Comprehensive 40-marker cyclic IF panel for spatial profiling of head and neck squamous cell carcinoma

Introduction Head and neck squamous cell carcinoma (HNSCC) incidence has increased by at least 23 % globally over the last ten years and is predicted to continue to rise by 30 % annually. Treatment for HNSCC often includes a multidisciplinary approach (i.e., chemotherapy + surgery) but success rates are still limited with less than half surviving more than two years post-treatment (Nieszporek et al., 2025). Significant improvements in the number of biomarkers that can be screened at once, while preserving tissue integrity, have been made over the last decade. These staining and imaging improvements have contributed to the identification of novel therapeutic targets while limiting the egregious use of precious tissues. This study aimed to interrogate the tumor microenvironment of head and neck squamous cell carcinoma using the same tissue slice using a fully automated cyclic IF system. Methods Two panels of 20 biomarkers each were designed using immune markers, tissue architectural markers, and specific targets of interest in head and neck squamous cell carcinoma. Panel 1 was comprised of I/O markers (CD3, CD4, CD8, FoxP3, CD56, CD20, CD68, CD11c, aSMA, PD-L1, PD-1, CD45, CD27, CTLA-4, CD19, PCNA, CD14, CD16, SOX10, CD79a) while Panel 2 was largely comprised of discovery markers (ALDH2, IL-8, CK17, MMP9, MAGE-A4, EpCAM, EGFR, CK14, CK19, CK5/6, p53, CD44, ZEB1, ZEB2, beta-Catenin, E-Cadherin, Vimentin, COL1A1, COL4A1, FAP). Optimization was achieved using FFPE tissue microarrays containing normal and cancerous skin tissues. Pre-processing steps were done in the Epredia© PT Module using Tris-EDTA ph9 solution, at 100°C, for 1 hour. Panel 1 was stained first followed by Panel 2 without removal of the tissue from the instrument. Automated immunofluorescent staining and imaging of the samples was performed on the Lunaphore COMET™ system. Results The sequential staining of two 20-plex protein panels on the same tissue demonstrates the COMET's capability of maximizing the number of biomarkers that can be evaluated. This protocol significantly reduces the use of tissue necessary, helps maintain tissue integrity for downstream processing (i.e., H&E staining), and minimizes costs by reducing chip and reagent use. Conclusions This comprehensive interrogation of the HNSCC tumor landscape with the use of high-plex, fully automated sequential immunofluorescent staining highlights many of the capabilities of the latest spatial technologies. In using this approach, deeper dives of tumor microenvironments can be achieved with minimal tissue use while keeping reagent and material costs low. References: 1. Nieszporek, A., Wierzbicka, M., Khan, A., Jeziorny, M., et al. Spatial profiling technologies for research and clinical application in head and neck squamous cell cancers. Current Research in Biotechnology. 2025. Vol 10: 100321.