Fluorescence Lifetime Imaging Enables <i>In Vivo</i> Quantification of PD-L1 Expression and Intertumoral Heterogeneity

Patient selection for cancer immunotherapy requires precise, quantitative readouts of biomarker expression in intact tumors that can be reliably compared across multiple subjects over time. The current clinical standard biomarker for assessing immunotherapy response is PD-L1 expression, typically quantified using IHC. This method, however, only provides snapshots of PD-L1 expression status in microscopic regions of ex vivo specimens. Although various targeted probes have been investigated for in vivo imaging of PD-L1, nonspecific probe accumulation within the tumor microenvironment has hindered accurate quantification, limiting the utility for preclinical and clinical studies. Here, we demonstrated that in vivo time-domain fluorescence imaging of an anti-PD-L1 antibody tagged with the near-infrared fluorophore IRDye 800CW (αPDL1-800) can yield quantitative estimates of baseline tumor PD-L1 heterogeneity across untreated mice, as well as variations in PD-L1 expression in mice undergoing clinically relevant anti-PD-1 treatment. The fluorescence lifetime (FLT) of PD-L1-bound αPDL1-800 was significantly longer than the FLT of nonspecifically accumulated αPDL1-800 in the tumor microenvironment. This FLT contrast allowed quantification of PD-L1 expression across mice both in superficial breast tumors using planar FLT imaging and in deep-seated liver tumors (>5 mm depth) using the asymptotic time-domain algorithm for fluorescence tomography. These findings suggest that FLT imaging can accelerate the preclinical investigation and clinical translation of new immunotherapy treatments by enabling robust quantification of receptor expression across subjects. Significance: Fluorescence lifetime imaging can quantify PD-L1 expression across multiple mice undergoing anti-PD-1 treatment, providing a critically needed noninvasive imaging method to quantify immunotherapy targets in vivo.