Introduction Chondrosarcoma (a malignant tumor of cartilage) is poorly vascularized and rich in proteoglycans (PGs).1 If the hypoxic and proteoglycan status of the tumor can be assessed by TEP imaging2 and scintigaphy3,4,5 it requires however 2 separated acquisitions. In this context we propose to develop an MRI strategy based on Chemical Exchange Saturation Transfer (CEST) to simultaneously co-register both hypoxia (pH) and PGs content in vivo. Methods The work hypotheses were tested in phantoms containing chondroitin sulfate A or creatine to validate PGs and pH imaging, respectively. NUDE mice (n=6) were implanted with HEMC-SS cells (human chondrosarcoma) in contact to tibia 7 weeks before imaging. MRI images were acquired at 11.7 T using a 40-mm quadratic volume coil. DWI was first performed to localize the tumor, then WASSR (B1=0,1μT for 1.5s, Δωsat=±1000Hz in 20Hz steps) and CEST Z-spectra (B1=1.5μT for 4s, Δωsat=±3000Hz in 50Hz steps) were acquired based on a RARE protocol.31P MRS was performed thanks to a volume coil. 23Na imaging was recorded by using a 20-mm surface coil. Data were analyzed using an in-house program written in Matlab®R2017a. After correction for B0 inhomogeneity, the CEST maps were generated. Results/Discussion Variations in PGs concentration and in pH (fig. 1) were observed in vitro by CEST MRI by monitoring the magnetization transfer ratio asymmetry at 450 (-OH) and 1000Hz (-NH), respectively. These moieties allow extracting the PG concentration and the pH, respectively. In vivo (fig. 2) results showed an increase of asymmetry at 450Hz in the chondrosarcoma compare to the muscle. This increase of asymmetry is expected as during the pathology development the PG concentration increase. In vivo, we also observed an increase of asymmetry at 1800Hz in the chondrosarcoma associated with acidosis in the hypoxic core. The two CEST images are currently being validated by 23Na imaging (GAG concentration) and 31P MRS (pH). Conclusions CEST MRI can be used as a new strategy for non-invasive assessment of chondrosarcoma. Indeed, CEST MRI offers the possibility to image in the same exam the 2 mains characteristics of this tumor: pH and PGs contain. CEST MRI is able to identify and differentiate zones of hypoxia in-vivo.