Abstract 3644: Uncover spatial signatures of tumor microenvironment and oncogenic pathways using 6,000-plex single-cell spatial molecular imaging on FFPE skin squamous cell carcinoma
Authors: Patrick Michael , He Shanshan , Ilcisin Saskia , Kroeppler David , Danaher Patrick , Reeves Jason , Gregory Mark , Zhai Haiyan , Rhodes Michael , Beechem Joseph 
Published: March 2024

Abstract
Background: Tumor progression and therapeutic response are regulated by the tumor microenvironment. 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. 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 immuno-oncology.
Methods: We performed an ultra-high-plex RNA assay to detect 6,000 targets simultaneously in situ on FFPE human skin squamous cell carcinoma 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 skin squamous cell carcinoma 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. The cell typing results revealed many cancerous subpopulations, which resolved spatially from cells that lacked any defining marker genes. 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. We found that some genes have elevated expression in macrophages which are near cancer compared to those are far from cancer cells, empowering the molecular investigation of novel signaling between immune and cancer cells in tumor immune microenvironment.
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. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
Citation Format: Michael Patrick, Shanshan He, Saskia Ilcisin, David Kroeppler, Patrick Danaher, Jason Reeves, Mark Gregory, Haiyan Zhai, Michael Rhodes, Joseph Beechem. Uncover spatial signatures of tumor microenvironment and oncogenic pathways using 6,000-plex single-cell spatial molecular imaging on FFPE skin squamous cell carcinoma [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 3644.