Abstract Programmed death ligand-1 (PD-L1) is the only clinically approved predictive biomarker for cancer immunotherapy; however, its expression is highly heterogeneous and routinely assessed by ex vivo immunohistochemistry. To enable spatially resolved, noninvasive PD-L1 quantification, we employed fluorescence lifetime (FLT) tomography with a normalization strategy designed to account for nonspecific probe uptake. C57BL/6 mice bearing orthotopic RIL-175 liver tumors (n=10) were injected with αPDL1-800 (anti-PD-L1 antibody conjugated to IRDye 800CW). 48 hours post-injection, we performed in vivo time-domain FLT imaging with co-registered CT, followed by in situ FLT imaging and western blotting to validate PD-L1 expression. Bi-exponential fitting of the time-resolved fluorescence data yielded spatial maps of FLT (τ) and amplitude (a). The top 10% of FLTs within the tumor were averaged to define the bound lifetime component (τb), while the mean FLT in the normal liver was considered as the FLT of the unbound component (τᵤ). Amplitudes of bound (ab) and unbound (aᵤ) probes were extracted and segmented into tumor (ab, T; aᵤ, T) and normal (ab, N; aᵤ, N) ROIs. Among four normalization strategies tested, one correcting for background cross-talk (ab, T - aᵤ, T) and normalizing by total uptake (ab, N - aᵤ, N) showed the strongest correlation with PD-L1 expression (R2 = 0. 77). 3D reconstructions using asymptotic time-domain analysis confirmed accurate tumor localization and size. FLT tomography distinguished bound from unbound αPDL1-800 and enabled quantitative assessment of PD-L1 expression in deep-seated tumors. The normalization method enabled accurate quantification of PD-L1 expression across tumors, supporting the utility of FLT imaging for preclinical immunotherapy evaluation and biomarker-guided therapy monitoring. Citation Format: Rahul Pal, Murali Krishnamoorthy, Xin Liu, Satoru Morita, Atsuyo Morita, Hak Soo Choi, Dan Duda, Anand T. Kumar. In-vivo fluorescence lifetime tomography for detection and quantification of programmed death ligand-1 abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (7 Suppl): Abstract nr 725.
Pal et al. (Fri,) studied this question.