Glioblastoma is an aggressive brain tumor characterized by a high heterogeneity that is assumed at the origin of failure of some therapies. This heterogeneity depends on different parameters among which the extracellular/extravascular pH (pHe) acidification. The assessment of such tumor hallmark could be useful to stage the tumor, to monitor its development, to follow the therapy outcome and, above all, to set innovative therapies specifically targeted against acidic tumor regions (responsive pH treatments). Recently, it had been reported that Yb-HPDO3A can be an efficient CEST (Chemical Exchange Saturation Transfer) pHe-responsive MRI probe¹ and here this method is applied for glioma mapping in U87-bearing mouse model at 4.7T. The feasibility of using such chemical has been herein reported and pHe-maps have been correlated with histology ( H/E, Hif-1a and Ki-67 staining).Yb-HPDO3A has been used as CEST agents for MRI pHe-mapping at 4.7T. This agent is an analogue of clinically Gd-HPDO3A (ProHance^®) obtained by replacing Gd by Yb. In vitro CEST-MRI has been performed on Yb-HPDO3A (20 mM) at different pHe for calibrations. In vivo experiments on U87-bearing glioblastoma nude mice (n=18) have been performed at late stage of tumor growth when the blood brain barrier (BBB) is break-down, since the Yb-HPDO3A cannot cross the intact BBB. Yb-HPDO3A has been intravenously injected at 1 mmol/Kg dose. CEST-MRI was a combination of presaturation pulse (rectangular pulse of 2s and 24μT) followed by RARE sequence with a short echo time TE=5.4ms and a repetition time TR=5s. Z-spectra have been acquired using 89 different presaturation frequency irradiations in the range of ±112ppm. Contrast enhancement T₂* images before and after Yb-HPDO3A administration (that is also a T₂* agent) have been acquired to detect tumor regions with BBB leakage and Yb-HPDO3A distribution in the brain. Ratiometric approach (signal difference between the two main isomers TSAP and SAP of Yb-HPDO3A versus pHe, was demonstrated independent from the probe concentration and it was used in vitro for Saturation Transfer (ST%) measurement and after for in vivo pHe mapping. Experimental CEST points of Z-spectrum have been interpolated according to standard method². At the end, upon mouse sacrifice and brain excision, histological analysis has been carried out by using Hematoxylin/Eosin (tumor size and staging) and staining for Hif-1a (hypoxia marker) and Ki-67 (proliferation marker). All these data have been evaluated to assess changes eventually occurring between smaller and bigger glioblastoma.The TSAP and SAP isomers of Yb-HPDO3A display OH chemical shifts at 71 ppm and 99 ppm respectively and were well resolved at 4.7T (Fig.1). Saturation Transfer (ST%) of the two hydroxyl protons display a different pH-dependence, thus it is possible to obtain a ratiometric curve in which the Ratio between ST% of the two isomers is plotted against the pH value (Fig.1C). In Fig.1D, a representative CEST image of glass capillaries filled with 20mM YbHPDO3A at different pH values has been reported. It shows that it is feasible to use this CEST probe for evaluating environmental pH at 4.7T. Glioblastoma mice have been analyzed to map pH and images of a representative mouse have been reported in Fig.2. For each mouse, T_2w images have been obtained to localize tumor (Fig.2A). Moreover, since YbHPDO3A is both a CEST and a T₂ agent, T₂* difference (post-pre) images have been obtained to evaluate the distribution of the probe in the tumor region (Fig.2B). Finally, CEST pH-maps are reported in Fig.2C. The analyzed tumor shows heterogeneous pHe distribution, in which the tumor core appears more acidic than the rim. At the end, representative histological analysis (H/E and Ki-67 staining) has been reported in Fig.2D. It has been demonstrated that cell proliferation rate is higher in bigger tumor than in smaller ones. Yb-HPDO3A can be used as CEST pHe probe at 4.7T. Herein it has been demonstrated as proof of concept that this probe can be applied for mapping glioblastoma extracellular/extravascular pH at 4.7T. It has potential in a clinical setting as this molecule has the same stability and the in vivo pharmacokinetic properties of the approved clinical Gd-HPDO3A. The assessment of glioblastoma pH can be useful for tumor staging and characterization as well as for developing innovative therapies targeted to acidic tumor regions. A relevant difference has been noted in pHe values between small and big glioblastoma tumors because the latter ones are highly heterogeneous, with an acidic core whereas the former ones are more homogenous with pHe closer to physiological values.