PEGPH20 is a PEGylated form of recombinant human hyaluronidase PH20. The purpose of this study is to investigate physiologic changes in pancreatic adenocarcinoma xenograft after PEGPH20 treatment using multi-modal imaging and further determine the utility of PEGPH20 as radiosensitizer. PEGPH20 treatment significantly increased intratumor pO2, blood perfusion and volume, and decreased glycolytic flux assessed by EPR, MRI with USPIO or Gd, and 13C DNP MRI, respectively. PEGPH20 enhanced treatment effect of radiotherapy. The results validated the utility of the imaging methods to monitor the changes in the tumor microenvironment after hyaluronan depletion and to predict the radio-sensitizing effect of PEGPH20.