Abstract 1726: Novel, high-throughput multiplexed immunofluorescence staining method for assessing spatial relationships of immuno-oncology biomarkers in whole slide FFPE tissue

Abstract

Abstract As cancer research has progressed, the role of the immune system in tumor development has been discovered to be increasingly more important. There is a need to explore the spatial relationships between tumor and immune cells in tissue to help better predict therapeutic treatment outcomes in patients in the growing field of immuno-oncology. We have developed a new single cell multiplexed immunofluorescence staining method along with our patented multi-spectral imaging (MSI) technology to allow scientists to detect up to six biomarkers simultaneously on one tissue section, which provides an end-to-end, fully automated workflow from staining to image analysis. In this study, we show a methodology that allows primary antibodies to be applied as a single cocktail in one incubation step followed by amplified detections using Opal fluorescent dye technology. Formalin-fixed paraffin-embedded (FFPE) samples were stained on the Leica BOND RX࣪ automated staining system using our new multiplex procedure and compared to 3,3′-Diaminobenzidine (DAB) chromogenic immunohistochemistry assay. Multispectral scans were acquired on the Akoya Vectra Polaris® imaging system with optimized acquisition parameters. Image analysis was performed with a phenotyping algorithm in inForm® analysis software and intensity analysis was performed in R using phenoptr and phenoptrReports. The six multiplexed biomarkers are shown to be comparable in staining patterns to their DAB counterparts. Whole slide image scans were successfully unmixed and treated as individual monoplexes, but combined at will to observe the spatial relationships. This easy-to-use staining method simplifies tissue-based multiplex immunofluorescence by providing primary antibody cocktailing and cyclic deposition of fluorescent Opal dyes while maintaining strong staining intensity and the ability to spectrally unmix different wavelengths from the image for visualization and spatial analysis. This novel method will provide researchers with a flexible and readily available solution for staining patient samples to investigate spatial phenotypic signatures, aiding in the development of targeted immune therapies. Citation Format: Rachel Schaefer, Linying Liu, Michael McLane, Oscar Perez, Jacob Circelli, Carla Coltharp, Yi Zheng. Novel, high-throughput multiplexed immunofluorescence staining method for assessing spatial relationships of immuno-oncology biomarkers in whole slide FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1726.