VectraPolarisData A Bioconductor ExperimentHub package providing formatted, open-source, experiment-scale single-cell multiplex immunofluorescence data 1

• Surprisingly little multiplex-imaging data is publicly available • VectraPolarisData package addresses this • Bioconductor ExperimentHub package as of April 2022 • Includes 2 experiment-scale single cell multiplex immunofluorescence datasets • Data provided in tabular format post segmentation and phenotyping VectraPolarisData Package 2

VectraPolarisData datasets HumanLungCancerV3 • Non-small cell lung carcinoma ROIs • Vectra3 mIHC, CU-AMC • 761 images from 153 patients • 1,604,786 • 7 markers • 5 phenotypic (CD3, CD8, CD14, CD19, CD68, ck) • 1 functional (HLADR) • Patient-level outcomes HumanOvarianCancerVP • High-grade serous ovarian cancer tumor microarray • VectraPolaris mIHC, CU-AMC • 128 patients, 1 image each • 1,610,431 cells • 8 markers • 5 phenotypic (CD3, CD8, CD68, CD19, CK) • 2 functional (pstat3, IER3) • Patient-level outcomes 3

• Leverage existing data structures in Bioconductor to store and analyze MI data 1. Data Packages through Bioconductor’s Experiment Hub 2. Tools for conversion for different MI platforms to SpatialExperiment class 3. End-to-end example analysis pipelines/tutorials Uses SpatialExperiment,an existing framework in Bioconductor for spatial transcriptomics (spatially-resolved RNA sequencing data) A Bioconductor framework for multiplex imaging data 4

Comparing Spatial Transcriptomics and Multiplex imaging Spatial Transcriptomics • Spatial analog of single cell RNA-seq • Targets mRNA • Lower spatial resolution • Spot-based, sometimes 10+ cells • NanoString has new single-cell technology • Very high dimensional Multiplex Imaging • Spatial analog of single cell flow/mass cytometry • Targets protein • Higher spatial resolution • Truly single-cell • Lower dimensional • Not as much emphasis on dimension reduction 5

• assays slot • Gene counts • spatialData slot • Cell/spot X and Y coordinates • colData slot • Information about specific cells/spots • rowData slot • Information about genes The Bioconductor SpatialExperiment Class 6 Righelli, Weber, Crowell, Pado, Collado-Torres, Ghazanfar, Lun, Hicks, and Risso, Bioinformatics, 2022

• assays slot • Stores mean marker intensity values for each cell • spatialData slot • Cell X and Y coordinates • colData slot • Cell size characteristics • Marker std dev, other summary stats • metaData slot • Patient characteristics Converting MI data to SpatialExperiment 7

• SpatialExperiment S4 object format • Enables immediate use with tools in the Bioconductor ecosystem that interact with SpatialExperiment, SingleCellExperiment, and SummarizedExperiment objects • Code in vignette to transform to a data.frame • 254 downloads and counting VectraPolarisData Package 8