , Satterfield Laura , Allen-Rhoades Wendy , Donehower Larry , Loeb David , Yustein Jason 
Published: May 2016

Our project tests whether metabolic inhibitors are able to target putative sarcoma stem cells in osteosarcoma and Ewing sarcoma. The cure rate for the two most common bone sarcomas affecting children and adolescents, osteosarcoma and Ewing sarcoma, has plateaued around 70% over the past twenty years, and the cure rate for patients with metastatic bone sarcomas has not significantly improved over 20% in several decades. The subpopulation of cells in these tumors referred to as sarcoma stem cells are thought to be responsible for resistance to current therapies, relapses and metastases. To date therapies targeting the sarcoma stem cell population have not been identified. We studied the ability of metabolic inhibitors to eliminate the sarcoma stem cell population.

A panel of established and verified osteosarcoma (HOS, 143B, MNNG, SAOS2) and Ewing sarcoma (TC71, TC32, MHH, A4573, CHLA9, CHLA10) cell lines was grown in 2-dimensional culture and treated with metabolic inhibitors, including 2-deoxyglucose, dichloroacetate, oxamate, lonidamine, 3-Bromopyruvate, and metformin for 72hrs establishing dose response curves for each individual agent. Most agents showed IC50s in the millimolar range. Combinations of two metabolic inhibitors at their IC50 were then tested and evaluated for synergy and if the combination would allow for dosing at physiologically attainable concentrations. The combination of 2-deoxyglucose and metformin proved toxic to the osteosarcoma and Ewing sarcoma cell lines (p &lt;0.0001 compared to untreated cells) with synergy detected as measured by combination index &lt;1. In particular this combination showed activity against the stem cell population in all cell lines tested, including reduction of aldehyde dehydrogenase expression as measured by ALDEFLUOR reagent system (Stem Cell Technologies, Vancouver, BC), and decreased sphere-forming efficiency using Image-Pro Plus (MediaCybernetics, Rockville, MD).

The cells were then grown in 3-dimensional cultures using Magnetic 3D Bioprinting kit(n3D Biosciences, Houston, TX) and exposed to physiologically attainable concentrations of 2-deoxyglucose (500uM) and metformin (5uM), based on Phase I studies of these individual agents in humans. The resulting 3-dimentional cell cultures were incubated for 10 days with 2-deoxyglucose and metformin and there was a significant reduction in cell proliferation as measured by trypan blue dye exclusion and reduced aldehyde dehydrogenase expression. Additionally, the functional assay of cancer stem cells, sphere-forming efficiency, was impeded in all cell lines treated with the combination of 2-deoxyglucose and metformin. Finally, the ability to establish orthotopic tumors in immune deficient mice was significantly reduced for intratibialy-injected cells pretreated with 2-deoxyglucose and metformin. In vivo testing of the combination of 2-deoxyglucose and metformin to treat established xenograft osteosarcoma and Ewing sarcomas is currently underway, and resulting tumors will be harvested and assessed for aldehyde dehydrogenase expression, sphere-forming efficiency, and further characterization of the stem cell properties and metabolic alterations. Our study will provide the preclinical basis for testing metabolic inhibitors, in particular the combination of 2-deoxyglucose and metformin, in clinical trials for pediatric bone sarcomas.

Citation Format: Matteo Trucco, Nino Rainsusso, Piti Techavichit, Ronald Bernardi, Ryan Shuck, Laura Satterfield, Wendy Allen-Rhoades, Larry Donehower, David Loeb, Jason Yustein. Targeting pediatric bone sarcoma stem cell with metabolic inhibitors. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A70.