Abstract The advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. While spatial transcriptomics has traditionally led the way in plex, multiple studies have demonstrated a poor correlation between RNA expression and protein abundance, owing to transcriptional and translational regulation, target turnover, and most critically, post-translational protein modifications. Therefore, a more holistic, ultra-high-plex, and high-throughput proteomic atlas approach becomes critical for the next phase of discovery biology. Here, we present a barrier-breaking, spatial proteomics panel that was designed to accelerate scientific discoveries. A Digital Spatial Profiler platform is uniquely suited to support high-plex proteomics, allowing for the simultaneous analyses of proteins from discrete regions of interest (ROIs) in FFPE tissue sections while preserving spatial context. The assay relies upon abcam antibodies coupled to photocleavable DNA barcodes readout with NGS sequencing, allowing for theoretically unlimited plex. Here we introduce the Human Immuno-Oncology Proteome Atlas (IPA), a 570+ antibody-based proteomic discovery panel, compatible with immunohistochemistry on FFPE tissues with NGS readout. IPA is the highest-plex, most comprehensive, antibody-based multi-omic panel to date focusing on key areas of immuno-oncology, oncology, immunology, epigenetics, metabolism, cell death, and signaling pathway regulation. Here we demonstrate the performance of IPA on various cell lines and tissue. Additionally, we show the power of IPA, using the spatial multi-omic assay along with the GeoMx® Whole Transcriptome Atlas (> 18,000 transcripts), a 40-plex custom antibody panel and microbiome-curated RNA custom spike-in (~220 transcripts) to evaluate 70 different colon disease samples across 5 pathologies including adenocarcinoma, hyperplasia, and chronic inflammation. This is the highest-plex multi-omic (~610-plex proteins and >18,220 genes) study ever implemented for spatial biology. When we compared the diseased tissue to normal tissue, we observed an upregulation of specific pathways associated with tumorigenesis and inflammation. Furthermore, we observed distinct differences in proteomic and transcriptomic landscape between pathologies. The cutting-edge, data-driven, expert-curated IPA panel is at the forefront of spatial proteomics, empowering the researcher for the acceleration of biological discoveries. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Alyssa Rosenbloom, Shilah Bonnett, Mark Conner, Christine Kang, Erin Piazza, Brian Filanoski, Rhonda Meredith, Hye Son Yi, Lori Hamanishi, Eduardo Ignacio, Nadine Nelson, Michael Prater, Vik Devgan, Rudy Van Eijsden, Melanie Moon, Lesley Isgur, Terence Theisen, Margaret Hoang, Gary Geiss, Joseph M. Beechem. A novel spatial multi-omic approach for biological discoveries in colonic diseased tissues using a comprehensive Immuno-Oncology Proteome Atlas and Whole Transcriptome Atlas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3649.

The brain is complex and heterogeneous where cell function and cell-to-cell communication are critical for rapid and accurate performance. The ability to explore protein-driven activities at high resolution within spatial context of their immediate environment is critical to gain comprehensive pictures of brain development, activity, aging, disease or dysfunction, and inflammatory responses. Many existing approaches for high-plex single cell spatial proteomics face issues around simplicity, speed, scalability, and big data analysis. Here, we present an integrated workflow from sample through analysis that addresses key concerns around high plex proteomics. The CosMx ™ Spatial Molecular Imager and AtoMx ™ Spatial Informatics Platform comprises an end-to-end workflow that efficiently handles highly multiplex protein analysis at plex sizes exceeding 68 targets. The CosMx protein assays uses oligonucleotide conjugated antibodies, detected using universal, multi-analyte CosMx readout reagents. The CosMx Mouse Neural Cell Typing and Alzheimer’s Pathology panel is optimized to comprehensively profile neural cell lineages across the brain as well as the progression of Alzheimer’s disease (AD), including specific antibodies for humanized mouse AD models. The AtoMx spatial informatics platform provides full analysis support, including whole-slide image viewer, and methods for performing built-in or fully customizable analyses for cell typing, ligand-receptor analysis, neighborhood analysis and spatial differential expression. The CosMx protein assay reagents were validated on FFPE adult mouse brain, mouse embryo, and Alzheimer’s positive human brain. We used the CosMx Mouse Neural Cell Typing and Alzheimer’s Pathology panel with the CosMx Spatial Molecular Imager to identify multiple neuronal subtypes, different reactive states of astrocytes and microglial, cell degeneration and proliferation. Single cell exploration of mitochondria showed distinct patterning of key immune targets based on their immediate microenvironment. CosMx SMI is a high-plex spatial multi-omics platform that enables detection of > 68 proteins at subcellular resolution. In combination with the high-plex CosMx Mouse Neural Cell Typing and Alzheimer’s Pathology panel, we present a flexible and scalable informatics platform, a robust solution for comprehensive neural and disease phenotyping that captures the complexity of neuronal and glial cellular activity with full spatial context. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Detecting and analyzing large numbers of proteins using whole-slide imaging is critical for a comprehensive picture of immune response to cancer. Many existing approaches for high-plex proteomics face issues around simplicity, speed, scalability, and big data analysis. Here, we present an integrated workflow from sample preparation through downstream analysis that addresses many key concerns around high plex proteomics. The CosMx Spatial Molecular Imager (SMI) and AtoMx Spatial Informatics Platform (SIP) comprise of a turnkey, end-to-end workflow that efficiently handles highly multiplex protein analysis at plex sizes exceeding 110 targets. We demonstrate an extension of our commercially available 64-plex human immuno-oncology panel to higher numbers of targets and show how the cloud computing-enabled AtoMx SIP allows flexible construction of analytic pipelines for cell typing and spatial analyses. The CosMx protein assay uses antibodies conjugated with oligonucleotides, which are detected using universal, multi-analyte CosMx readout reagents. The CosMx Human Immuno-oncology panel was optimized to comprehensively profile lymphoid and stromal lineages within the tumor microenvironment as well as markers of cancer signaling and progression. Each CosMx SMI antibody was validated on multi-organ FFPE tissue microarrays covering prevalent solid tumor types with matched controls, and 52 human FFPE cell lines, including overexpression lines for key targets such as GITR, CD278, PD-L1, and PD-1. CosMx SMI uses a deep learning algorithm to segment whole cells and a semi-supervised algorithm to classify cell types. The AtoMx SIP provides full analysis support, including a whole-slide image viewer, and methods for performing built-in or fully customizable analyses for cell typing, ligand-receptor analysis, neighborhood analysis and spatial differential expression. Within the cancer sample profiled, we performed in-depth single-cell proteomic profiling across different cell populations. We detected TLS, characterized TLS maturation, and identified immune interactions with the tumor microenvironment. The CosMx SMI assay profiled the composition and spatial organization of infiltrating immune cells within and around the tumor microenvironment. We found that markers of T cell activation and exhaustion varied across the tumor landscape. CosMx SMI is a high-plex spatial multi-omics platform that enables detection of more than 110 proteins at subcellular resolution in real-world FFPE tissues. The extensibility of the CosMx protein assay to large numbers of protein targets and our flexible, scalable bioinformatic platform provides a straightforward and robust solution for comprehensive immune phenotyping with full spatial context. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Tien Phan-Everson, Zachary Lewis, Giang Ong, Yan Liang, Emily Brown, Liuliu Pan, Aster Wardhani, Mithra Korukonda, Carl Brown, Dwayne Dunaway, Edward Zhao, Dan McGuire, Sangsoon Woo, Alyssa Rosenbloom, Brian Filanoski, Rhonda Meredith, Kan Chantranuvatana, Brian Birditt, Hye Son Yi, Erin Piazza, Jason Reeves, John Lyssand, Vik Devgan, Michael Rhodes, Gary Geiss, Joseph Beechem. A complete pipeline for high-plex spatial proteomic profiling and analysis on the cosmxtm spatial molecular imager and atomtm spatial informatics platform. [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 4617.

Background Patient response to immunotherapy has been revolutionary but remains limited due to the inability to convert excluded or cold tumors into ones which would be permissive to therapeutic intervention. To treat patients that evade immune therapy, comprehensive understanding of their tumor microenvironment (TME) is needed. To date, most profiling efforts have lacked the ability to capture high-plex 'omics data while retaining the spatial architecture of the TME. We developed the CosMx™ Spatial Molecular Imager for analyzing formalin-fixed paraffin-embedded (FFPE) or fresh-frozen (FF) tissue and capturing the expression of over 1000 RNA targets simultaneously with subcellular resolution from a single histopathology slide. Methods We profiled a cohort of 10 patient samples with CosMx using the Human Universal Cell Characterization Panel across a range of solid tumors. This cohort represents a diverse array of patients which include both infiltrated and excluded tumors. We included technical replicates for 5 of the samples to better understand reproducibility of the assay. We were able to characterize over 1.8 million cells, and detected on average over 80% of the panel per patient and assigning more than 95% of the transcripts profiled to unique cells across these samples. Results We were able to robustly identify more than 20 cell types by integrating our data with previous single-cell sequencing projects from the human cell atlas. We demonstrate robust delineation of critical immune cell populations from across lymphoid and myeloid lineages, as well as stromal cell populations, including cell types frequently missed using dissociated cell sequencing, such as vascular endothelium associated with immune cell migration into the tumor bed. We leveraged 450+ genes from our panel dedicated to cell lineage, cell-cell interaction, and ligand-receptor signaling to identify unique interactions happening at different scales between the tumor and the TME. These include evidence of direct inhibition of T-cell function through the PDL1 axis between tumor, and tumor intrinsic and extrinsic interactions that mediate tissue architecture and T-cell exclusion. Conclusions The CosMx platform for profiling tissue allows for robust resolution of critical immunogenic signaling cascades and cellular interactions that are necessary to truly understand the tumor architecture. By maintaining the tissue structure, we can directly measure cellular interactions and capture cells commonly missed during dissociative studies. With this new platform, we are better poised than ever to truly understand the molecular mechanisms which drive tumor response to intervention. FOR RESEARCH USE ONLY Not for use in diagnostic procedures.

Background The spatial interactions between the immune system and tumor cells greatly influence antitumoral immunity, patient prognosis, and therapeutic efficacy. However, few methods exist to query large numbers of immune biomarkers at subcellular spatial resolution. Launched earlier this year, the CosMx™ Spatial Molecular Imager (SMI) platform captures high-plex single cell and subcellular detection of proteins from FFPE tissues. To detect these key drivers of cancer progression and immune cell activation states and functions, we designed and validated a high-plex protein panel for the CosMx SMI platform. This panel contains 4 markers for cell segmentation and pre-experimental imaging and 64 barcoded antibodies emphasizing immuno-oncology focused targets. Methods The CosMx protein assay uses antibodies conjugated with oligonucleotides, which are detected using universal, multi-analyte CosMx readout reagents. The fully automated CosMx instrument carries a widefield water immersion objective with 1.1 NA. The CosMx Human Immuno-oncology panel was optimized to comprehensively phenotype lymphoid and stromal lineages within the tumor microenvironment. The CosMx protein assay reagents were validated on multi-organ FFPE tissue microarrays covering prevalent solid tumor types and matched controls, and 52 human FFPE cell lines, including overexpression lines for key targets such as GITR, CD278, PD-L1, and PD-1. Results We achieved 86% sensitivity and 90% specificity across well-characterized human cell lines compared to GeoMx spatial profiling, and further benchmarked to multiple orthogonal datasets (e.g., the Human Protein Atlas, low-plex IHC). Within the tissue sample profiled, we captured immune cell localization across and within the tumor, key signaling markers related to lymphoid and myeloid activation such as checkpoint engagement, and myeloid cell polarization and antigen cross-presentation markers. We made the data from the study profiling cancer tissue freely available online. This includes the protein images, AI-based cell segmentation, and the per-cell protein abundance profiles. Conclusions CosMx SMI is a high-plex spatial multiomics platform that enables detection of more than 64 proteins at subcellular resolution in real-world FFPE tissues. Our 64-plex human immuno-oncology protein panel enables in-depth study of the tumor microenvironment, including markers covering cell typing and lineage, immune activation, and checkpoints. This new platform will enable researchers to collect high-plex protein immunophenotyping data and understand immune molecular mechanisms with full spatial context. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.