HSIEN-NENG HUANGCHUN-WEI KUOHung, Yu-LingYu-LingHungYang, Chia-HungChia-HungYangHsieh, Yu-HanYu-HanHsiehLin, Yu-ChiehYu-ChiehLinChang, Margaret Dah-TsyrMargaret Dah-TsyrChangLin, Yen-YinYen-YinLinJEN-CHUNG KO2024-07-152024-07-152024-07-02https://pubmed.ncbi.nlm.nih.gov/38956114/https://scholars.lib.ntu.edu.tw/handle/123456789/719769Assessing programmed death ligand 1 (PD-L1) expression through immunohistochemistry (IHC) is the golden standard in predicting immunotherapy response of non-small cell lung cancer (NSCLC). However, observation of heterogeneous PD-L1 distribution in tumor space is a challenge using IHC only. Meanwhile, immunofluorescence (IF) could support both planar and three-dimensional (3D) histological analyses by combining tissue optical clearing with confocal microscopy. We optimized clinical tissue preparation for the IF assay focusing on staining, imaging, and post-processing to achieve quality identical to traditional IHC assay. To overcome limited dynamic range of the fluorescence microscope's detection system, we incorporated a high dynamic range (HDR) algorithm to restore the post imaging IF expression pattern and further 3D IF images. Following HDR processing, a noticeable improvement in the accuracy of diagnosis (85.7%) was achieved using IF images by pathologists. Moreover, 3D IF images revealed a 25% change in tumor proportion score for PD-L1 expression at various depths within tumors. We have established an optimal and reproducible process for PD-L1 IF images in NSCLC, yielding high quality data comparable to traditional IHC assays. The ability to discern accurate spatial PD-L1 distribution through 3D pathology analysis could provide more precise evaluation and prediction for immunotherapy targeting advanced NSCLC.enHigh dynamic rangeImmunofluorescenceNon-small cell lung cancerProgrammed death ligand 1Three-dimensional pathology[SDGs]SDG3journal article10.1038/s41598-024-65187-x38956114