Introduction: Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain cancer. A subset of glioblastoma cells, glioblastoma stem cells (GSCs), is treatment resistant and implicated in disease recurrence. Currently, there is debate over metabolic pathways—glycolysis versus oxidative phosphorylation—employed by GSCs (1,2). Therefore, we sought to perform ultrastructure analysis of GSCs with electron microcopy (EM) to determine metabolic preference and evaluate therapeutic targets as potential treatments necessary to eliminate GSCs.
Methods: GSCs were generated from patient-derived tumors, propagated in neurosphere media, and examined for stem-cell markers (Nestin, Musashi, Sox2, CD44, GFAP, BMI1) and ability to generate tumors in nude mice. Prior to analysis using Jeol 1400 TEM, cells were prepared for imaging based on cell culture EM protocol. Effect of glycolytic inhibitors, 2-deoxyglucose (2-DG), 3-bromopyruvate, and dichloroacetate, on cell viability was determined by MTS assay.
Results: EM analysis of multiple patient-derived GSCs confirmed a majority (>50%) of mitochondria exhibit either cristae loss or inner-fold polarization, indicating less available surface area for oxidative phosphorylation suggesting a metabolic dependence on glycolysis. Glycolytic inhibition significantly reduced cell viability of all GSCs after 72h. 2-DG showed the most consistent reduction in GSC viability compared to non-treated controls at 0.5mM, 2mM, and 10mM (average viability at 80.5%, 40.4%, and 5.3%, respectively). Longer duration studies (7days) using all three inhibitors showed a remarkable loss in cell viability, signifying time dependent effects, along with dose dependent.
Conclusions: Targeting GSCs is vital in preventing tumor regeneration. EM provides a useful tool in developing experimental therapies. Our EM results indicate that oxidative phosphorylation is severely compromised in GSCs. Glycolytic inhibition proved effective in targeting GSCs and may represent an adjuvant therapy for a disease with minimal survival.
Patient Care: The high rate of GBM recurrence in patients has been attributed to the presence of Glioblastoma stem cells (GSCs), but unfortunately, these GSCs are incredibly resistant to both chemotherapy and radiotherapy. As a result, it is imperative to explore novel treatments that can target GSCs and reduce their viability. Our research will improve patient care by suggesting a new paradigm in treating GSCs, specifically by utilizing glycolytic inhibition. According to a review article published in Cell Death and Disease by Zhao Y, Butler EB, and Tan M (2013), the metabolic pathways utilized by cancer cells are remarkably different than those used by normal cells. However, it is this dysregulation of cellular metabolism that may provide drug resistance to cancer cells. Therefore, we believe that targeting the energy pathway of GSCs can provide a very novel, effective, and safe way to combat cancer. Many of the glycolytic inhibitors utilized have been studied in other cancers, with little cytotoxic effects to normal tissue at reasonable doses, therefore translational research to patients suffering from GBM tumors can be feasible.
Learning Objectives: By the conclusion of this session, participants should be able to: (1) Describe the use of Transmission Electron Microscopy in analyzing cancer stem cell morphology to find potential therapeutic targets, (2) Identify the importance and efficacy of treating GSCs by inhibiting glycolysis, and (3) Discuss, with other physicians and researchers, the clinical relevance of targeting GSCs to prevent tumor regrowth.
References: 1. Yuan S, Wang F, Chen G et al. Effective elimination of cancer stem cells by a novel drug combination strategy. Stem Cells 31(1): 23-34.
2. Vlashi E, Lagadec C, Vergnes L et al. Metabolic state of glioma stem cells and nontumorigenic cells. Proc Natl Acad Sci 108(38): 16062 – 16067.