Abstract 4658: Flexible multiplexed immunofluorescent panels for accelerated identification of spatial signatures for immunotherapy checkpoint investigations
Authors: Circelli Jacob , Schaefer Rachel , Perez Oscar , Liu Linying , McLane Michael , Zheng Yi 
Published: June 2023

Abstract
A fundamental step in the characterization of the tumor microenvironment (TME) is the identification of the distinct immunologic phenotypes in various cancer types. The spatial arrangement of these cells and their co-expression patterns serve as an increasingly important tool for the identification of a novel class of highly predictive biomarkers for immunotherapy response knows as spatial signatures. To study the complex biological processes within the TME and develop clinically useful spatial signatures, it is imperative to take an approach that combines relevant content with flexibility, speed, and throughput. We recently introduced PhenoCodeTM Signature Panels that offer researchers the ability to stain for multiple biomarkers including their marker of choice at a single cell resolution on a single tissue in a scalable end-to-end automated workflow. Incorporating Akoya’s novel barcoded antibody labeling chemistry, these panels enable all primary antibodies to be applied as a cocktail in a single incubation step, followed by the sequential amplified detection of each marker via Opal fluorescent dye technology. Previously, we have presented data on 3 panels that address key questions for the characterization of the TME, including whether the tumor is “hot” or “cold,” the identity of the immune cells comprising the TME, the proliferative state of tumor cells and the activation state of the immune cells. Here we showcase two new PhenoCode Signature panels that allow researchers to further characterize the TME by examining immune cell exhaustion and macrophage polarization, key events that contribute to an immunosuppressive TME. In this study, staining was performed using all five PhenoCode Signature panels and DAB on the Leica BOND RXTM automated stainer. Human formalin-fixed, paraffin embedded (FFPE) lung cancer tissues were stained and whole slide multispectral scans were acquired on the PhenoImager HT® platform. Image analysis was performed with a phenotyping algorithm in inForm® and staining intensities were analyzed in R using Phenoptr and PhenoptrReports. The fluorescent staining of each marker was benchmarked to the DAB staining. Each panel was proven to be both highly accurate and reproducible. Together these five PhenoCode Signature panels provide researchers a rapid and robust method for characterizing the TME to aid the development of spatial signatures which have been shown to be highly effective at predicting therapeutic outcomes
Citation Format: Jacob Circelli, Rachel Schaefer, Oscar Perez, Linying Liu, Michael McLane, Yi Zheng. Flexible multiplexed immunofluorescent panels for accelerated identification of spatial signatures for immunotherapy checkpoint investigations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4658.