Abstract: Immunotherapy using programmed death 1 receptor (PD-1) blockade, either alone or in combination with existing therapies, has been proven to significantly improve survival rates for many cancers, including triple negative breast cancer (TNBC). However, only about a quarter of the patients respond to treatment, typically those with programmed death ligand 1 (PD-L1)-positive tumors, while a majority experience serious drug related side effects. The efficient selection of likely responders to immunotherapy is limited by the fact that biopsy, the current screening standard for PD-L1 expression, provides only a snapshot of biomarker status at a single time point, while it is known that PD-L1 expression can dynamically change during therapy. Additionally, tumor vascular “normalization” indicators, such as perfusion, hypoxia and angiogenesis can dynamically change during treatment, potentially serving as early indicators of treatment efficacy. There is therefore an urgent need for non-invasive imaging techniques that can longitudinally quantify molecular and physiological predictive tumor biomarkers before and during treatment. Such techniques can potentially save non-responders from ineffective treatment and life-threatening effects and can also facilitate a robust evaluation of new combination therapies that improve survival and prove effective in a larger patient population. Our preliminary studies using time domain fluorescence imaging indicate that the fluorescence lifetime (FLT) of immune-receptor targeted near infrared probes is longer in PD- L1 positive tumors compared to non-specific probe in normal tissue, thereby dramatically improving sensitivity and specificity compared to fluorescence intensity-based imaging. Furthermore, time domain imaging allows the simultaneous detection and quantification of multiple fluorophores using spectral and lifetime contrast (multiplexing) and is therefore ideal for imaging multiple molecular and physiologic parameters of treatment response. The goal of this proposal is to translate these powerful benefits of FLT to validate tomographic FLT imaging as a new tool for multiplexed longitudinal monitoring of biomarkers during immunotherapy. We will validate the accuracy of the optical readouts for monitoring therapeutic response longitudinally in TNBC-bearing mice by comparison with histology. The feasibility of fluorescence imaging has previously been demonstrated for superficial lymph nodes and for organs such as the breast. Therefore, validation of FLT multiplexing in preclinical models is a fundamental step that will lead to targeted clinical trials to evaluate TD technology for non-invasive functional immunotherapy screening in TNBC patients.