Abstract 5642: Validation of a novel multiplex immuno-fluorescence panel for the spatial analysis of the tumor microenvironment
Authors: Rivest François , de Gautard Victor , Navikas Vytautas , Brajkovic Saska , Nicolai Bastian 
Published: April 2023

Abstract
IHC counterparts. Subsequently, the panel was transferred on a multi-organ TMA including several tumoral and non-tumoral specimens, showing robust performance across multiple tissue types. The protocol was optimized to achieve high staining quality for all 13 markers in terms of signal specificity Investigation of the tumor microenvironment (TME) by multiplex immunofluorescence (mIF) has accelerated the understanding of the spatial immune context in tumors. mIF has proven to be a powerful technique for the identification of new potential biomarkers and therapeutical targets. Despite increased application of mIF assays to characterize the TME, state-of-the art protocols remain technically challenging. Manual execution and use of dedicated reagents render them lengthy and costly. Moreover, their reproducibility is often questioned together with their transferability between different tissue types.
Here, we show the development and validation of an Immuno-Oncology (IO) Core Panel of 13 clinically relevant biomarkers to enable spatial analysis of the immune TME on the COMET™ platform across various tissue types. Formalin-fixed paraffin-embedded human tissue sections from tonsil and a 24-cores multi-organ tissue microarray (TMA) were stained using the IO Core Panel from Lunaphore on the COMET™ platform by fully automated sequential immunofluorescence (seqIF™), which consists of cycles of staining, imaging, and elution. The panel allows for simultaneous detection of CD3, CD4, CD8, CD45, FoxP3, PD1, PD-L1, CD11c, CD20, CD56, CD68, aSMA and Ki-67 by indirect immunofluorescence using unlabeled primary antibodies and Alexa Fluor™ Plus secondary antibodies. The 13-plex IO Core panel was initially developed and validated on tonsil as positive control tissue. To compare immunofluorescent (IF) and immunohistochemistry (IHC) staining patterns, the sections retrieved from COMET™ after seqIF™, were stained by a histology facility with standard IHC established for routine pathological diagnosis. All markers demonstrate accurate detection with specific IF staining, comparably to gold-standard, sensitivity, ratio to background and dynamic range. The repeatability and reproducibility of the automated IO Core Panel on the COMET™ platform was verified by day-to-day tests on one platform and tests among multiple platforms, respectively.
Our study demonstrated the robustness of the validated IO Core Panel across multiple tissue types with highly specific and reproducible results. The marker detection with standard indirect immunofluorescence on the COMET™ instrument allows for future, rapid expansion and customization of the panel including additional primary antibodies towards the need of the individual underlying scientific question.
Citation Format: François Rivest, Victor de Gautard, Vytautas Navikas, Saska Brajkovic, Bastian Nicolai. Validation of a novel multiplex immuno-fluorescence panel for the spatial analysis of the tumor microenvironment. [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 5642.