Animal studies

The Institutional Animal Care and Use Committees (IACUC) of the University of Pennsylvania approved experimental protocols, and all experiments were carried out in accordance with approved IACUC guidelines.

Lymphoma tumor model: Preparation of lymphoma xenografts. Male athymic nude mice (n=4) (01B74) 4-6 weeks of age were obtained from the National Cancer Institute, Frederick, MD, USA. The mice were housed in microisolator cages and had access to water and autoclaved mouse chow ad libitum.

The tumor cells used in this study were diffuse large B-cell lymphoma cells from the WSU-DLCL2 cell line, and were kindly provided by Drs Mohammad and Al-Katib (Wayne State University, Detroit, MI, USA). The cells were grown as described in detail by Al-Katib et al. (Mohammad, Wall et al. 2000): briefly, the cells were “maintained in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 1% l-glutamine, 100

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units/ml penicillin G, and 100ug/ml streptomycin. The cells were incubated in a humidified 5% Co2 atmosphere at 37°C.”

Cells were implanted subcutaneously into the right thigh of 4-6-week-old male athymic nude mice (01B74) 4-6 weeks (National Cancer Institute) by injecting ten million WSU- DLCL2 cells in 0.1mL Hanks’ Balanced Salt Solution (without calcium or magnesium; Invitrogen/Gibco, Carlsbad, CA, USA). Lymphoma xenografts were allowed to grow until the tumor volume reached ~500 mm3. The tumor dimensions were measured with calipers in three orthogonal directions, and the volume was calculated using the equation, V= π(a×b×c)/6, where a, b, and c are the length, width, and depth of the tumor.

Lymphoma tumor model: Imaging and spectroscopy. For imaging and spectroscopy experiments at 9.4T (horizontal bore) of the lymphoma tumor mice, we used a custom- built single frequency (1H) slotted tube resonator (inner diameter = 13 mm, outer diameter = 15 mm, depth = 16.5 mm).

The mice were anaesthetized and maintained under 1% isoflurane in 100% oxygen, supplied at 1 L/min for the duration of the experiment, which did not exceed three hours per IACUC guidelines. The animal body temperature was maintained at 37 ± 1°C with the air generated and blowing through a heater (SA Instruments, Inc., Stony Brook, NY). Respiration and body temperature were continuously monitored using a MRI-compatible small animal monitor system (SA Instruments, Inc., Stony Brook, NY). CEST imaging was performed as described for phantoms at 9.4T with the following sequence parameters: field of view = 25 × 25 mm2, slice thickness = 3 mm, flip angle = 15°, TR =

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6.2 ms, TE = 2.9 ms, matrix size = 128 × 128. CEST images were collected with B1rms =

1.17 µT for frequencies ranging from -1.5 ppm to + 1.5 ppm from bulk water in step size of 0.1 ppm. B1 and WASSR B0 field maps were also acquired and used to correct the

CEST maps as described previously (Kogan, Haris et al. 2013).

Following acquisition of the baseline CEST images, sodium pyruvate (300 mM) was delivered through a tail vein catheter (26 Gauge, I.V. Catheters FEP, Tyco Healthcare, Tyco International Ltd., Schaffhausen, Switzerland) at a variable rate using a syringe pump (Harvard Apparatus, Holliston, MA, USA) using the following protocol: (10ml/hr, 1min; 3ml/hr, 4min; 2.5ml/hr, 2min; 2.0ml/hr, 2min; 1.5ml/hr, 2min; 1.0ml/hr, 2min; 0.5ml/hr, 57min) making a constant blood pyruvate concentration of 13 mM during the experiment. CEST images were then acquired after the infusion.

Tumor lactate was also measured using HADAMARD SEL-MQC 1H-MRS (He, Shungu et al. 1995, Pickup, Lee et al. 2008) in separate experimental sessions. For lactate measurements following pyruvate infusion, tumors were positioned in a home-built, single-frequency (1H), slotted-tube resonator (inner diameter, 13 mm; outer diameter, 15 mm; depth, 16.5 mm). A slice-selective double-frequency Hadamard-selective multiple quantum coherence transfer pulse sequence was used to detect lactate and to filter out overlapping lipid signals. The acquisition parameters were as follows: sweep width = 4 kHz; 2048 data points; TR = 8 s; 128 scans. Since there is no published lactate visibility data on this tumor model we have chosen to correlate the lactate peak amplitude values with the LATEST results.

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9L brain tumor model in rats. LATEST was also implemented to probe the lactate dehydrogenase (LDH) activity in vivo in a rat glioma model. Syngeneic female Fisher F344/NCR rats were used to generate the model. MRI experiments were performed on the 9.4T MRI scanner on rats that had been injected with 9L cells intracranially 3 weeks prior. During the imaging experiments, tumor-bearing rats were maintained under 1-1.5% isourane in O2, supplied at 1 L/min. Throughout the experiment, the body temperature

was maintained at 37º C. T2 weighted imaging was performed on the brain to locate the

tumor region in coronal slices. For these experiments, the LATEST sequence parameters were: slice thickness=2 mm, GRE flip angle=5º, GRE readout TR=5.6 ms, TE=2.7 ms, FOV=30×30 mm, matrix size=128×128, number of averages=4. LATEST images from 0 to ±1.5 ppm (step size 0.25 ppm) were collected at 1.17 µT saturation pulse power (B1)

and saturation duration of 4s. B0 correction on the LATEST images was performed by

acquiring WASSR images at 0.24 µT from -1 to +1 ppm in steps of 0.1 ppm. LATEST maps were computed using the method described above. Following the acquisition of the baseline CEST signal, sodium pyruvate (300 mM) was delivered through a tail vein at a rate of 100uL/min via a syringe pump. Following the 15-min infusion of pyruvate (for a total volume of 1.5mL), the CEST acquisitions were performed over a period of 90 minutes.