FULL PAPER CEST MRI of 3-O-Methyl-D-Glucose on Different Breast Cancer Models Michal Rivlin and Gil Navon* Purpose: To test the ability of chemical exchange saturation transfer (CEST) MRI of 3-O-methyl-D-glucose (3OMG) to detect tumors in several breast cancer models of murine and human origin, for different routes of administration of the agent and to compare the method with glucoCEST and with 18 FDG- PET on the same animals. Methods: In vivo CEST MRI experiments were performed with a 7T Biospec animal MRI scanner on implanted orthotopic mammary tumors of mice before and after administration of 3OMG. Results: A marked 3OMG-CEST MRI contrast that was corre- lated with the administrated dose was obtained in different breast cancer models and by intravenous, intraperitoneal, and per os methods of administration. The most aggressive breast cancer model yielded the highest CEST contrast. 3OMG-CEST contrast reached its maximum at 20 min after administration and lasted for more than an hour, while that of glucose was lower and diminished after 20 min. 3OMG-CEST showed com- parable results to that of FDG PET. Conclusion: The sensitivity of the 3OMG-CEST MRI method indicates its potential for the detection of tumors in the clinic. Magn Reson Med 000:000–000, 2017. V C 2017 International Society for Magnetic Resonance in Medicine. Key words: MRI; breast cancer; chemical exchange saturation transfer; CEST; 3-O-Methyl-D-glucose; PET/CT INTRODUCTION Current clinical MRI assessment of tumors is based on volumetric measurements and morphological criteria, but fails to reflect metabolic changes that often occur without apparent morphological changes. Thus, there is an increasing need to improve the prognosis of early disease detection that takes these changes into account. Alteration in glucose metabolism is considered a hall- mark of cancer (1). Enhanced tumor uptake of glucose or its analogues occurs by means of the overexpression of glucose transporter proteins (GLUTs) widely present in tumor tissues (2,3). The ability of MRI to obtain images of both anatomy and cellular responses led recently to the suggestion that it can function as an in vivo molecu- lar imaging technique. Chemical exchange-dependent saturation transfer (CEST) (4–7) has been proposed as a new molecular imaging approach to detect glucose (or its analogues) for diagnosing tumors in view of its high sen- sitivity and specific molecular targeting (8–12). The CEST MRI method permits direct detection of millimolar concentrations of exchangeable hydroxyl protons of glu- cose (or its analogues) by means of the water signal (110M), enabling the monitoring of its uptake. 2-deoxy-D-glucose (2DG) and 2-deoxy-fluoro-D-glucose (FDG) exhibit CEST MRI enhancements in tumors that are larger and longer lasting than those of D-glucose (10,11). However, their toxicity at high concentrations precludes their clinical use in humans and limits them to experiments in laboratory animals. The nonmetaboliz- able glucose analogue 3-O-methyl-D-glucose (3OMG), which is considered nontoxic, was recently suggested as an alternative (12). This agent exhibited pronounced uptake and produced a significant CEST MRI signal in a tumor from a D1-DMBA-3 cell line derived from a poorly differentiated mammary adenocarcinoma induced in BALB/C mice by dimethylbenzanthracene (13). The pre- sent work examined the validity of 3OMG-CEST MRI to detect tumors in several other murine and human breast cancer models in which the agent was administered intravenously (IV), intraperitoneally (IP), or per os (PO), and compared the results with those obtained from glu- coCEST as well as 18 FDG-PET on the same mice. METHODS Chemicals and Media 3OMG and D-glucose were obtained from Sigma-Aldrich, Israel. Solutions of 3OMG and D-glucose were prepared in 10 mM phosphate-buffered saline (PBS). Tumor-Bearing Animals Cell Culture 4T 1 (mouse mammary cancer cells), MDA-MB-231 and MCF7 (human mammary cancer cells) were obtained from American Type Culture Collection (ATCC, Manas- sas, VA). Cells were cultured in high glucose DMEM supplemented with 10% PBS, 100 mg/mL penicillin, 100 m/mL streptomycin, 12.5 m/mL nystatin, and 2 mM L-glutamine (Sigma, Israel) at 37  C in a humidified atmosphere containing 5% CO 2 . Orthotropic Mammary Fat Pad Implantation BALB/C or nu/nu or severe combined immune defi- ciency (SCID-ICR) female mice were purchased and housed in the breeding facility of Tel Aviv University. Orthotropic xenograft tumors were induced in the mice by injecting 4T 1 cells or human MDA-MB-231 or MCF7 cells (10 6 /100 mL cells) into the lower left mammary School of Chemistry, Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, Israel. *Correspondence to: Gil Navon, School of Chemistry, Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978. E-mail: navon@post.tau.ac.il Received 14 December 2016; revised 15 April 2017; accepted 20 April 2017 DOI 10.1002/mrm.26752 Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary. com). Magnetic Resonance in Medicine 00:00–00 (2017) V C 2017 International Society for Magnetic Resonance in Medicine 1