Abstract 5307: New developments in spatial transcriptomics unlock insights into the tumor microenvironment

Abstract The emergence of tools enabling researchers to perform high-plex spatial transcriptomics with single-cell resolution has revolutionized our understanding of tumor development. However, tissue samples with degraded RNA or extensive crosslinking present challenges for gene expression measurements. For decades, cancer tissue samples have been collected and preserved using formalin-fixation and embedding in paraffin (FFPE), which is not optimal for preserving RNA integrity. This has limited the depth of insights researchers can obtain from archival samples. The Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) technology facilitates direct RNA profiling in situ with high sensitivity and resolution. The high accuracy and high plexity are enabled by tiling probes along the length of a transcript, which is particularly challenging in samples with fragmented RNA, such as in FFPE. The MERFISH 2.0 chemistry and sample preparation workflow was developed specifically to improve transcript detection efficiency in measurement of up to 1000 genes in low quality tissues. These improvements can be applied to tissues of up to 3cm2 using the MERSCOPE® Ultra™ Platform. Here we applied the improved capabilities of MERFISH 2.0 to profile human breast cancer and human lung cancer tumors over a range of sample qualities. Breast and lung tumor samples, both whole sections and tissue microarrays (TMA), were run on the MERSCOPE Ultra Platform using a panel of 815 immuno-oncology genes with both MERFISH 1.0 and MERFISH 2.0. The resulting data was used for spatial and single-cell analyses. We show that in all tissues, particularly in lower quality tissues, MERFISH 2.0 increases the counts and subsequent quality of data analysis, such as increased detection of cell types and increased neighborhood associations. Inter- and intra-tumor immune profiling showed improved detection of low-expressing markers, enabling deeper study of the tumor microenvironment. Spatially resolved transcriptomic profiling of low-quality samples at single-cell level offers significant opportunities for understanding how cancers develop in situ. These improvements will enable new genomic inquiries into how tumors develop and modulate their environment, which in turn will open up new areas of therapeutic research. Citation Format: Manisha Ray, Justin He, Bin Wang, Bing Yang, Sudhir Tattikota, Timothy Wiggin, Hao Wang, Jichuan Zhang, Lizi Maziashvili, Alexander Genshaft, Peter Reinhold, Brittany Auclair, Robert Mathis, Shawn Wang, Jiang He, George Emanuel. New developments in spatial transcriptomics unlock insights into the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5307.