Lactate and Choline Metabolites Detected In Vitro by
Nuclear Magnetic Resonance Spectroscopy Are Potential
Metabolic Biomarkers for PI3K Inhibition in Pediatric
Glioblastoma
Nada M. S. Al-Saffar1*, Lynley V. Marshall2,3,4, L. Elizabeth Jackson1, Geetha Balarajah1¤,
Thomas R. Eykyn1,5, Alice Agliano1, Paul A. Clarke3,6, Chris Jones2,3, Paul Workman3,6,
Andrew D. J. Pearson3,4, Martin O. Leach1
1 Cancer Research UK and EPSRC Cancer Imaging Centre, Division of Radiotherapy and Imaging, The Institute of Cancer Research and The Royal Marsden NHS Foundation
Trust, London, United Kingdom, 2 Division of Molecular Pathology, The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, London, United
Kingdom, 3 Division of Cancer Therapeutics, The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, London, United Kingdom, 4 Division of
Clinical Studies. The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, London, United Kingdom, 5 Division of Imaging Sciences and Biomedical
Engineering, King’s College London, St Thomas’ Hospital, London, United Kingdom, 6 Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research,
London, United Kingdom

Abstract
The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/ mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline
(tCho) and lactate levels (p,0.02) as detected by phosphorus (31P)- and proton (1H)-NMR. Similar changes were also detected using the pan–Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p,0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNAdamaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential noninvasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma.
Citation: Al-Saffar NMS, Marshall LV, Jackson LE, Balarajah G, Eykyn TR, et al. (2014) Lactate and Choline Metabolites Detected In Vitro by Nuclear Magnetic
Resonance Spectroscopy Are Potential Metabolic Biomarkers for PI3K Inhibition in Pediatric Glioblastoma. PLoS ONE 9(8): e103835. doi:10.1371/journal.pone.
0103835
Editor: Ramo´n Campos-Olivas, Spanish National Cancer Center, Spain
Received March 17, 2014; Accepted July 2, 2014; Published August 1, 2014
Copyright: ß 2014 Al-Saffar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Funding: This work was supported by The