Tumor and Stem Cell Biology Hyperthermia Sensitizes Glioma Stem-like Cells to Radiation by Inhibiting AKT Signaling

Glioma stem-like cells (GSC) are a subpopulation of cells in tumors that are believed to mediate self-renewal and relapse in glioblastoma (GBM), the most deadly form of primary brain cancer. In radiation oncology, hyperthermia is known to radiosensitize cells, and it is reemerging as a treatment option for patients with GBM. In this study, we investigated themechanisms of hyperthermic radiosensitization in GSCs by a phospho-kinase array that revealed the survival kinase AKT as a critical sensitization determinant. GSCs treated with radiation alone exhibited increased AKT activation, but the addition of hyperthermia before radiotherapy reduced AKT activation and impaired GSC proliferation. Introduction of constitutively active AKT in GSCs compromised hyperthermic radiosensitization. Pharmacologic inhibition of PI3K further enhanced the radiosensitizing effects of hyperthermia. In a preclinical orthotopic transplant model of human GBM, thermoradiotherapy reduced pS6 levels, delayed tumor growth, and extended animal survival. Together, our results offer a preclinical proof-of-concept for further evaluation of combined hyperthermia and radiation for GBM treatment. Cancer Res; 75(8); 1760–9. 2015 AACR. Introduction Glioblastoma (GBM) is the most common malignant primary brain tumor (1). The median survival for patients with newly diagnosed GBM is about 1 year despite aggressive therapy with surgery, radio-, and chemotherapy (2). Radiation is the most efficacious, nonsurgical treatment but control rates remain poor (3). Glioma stem-like cells (GSC) are increasingly recognized as playing important roles in tumor progression and therapeutic resistance (4–10). Therefore, treatments that improve targeting of GSCs are needed to provide durable tumor control. Hyperthermia is one of the oldest and most potent radiosensitizers (11, 12) and has been shown to improve cancer control in numerous phase III clinical trials (13–17). When used in conjunction with brachytherapy, hyperthermia significantly improves the survival of patients with GBM (15). However, widespread adoption of hyperthermia has been limited because of technical challenges in its administration. In addition, brachytherapy has failed to demonstrate a clinical benefit over standard external beam irradiation (18, 19). Laser interstitial hyperthermia is emerging as an innovative, minimally invasive surgery that permits real-time three-dimensional thermometry within the operating theater. Initial studies of interstitial hyperthermia for patients with GBM have been promising, and this technique is gaining momentum as a new therapeutic option (20–22). In light of this developing treatment modality, we sought to determine the efficacy of hyperthermia and external beam radiation in treating GBM and to characterize the molecular mechanisms of radiosensitization by hyperthermia to uncover potential targets for improving GSC radiosensitivity. Materials and Methods Cell culture and treatment conditions Glioma stem cells (specimens 3691 and 387)were isolated and functionally validated as previously described (23, 24) and cultured as neurospheres in neural basal medium enriched with B27 supplement (Gibco), 10ng/mLbasicfibroblast growth factor, and 10 ng/mL EGF (R&D Systems; ref. 24). Only low-passage GSCs were used (<5 passages). Non-stem tumor cells (NSTC) were cultured in DMEM containing 10% FBS. GSCs were sham-treated or treated with hyperthermia (HT, incubated in a humidified incubator, 5% CO2, temperature set at 42.5 C for 1 hour), radiation (RT, 2 Gy), or combined hyperthermia and radiation treatment (HTþRT, incubated in a 42.5 C incubator for 1 hour followed by 2 Gy irradiation within 30 minutes of completion of HT). We chose to use a clinically relevant dose of 2 Gy, the standard fraction size used for radiotherapy for patients with GBM (2) to investigate the effects of hyperthermia on GSCs. Cells were treated with a Cesium-137 irradiator. The pCDH-T2A-PuroMSCV-GFP and pCDH-T2A-Puro-MSCV-MYR-AKT1 plasmids were introduced into GSCs as previously described (24). GSCs were treated with 0 or 5 mmol/L of the small-molecule inhibitor Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic, Cleveland, Ohio. Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic, Cleveland, Ohio. Department of Radiation Oncology, Cleveland Clinic, Cleveland,Ohio. Department of Neurosurgery, Cleveland Clinic, Cleveland, Ohio. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J. Man and J.D. Shoemake contributed equally to this article. Corresponding Author: Jennifer S. Yu, Cleveland Clinic, 9500 Euclid Avenue, T28, Cleveland, OH 44195. Phone: 216-445-9799; Fax: 216-445-1068; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-14-3621 2015 American Association for Cancer Research. Cancer Research Cancer Res; 75(8) April 15, 2015 1760 on April 13, 2017. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst February 20, 2015; DOI: 10.1158/0008-5472.CAN-14-3621