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Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .

Cell annotation is a crucial methodological component to interpreting single cell and spatial omics data. These approaches are often biased and manually curated. Here we harness an existing stemness model for assessing oncogenic states to transform its application to single cell and spatial omic datasets. This one-class logistic regression machine learning algorithm is used to extract transcriptomic or proteomic features from non-transformed stem cells to identify dedifferentiated cell states. We found this method identifies single cell states in metastatic tumor cell populations without the requirement of cell annotation. Finally these stemness indices are applicable across a variety of spatial transcriptomic and proteomic technologies for the identification of oncogenic cell types in the tumor microenvironment.Graphical Abstract

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.

The spatial interactions between the immune system and tumor cells greatly influence antitumoral immunity. Characterization of immune cell composition and infiltration within the tumor niche informs prognosis, drug delivery efficiency, and therapeutic efficacy. However, few methods exist to query large numbers of immune biomarkers at subcellular spatial resolution. The CosMx™ Spatial Molecular Imager is the first platform to demonstrate simultaneous single-cell and subcellular detection of over 100 proteins on standard, biobanked, FFPE tissue samples. This high-plex protein panel detects key drivers of cancer progression and immune cell activation states. Here, we apply the CosMx 100-plex immuno-oncology assay on a set of breast cancer biopsies and demonstrate its quantitative and spatial capabilities. Key to CosMx protein technology is an antibody-oligonucleotide-conjugate 64-bit encoding method, not a cyclic exchange method. The encoding scheme is enabled by a 20nm hybridization-based optical barcode. The CosMx system uses a fully automated, cyclic microfluidics imaging system, high-resolution optics and 3D capability. The raw cyclic encoded 4-color tissue images are decoded using a robust automated decoding algorithm that detects protein sub-cellular localization and quantifies expression level. CosMx SMI produces protein localization maps for each target, which characterizes tissue microenvironment heterogeneity while providing spatial information. Additionally, accurate segmentation of individual cells enables spatial single-cell protein expression analysis, facilitating further mining and analyses of cellular subpopulations. The CosMx protein assay reagents were validated on multi-organ FFPE tissue microarrays and 35 human FFPE cell lines, including overexpression lines for key targets and cellular activation states, such as GITR, CD278, PD-L1, and PD-1. Benchmarking to multiple orthogonal datasets (e.g., the Human Protein Atlas, Cancer Cell Line Encyclopedia, and low-plex IHC) demonstrates that the assay is highly sensitive and specific. CosMx SMI protein assay can be coupled with SMI’s 1000-plex RNA-detection assay; together, such a multi-omics platform can generate an unprecedented information-rich view of spatial biology that could usher in novel discoveries about health and disease. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Zachary R. Lewis, Tien Phan-Everson, Gary Geiss, Mithra Korukonda, Ruchir Bhatt, Carl Brown, Dwayne Dunaway, Joseph Phan, Alyssa Rosenbloom, Brian Filanoski, Rhonda Meredith, Kan Chantranuvatana, Yan Liang, Emily Brown, Brian Birditt, Giang Ong, Hye Son Yi, Erin Piazza, Vikram Devgan, Nicole Ortogero, Patrick Danaher, Sarah Warren, Michael Rhodes, Joseph Beechem. Subcellular characterization of over 100 proteins in FFPE tumor biopsies with CosMx Spatial Molecular Imager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3878.

The Spatial Molecular Imaging platform (CosMxTM SMI, NanoString Technologies, Seattle, WA) utilizes high-plex in-situ imaging chemistry for both RNA and protein detection. This automated instrument provides 1000’s of plex, at high sensitivity (1 to 2 copies/cell), very low error rate (0.0092 false calls/cell) and background (∼0.04 counts/cell). The imaging system generates three-dimensional super-resolution localization of analytes at ∼2 million cells per sample, four samples per run. Cell segmentation is morphology-based using antibodies, compatible with FFPE samples. Multiomic data (980 RNAs, 108 proteins) were measured at subcellular resolution using FFPE tissues (non-small cell lung (NSCLC) and breast cancer) and allowed identification of over 18 distinct cell types, 10 unique tumor microenvironments, and 100 pairwise ligand-receptor interactions. Over 800,000 single cells and ∼260 million transcripts data are released into the public domain allowing extended data analysis by the entire spatial biology research community.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the huntingtin gene (HTT). Although HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced activin signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFβ signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of activin response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the 2D gastruloid, leading to ectopic subcellular localization of TGFβ receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first 2 weeks of human development, as modeled by our 2D gastruloids., Summary: 2D gastruloids of isogenic human embryonic stem cells modeling Huntington's Disease reveal that huntingtin CAG expansion perturbs the spatial restriction of the activin response in the context of the polarized epithelium.

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the Huntingtin gene (HTT). While HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced ACTIVIN signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFβ signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of ACTIVIN response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the gastruloid, leading to ectopic subcellular localization of TGFβ receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first two weeks of human development as modeled by our gastruloids.

Using self-organizing human models of gastrulation, we previously showed that (i) BMP4 initiates the cascade of events leading to gastrulation; (ii) BMP4 signal-reception is restricted to the basolateral domain; and (iii) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically-presented NOGGIN could inhibit basally-delivered BMP4. Apically-imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-to-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.

Using self-organizing human models of gastrulation, we previously showed that (i) BMP4 initiates the cascade of events leading to gastrulation; (ii) BMP4 signal-reception is restricted to the basolateral domain; and (iii) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically-presented NOGGIN could inhibit basally-delivered BMP4. Apically-imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-to-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.