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86 Reveal spatial signatures of tumor microenvironment and oncogenic pathways using 6,000-plex single-cell spatial molecular imaging on FFPE tissue
  1. Shanshan He1,
  2. Michael Patrick2,
  3. David Kroeppler2,
  4. Patrick Danaher2,
  5. Jason Reeves2,
  6. Mark Gregory2,
  7. Julian Preciado2,
  8. Olivia Ho-shing2,
  9. Haiyan Zhai2,
  10. Michael Rhodes2 and
  11. Joseph Beechem2
  1. 1NanoString Technologies, Inc., Redmond, WA, USA
  2. 2NanoString Technologies, Inc., Seattle, WA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Tumor progression and therapeutic response are regulated by the tumor microenvironment.1 Understanding the spatial association and architecture of molecular characteristics and composition of tumor immune microenvironment at single-cell and subcellular resolution encourages improvements in clinical prognosis and immunotherapy benefits.2 Single-cell Spatial profiling technologies permit the study of transcriptional activity at the spatial, single-cell level and provide abundant, high-resolution information required for the identification of clinical-related features in immune-oncology.

Methods We performed an ultra-high-plex RNA assay to detect 6,000 targets simultaneously in situ on FFPE human cancerous tissues using the CosMx™ Spatial Molecular Imager (SMI). For the selection of regions of interest, four protein markers and DAPI were co-detected on the same tissue slide. Tertiary analysis algorithms were developed for cell typing, co-localization of genes and ligands, cell-cell interaction, and pathway analysis.

Results Thousands of transcripts were simultaneously detected with high sensitivity and specificity on the FFPE cancerous tissue section at single-cell subcellular resolution. We investigated the tumor microenvironment including cell types, their spatial distribution and proportion of diversified immune cells in the tumor compartment. We also assessed distances between immune cells and their nearest functional-related neighbors. Moreover, we revealed the ligands co-localization, as well as spatial patterns of direct cell-cell interactions and signaling pathways.

Conclusions Single-cell spatial measurements of 6,000 RNA and four proteins on the same tissue section, along with a large viewing area on archival FFPE tissue, provide a novel tool to reveal the spatial signature of tumor microenvironment and oncogenic pathways, facilitating the next level of cancer and therapeutic research.

References 1. Wang Q, et al. Role of tumor microenvironment in cancer progression and therapeutic strategy. Cancer Med. 2023;00:1–17.

2. Hu B, et al. A review of spatial profiling technologies for characterizing the tumor microenvironment in immuno-oncology. Front Immunol. 2022 Oct 31;13:996721.

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