Abstract Introduction Metformin (MET) may improve tumor oxygenation and thus radiotherapy (RT) response. However, appropriate imaging biomarkers for patient stratification, critically needed to be able to advance to clinical trials with MET as a radiosensitizer, are still lacking. In this proof-of-concept (POC) study, we first assessed the effect of acute MET administration on NSCLC xenograft tumor hypoxia with PET using the novel hypoxia PET tracer [18F]HX4. Second, we verified the influence of a single dose of MET prior to RT on treatment outcome. Experimental procedures A549 tumor-bearing mice (n=9; animals were inoculated in both hind legs) underwent a [18F]HX4 PET/CT scan to determine baseline tumor hypoxia. The next day, mice were administered 100 mg/kg MET IV. [18F]HX4 was administered IV 30 min. later, whereupon a second PET/CT scan was performed to assess changes in tumor hypoxia. Two days after this second scan, mice were randomized into three groups (n=3/group): a control group (1), a RT group (2), and a MET+RT group (3) with comparable tumor volumes (259±103; 218±81 and 247±121 mm3, resp.). Animals were administered 0.9% saline (groups 1-2) or 100 mg/kg MET (group 3) IV, followed by a single dose of 10 Gy 30 min. after administration (groups 2-3). Control animals of group 1 underwent sham RT. During the entire study, tumor growth was monitored triweekly by caliper measurements. Calculation of the relative tumor volumes (RTV) was initiated when tumors reached a mean baseline volume of 100 mm3 with the formula RTV=Vtime x/Vbaseline. Tumor growth inhibition (TGI) was calculated for each tumor in both treatment groups with the formula TGI=1-(RTVtreated/RTVcontrol). Results MET significantly altered A549 tumor hypoxia, as the mean [18F]HX4 tumor-to-blood ratio (TBR) was reduced from 3.03±0.27 to 2.82±0.25 (p=0.040) after MET administration. The tempering influence of MET on tumor hypoxia improved RT response, as fifteen days after irradiation TGI was 63±7% in the MET+RT group as compared to only 36±9% in the RT group, however significance was not reached. To date, tumors are still being measured triweekly and this will be continued until a volume of 1500 mm3 is reached. Then, animals will be sacrificed for histological validations on tumor tissue to assess differences in a.o. proliferation and hypoxia. Conclusions Using non-invasive imaging, we showed in A549 xenograft tumors that MET acts as a radiosensitizer possibly by decreasing tumor hypoxia. Our results imply that [18F]HX4 PET imaging holds potential as a predictive biomarker for the beneficial effect of MET in the treatment of NSCLC; however, the small group sizes in this POC study did not allow us to draw sound conclusions. These promising findings will be validated in a larger follow-up study in order to validate [18F]HX4 PET as a predictive biomarker for MET and RT response. Citation Format: Sven De Bruycker, Christel Vangestel, Tim Van den Wyngaert, Steven Deleye, Dominique Vanderghinste, Steven Staelens, Sigrid Stroobants. [18F]HX4 shows potential as a predictive biomarker for the radiosensitizing capacities of metformin in a NSCLC xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1875. doi:10.1158/1538-7445.AM2017-1875