Our acidoCEST MRI protocol has been described in a previous report [21 (link)]. Briefly, each mouse was anesthetized with 1.5–2.5 % isoflurane delivered in 1 l/min oxygen gas ventilation and then secured to a customized cradle. A 27-g catheter was inserted into the tail vein. The cradle was inserted into a 7-T Biospec MRI scanner with a 72-mm-diameter quadrature transceiver coil (Bruker Biospin, Inc, Billerica, MA). The breathing rate was monitored, and the core body temperature was regulated at 37.0±0.2 °C using an automated feedback loop between the temperature probe and an air heater (SA Instruments, Inc., Stony Brook, NY). A FLASH MRI acquisition sequence was performed to determine the location of the tumor (500 ms repetition time (TR), 10 ms echo time (TE), 625×625 μm in-plane resolution, 2 mm slice thickness, 64×64 pixels, 4.0×4.0 cm field of view (FOV), 32 s total acquisition time). A spin-echo MRI acquisition sequence was performed for the tumor volume measurements (2,000 ms TR, 35 ms TE, 312.5×312.5 μm in-plane resolution, 1 mm slice thickness, 128×128 pixels, 4.0×4.0 cm FOV, 4:20 min total acquisition time). A bolus of 200 μl iopromide was injected via the catheter within 30 s. The catheter was then connected to an infusion pump, and iopromide was pumped at a rate of 150 μl/h. Our acidoCEST MRI protocol consisted of 54 CEST-Fast imaging with steady-state precession (FISP) MR images acquired at different saturation frequencies using 2.8 μT saturation power and a saturation period of 5 s, which required 4:50 min of acquisition time. The CEST saturation period consisted of a series of Gaussian-shaped radio frequency pulses with an interpulse delay of 10 μs and no additional spoiling of fat saturation pulses. The FISP acquisition used the following parameters: 3.218 ms TR; 1.609 ms TE; 60° excitation angle; 2 mm slice thickness; 250×250 μm in-plane resolution; 3.2×3.2 cm FOV; linear encoding order; unbalanced “FID” mode; and 418.54 ms scan time. This process was repeated six times, for a total acquisition time of 29 min. In a previous study, the concentration of iopromide in tumor tissue was found to be relatively stable with a change of only 6.69 % over the acquisition time frame of 4:50 min. Notably, the images acquired with selective saturation from 7.0 to 2.8 ppm, which primarily impacts the measurements of the CEST effects at 5.6 and 4.2 ppm, were acquired within 1:21 min, which had a relatively stable concentration with a change of only 1.86 % over the acquisition time frame in this previous study. The concentration of water in tumor tissue and the T1 relaxation time of the tumor tissue were assumed to remain constant during the study. At the conclusion of the imaging scan, the mouse was removed from the scanner and cradle and allowed to recover.