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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.

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.