Anand T. N. Kumar

This file contains all the supplementary figures and tables related to this manuscript.

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.

Indocyanine green (ICG) is a well‐established near‐infrared dye which has been used clinically for several decades. Recently, it has been utilised for fluorescence‐guided surgery in a range of solid cancer types, including sarcoma, with the aim of reducing the positive margin rate. The increased uptake and retention of ICG within tumours, compared with normal tissue, gives surgeons a visual reference to aid resection when viewed through a near‐infrared camera. However, the mechanisms of this process are poorly understood. We performed in vitro ICG cellular uptake studies across a panel of sarcoma cell lines exhibiting varying proliferation rates and phenotypes. The effects of ICG concentration, incubation time, inhibition of clathrin‐mediated endocytosis, and cell line proliferation rate on the cellular uptake of ICG were investigated using fluorescence microscopy and flow cytometry. Subcellular localisation of intracellular ICG was assessed via colocalization with a lysosomal marker. The spatial distribution of ICG in patient tumour tissue following fluorescence‐guided surgery was assessed by high‐resolution tissue imaging and quantified using fluorescence lifetime imaging. In vitro results showed that the cell line proliferation rate correlated significantly with ICG uptake (Spearman's rank correlation coefficient = 1.00, p < 0.001), and maximum ICG uptake was observed after 24 h incubation. ICG cellular uptake was significantly reduced by inhibition of clathrin‐mediated endocytosis (p = 0.0004), and intracellular ICG significantly colocalized with a lysosomal marker within 30 min (Pearson's r = 0.8). On histological analysis of tumour tissue from three different sarcoma subtypes, ICG was observed within sarcoma cells as well as accumulating in paucicellular areas of haemorrhage and necrosis within the tumour microenvironment. Through quantification of fluorescence lifetime imaging of ICG, we were able to differentiate sarcoma cells from haemorrhage and necrosis within tumour tissue. Combining in vitro data with analysis of patient tissue, we propose that the uptake and accumulation of ICG in sarcomas is driven by a synergistic mechanism involving the enhanced permeability and retention effect combined with active tumour cell endocytosis of the dye. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

We present a technique for fast wide-field fluorescence lifetime (FLT) imaging using simultaneous steady-state and time- (or frequency-) domain measurements acquired in a single shot and exploiting the theoretical dependence of fluorescence intensity on FLT. Using theory, simulations, and in vitro experiments, we show that the single-shot FLT (S-FLT) method can provide absolute FLTs at a higher signal-to-noise ratio compared to other high-speed FLT imaging techniques, without the need for sample-dependent system calibration or model training. We demonstrate that S-FLT can provide accurate tumor delineation in real-time during surgical resections in preclinical tumor models in vivo and in clinical patient specimens ex vivo. We also extend S-FLT for real-time multiplexing and demonstrate this technique for quantifying dynamic FLT changes in vitro and for multiplexing of organ-targeted NIR fluorophores in vivo. Our results suggest that S-FLT can enable real-time quantitative high-throughput studies in preclinical applications and accurate tumor margin delineation in real-time during surgeries.

This file contains all the supplementary figures and tables related to this manuscript.

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.

Cancer patient selection for immunotherapy is often based on programmed death-ligand-1 (PD-L1) expression as a biomarker. PD-L1 expression is currently quantified using immunohistochemistry, which can only provide snapshots of PD-L1 expression status in microscopic regions of ex vivo specimens. In vivo imaging using targeted agents can capture dynamic variations of PD-L1 expression in entire tumors within and across multiple subjects. Towards this goal, several PD-L1 targeted molecular imaging probes have been evaluated in murine models and humans. However, clinical translation of these probes has been limited due to a significant non-specific accumulation of the imaging probes and the inability of conventional imaging modalities to provide quantitative readouts that can be compared across multiple subjects. Here we report that in vivo time-domain (TD) fluorescence imaging can provide quantitative estimates of baseline tumor PD-L1 heterogeneity across untreated mice and variations in PD-L1 expression across mice undergoing clinically relevant anti-PD1 treatment. This approach relies on a significantly longer fluorescence lifetime (FLT) of PD-L1 specific anti-PD-L1 antibody tagged to IRDye 800CW (αPDL1-800) compared to nonspecific αPDL1-800. Leveraging this unique FLT contrast, we show that PD-L1 expression can be quantified across mice both in superficial breast tumors using planar FLT imaging, and in deep-seated liver tumors (> 5 mm depth) using the asymptotic TD algorithm for fluorescence tomography. Our results suggest that FLT contrast can accelerate the preclinical investigation and clinical translation of novel molecular imaging probes by providing robust quantitative readouts of receptor expression that can be readily compared across subjects.

The surgical resection of solid tumours can be enhanced by fluorescence-guided imaging. However, variable tumour uptake and incomplete clearance of fluorescent dyes reduces the accuracy of distinguishing tumour from normal tissue via conventional fluorescence intensity-based imaging. Here we show that, after systemic injection of the near-infrared dye indocyanine green in patients with various types of solid tumour, the fluorescence lifetime (FLT) of tumour tissue is longer than the FLT of non-cancerous tissue. This tumour-specific shift in FLT can be used to distinguish tumours from normal tissue with an accuracy of over 97% across tumour types, and can be visualized at the cellular level using microscopy and in larger specimens through wide-field imaging. Unlike fluorescence intensity, which depends on imaging-system parameters, tissue depth and the amount of dye taken up by tumours, FLT is a photophysical property that is largely independent of these factors. FLT imaging with indocyanine green may improve the accuracy of cancer surgeries.