Project Summary Positron emitters result in the detection of coincident 511 keV annihilation photons. Thus, it is not possible to perform dual radiotracer studies in PET as is done SPECT, where energy discrimination may be performed. This grant application's significance lies in that it proposes a method to separate two PET radiotracers injected during the same PET imaging session. As a proof of principle, we shall inject two radiotracers commonly employed in cancer imaging: FMISO (tumor hypoxia) and FLT (cellular proliferation). If successful, our study would show the feasibility to provide the spatial delineation of tumor hypoxia and proliferation within the same imaging session. Although it will be applied to both, our approach is not restricted to FMISO and FLT; it could be applied to a number of radiotracer pairs. As is the case of PET/MR, dual tracer imaging makes it possible to measure two phenotypic properties of the tumor, which may vary temporally when the tumor is in the same state. Moreover, it offers greater patient convenience than sequential acquisitions, e.g., on separate days. In this project, therefore, we propose to validate a method to simultaneously image tumor hypoxia and proliferation within a single PET study in an animal tumor model. A dynamic PET acquisition is performed while the two radiotracers, 18FMISO and 18FLT, are injected sequentially, closely staggered in time. Retrospective 18FMISO /18FLT separation of imaging signals will be based on a priori knowledge of tissue pharmacokinetics and the ability to distinguish these two radiotracers in blood samples by thin-layer chromatography (TLC). If successful, this approach, which is immediately translatable into the clinic, would set the stage for patient studies in which dual radiotracer studies would be useful.