Abstract 4616: Automated multiplex immunofluorescence enables single cell analysis of tumor stroma
Authors: Rivest François , de Gautard Victor , Navikas Vytautas , Nelson Nadine , Nicolai Bastian , Kowal Joanna , Brajkovic Saska 
Published: April 2023

Abstract
Background: The tumor microenvironment (TME) consists of malignant cells and supporting non-malignant cellular and non-cellular components that form the tumor stroma. The tumor stroma plays an important role in tumor progression and has emerged as a modulator of anti-tumor immunity (Salmon et al., Nat Rev Cancer 2019) and responses to therapy (Hirata and Sahai, Cold Spring Harb Perspect Med 2017). As such, several therapeutic approaches have recently been developed to target stromal cells as anti-cancer treatments (Valkenburg et al., Nat Rev Clin Oncol 2018, Bejarano et al., Cancer Discov 2021). In addition, the composition of the TME has been recognized as a prognostic factor for survival in cancer patients (Pagès et al., Oncogene 2011). Current protein-based approaches to characterize and better understand the cell composition of the tumor stroma face many limitations such as reagent availability and lengthy protocols. In this study, we identified a list of 22 markers to characterize non-tumoral immune cells, fibroblasts and endothelial cells in the TME, in a single tissue slide. We propose an approach that overcomes reagent incompatibility and opens new avenues of research of tumor stroma.
Method: Multiorgan Tumor Microarray (TMA) was interrogated with a sequential immunofluorescence (seqIF™) panel encompassing protein markers enabling characterization of TME. A 22-plex panel was created based on expanding an already established 13-plex panel (CD3, CD4, CD8, CD11c, CD20, CD45, CD56, CD68, aSMA, FoxP3, Ki67, PD1, PD-L1) by adding 9 additional antibodies (CD11b, CD14, CD31, CD47, CK, FAP, LaminB1, SIRPα, Vimentin). Hyperplex immunofluorescent staining was performed using automated staining-imaging COMET™ platform generating ome-tiff images containing 25 layers: DAPI, 2 autofluorescent and 22 marker channels. Postprocessing of images was done with HORIZON™ image analysis software.
Results: We established a panel of 22 markers that can be analyzed simultaneously on a single tissue slide despite limited variability in primary antibody species. Using seqIF™ protocol allowed the study of colocalization and co-expression of markers not compatible to study simultaneously in the traditional immunofluorescence approach. Our hyperplex data revealed distinct composition of the stromal compartment between different tumor types, highlighting high heterogeneity in the tissue composition and stromal architecture.
Conclusion: COMET™ platform enabled studying in detail TME components and highlighted heterogeneity of tumor stroma across different tissue types. SeqIF™ protocol lifted the limitation imposed by same species antibodies and allowed simultaneous interrogation of markers’ expression preserving their spatial relationship.
Citation Format: François Rivest, Victor de Gautard, Vytautas Navikas, Nadine Nelson, Bastian Nicolai, Joanna Kowal, Saska Brajkovic. Automated multiplex immunofluorescence enables single cell analysis of tumor stroma. [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 4616.