78 Mapping the tissue composition with hyperplex immunofluorescence on delicate samples

BackgroundIn health and disease, cells are interacting with their microenvironment, which shapes their homeostasis and response to any stimuli.1 Deciphering the tissue architecture emerged as a crucial step to better understand tissue biology and harness it in a future therapeutic intervention.2 Recently, advancements were made to interrogate the tissue spatial composition with the development of immunofluorescence-based hyperplex assays,3,4 allowing the investigation of dozens of biomarkers on a single tissue slide. There is a growing interest to apply hyperplex assay on fresh frozen sections (FS), yet harsh procedures used in manual protocols are detrimental to tissue morphology and limit the use of this application.5 Furthermore, manual protocols are laborious and time-consuming, allowing to process a limited number of samples. Here, we describe the capability to perform automated hyperplex assays for up to 32 biomarkers on fragile FS tissues as a cogent case combining staining quality with tissue preservation.MethodsCOMET™ platform allows performing sequential immunofluorescence (seqIF™) assays based on an iterative series of fast tissue staining, imaging, and antibody elution cycles. Fresh frozen tissue sections of human and murine origins were fixed and permeabilized prior to loading on the device for automated staining protocols. Multiplex assay was performed for up to 32 biomarkers on a single tissue slide. To assess the tissue morphology preservation, standard hematoxylin and eosin (H&E) staining was performed on FS freshly processed or post-staining protocol.ResultsIn this study, a sequential immunofluorescence protocol was automated and optimized on COMET™ for both human and mouse frozen tissue sections. Murine spleen, brain and lung tissues were stained with multiplex panels of up to 6 proteins and showed an accurate detection of both common immune and organ-specific biomarkers. For a deep characterization of human lung cancer, 32 biomarkers were simultaneously detected on a single FS with optimal staining and a total procedure time of 23 hours. An H&E staining performed on the slide retrieved from the platform after the multiplex protocol showed excellent preservation of the tissue architecture in comparison with an unprocessed FS slide.ConclusionsThis work proves the feasibility of performing automated hyper-plex assays on a variety of delicate frozen samples with high-quality results. With this new toolbox, we aim to support and hasten discovery studies across multiple research fields overcoming current limitations in tissue spatial biology.ReferencesQuail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 2013; 19(11):1423–37. Allam M, Cai S, Coskun AF. Multiplex bioimaging of single-cell spatial profiles for precision cancer diagnostics and therapeutics. npj Precis. Onc. 2020; 4(11). Mund A, Brunner AD, Mann Unbiased spatial proteomics with single-cell resolution in tissues. Molecular Cell 2022; 82:2335–2349. Palla G, Fischer DS, Regev A, Theis FB. Spatial component of molecular tissue biology. Nat Biotech. 2022; 40: 308–318. Hickey JK, Neumann EK, Radtke AJ, Camarillo JM, Beuschel RT, Albanese A, McDonough E, Hatler J, Wiblin AE, Fisher J, Croteau J, Small EC, Sood A, Caprioli RM, Angelo RM, Nolan GP, Chung K, Hewitt SM, Germain RN, Spraggins JM, Lundberg E, Snyder MP, Kelleher NL, Saka SK. Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging. Nat Methods. 2022; 19: 284–295.