Abstract 3878: Subcellular characterization of over 100 proteins in FFPE tumor biopsies with CosMx Spatial Molecular Imager

Abstract 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.