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Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells are largely based on transcriptomic single-cell approaches that lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient coverage depth, several multiplexed protein imaging methods have recently been developed. Though these antibody-based technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this perspective, we provide essential resources and key considerations for obtaining robust and reproducible multiplexed antibody-based imaging data compiling specialized knowledge from domain experts and technology developers., Comment: {\ddag}authors contributed equally to this work

Understanding the role of distinct cellular phenotypes in tissue function, development, and pathogenesis requires tools that can rapidly and consistently quantify the expression of multiple proteins while preserving spatial information. To meet this need, we have developed Multiplexed Ion Beam Imaging by Time-Of-Flight (MIBI-TOF). MIBI-TOF uses secondary ion mass spectrometry to visualize up to 50 metal labeled antibodies at subcellular resolution simultaneously with histochemical stains and native biological elements. We have used this capability to study how single cell function and tissue structure interact at the tumor immune microenvironment, the human maternal-fetal interface, and in infectious disease. These studies have led us to develop scalable, automated, and extensible tools for image thresholding, single cell segmentation, cell clustering, and cell neighborhood analyses that can be deployed on any tissue. Through NIH HuBMAP and CIMAC initiatives, we are currently working to deploy this computational pipeline as well as preformulated antibody staining master mixes as a streamlined, end-to-end workflow for routine high dimensional tissue analysis in basic and translational research. Lastly, we are currently working to develop brighter primary ion sources and new metal reporter constructs that will permit full tissue section scanning while extending multiplexed detection to include RNA and DNA. Citation Format: R. Michael Angelo. Relating single cell function to tissue structure in human tumors using MIBI-TOF [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA04.