3D NIR-II FMT-XCT imaging for quantitative analysis of PD-L1 expression in lung cancer and tumor-draining lymph nodes facilitating immunotherapy efficacy assessment: an exploratory study

Abstract Background Accurate evaluation of programmed death-ligand 1 (PD-L1) expression is crucial for guiding immunotherapy in lung cancer. Conventional biopsy-based immunohistochemistry (IHC) fails to capture the spatial and temporal heterogeneity of PD-L1 expression. This study aims to develop a near-infrared II fluorescence molecular tomography system integrated with X-ray CT (NIR-II FMT-XCT) and a PD-L1-targeted fluorescence probe (aPD-L1-ICG) for three-dimensional, quantitative imaging of PD-L1 in lung cancer and lymph nodes. Methods We integrated the NIR-II FMT-XCT system with the PD-L1-targeted probe (aPD-L1-ICG) for deep tissue penetration and three- dimensional (3D) imaging of PD-L1 expression in lung cancer models. This approach was tested on lung cancer cell lines, patient-derived xenografts (PDXs), resected human lung tumors, and lymph nodes. NIR-II FMT-XCT performance was compared with 2D NIR-II FMI and immunohistochemistry. In patients receiving neoadjuvant chemoimmunotherapy, we assessed the associations of post-treatment ex vivo NIR-II FMT signal-to-background ratio (SBR) and PD-L1 IHC measurements in resected specimens with the preceding radiographic response and post-treatment pathological response. Results 3D NIR-II FMT achieved a 1.73 ± 0.27-fold higher SBR than 3D NIR-I FMT. Reconstructed signals were associated with PD-L1 immunohistochemistry and demonstrated spatial variation within tumors and lymph nodes. In 28 patients receiving neoadjuvant chemoimmunotherapy, tumor NIR-II FMT SBR correlated with CT-based tumor shrinkage (r = 0.6076, P < 0.001). Post-treatment ex vivo NIR-II FMT SBR was inversely associated with residual viable tumor proportion (Pearson r = −0.5332, P = 0.0035), whereas tumor-cell PD-L1 expression was not (r = −0.1119, P = 0.571). Conclusions NIR-II FMT-XCT provides a three-dimensional platform for quantifying PD-L1-targeted fluorescence in preclinical models and resected human tissues. In the exploratory neoadjuvant cohort, post-treatment ex vivo tumor SBR was associated with the preceding radiographic tumor shrinkage and lower residual viable tumor proportion. These retrospective post-treatment associations support further prospective investigation but do not establish pretreatment predictive utility or clinical net benefit.