Understanding the interactions between cells within their natural tissue environment is crucial for uncovering both normal physiological processes and disease mechanisms. Historically, capturing the full gene expression profile of a cell while preserving the spatial location of transcripts within tissues was a significant challenge. Traditional single-cell RNA sequencing (scRNA-seq) techniques, while powerful, lose spatial context, and earlier spatial transcriptomics approaches were limited in resolution or scope. Today, Bruker Spatial Biology offers an innovative solution that combines subcellular resolution with comprehensive human protein-coding genome coverage, all within an easy-to-implement workflow compatible with archival samples.

Their latest preprint on bioRxiv introduces the CosMx Whole Transcriptome (WTX) assay. This method provides highly detailed subcellular imaging of nearly the entire human protein-coding transcriptome (over 18,000 targets) in formalin-fixed, paraffin-embedded (FFPE) tissues. Leveraging ultra-high-plex barcoded in situ hybridization (ISH) probes alongside advanced imaging and computational techniques, this technology allows for transcript identification and quantification across thousands to millions of cells on a single slide, retaining precise spatial coordinates for each transcript.

Advancing Beyond Single-Cell RNA Sequencing

Single-cell RNA sequencing has transformed how we study cellular heterogeneity, but it comes with limitations. It necessitates enzymatic dissociation of tissues, which can result in the loss of delicate or tightly packed cells and disrupts the spatial organization of tissues. Conversely, the CosMx Spatial Molecular Imager (SMI) workflow maintains the integrity of tissue architecture, enabling the analysis of individual cells in their original locations. This approach supports the exploration of cell-to-cell communication, microenvironmental gradients, and tissue structure without sacrificing transcriptomic complexity.

A comparative study using matched FFPE colorectal carcinoma samples revealed that scRNA-seq underestimated certain cell populations, such as those that were larger or more difficult to isolate. In contrast, the CosMx WTX panel identified a more diverse array of cell types, including epithelial, stromal, immune, and tumor cells, in their natural contexts. This highlights two key advantages of spatial transcriptomics: unbiased capture of all cell types, and preservation of spatial position of every cell for a comprehensive analysis.

Yi Cui (2025), CosMx® SMI Spatially Resolved Whole Transcriptome in FFPE Tissue – A Paradigm Shift for Tissue Analysis, Bruker Spatial Biology, Seattle, WA

A Versatile Tool for Complex Biological Studies

The CosMx WTX assay detects thousands of transcripts per cell across various FFPE tissue types, such as colon, pancreas, breast, brain, kidney, and skin. This method consistently delivered high-quality, reproducible data with four orders of dynamic range and strong alignment with bulk RNA-seq results above a practical detection threshold.

Sub-cellular Imaging of the Entire Protein-Coding Human Transcriptome (18933-plex) on FFPE Tissue Using Spatial Molecular Imaging. Rustem Khafizov, Erin Piazza, Yi Cui, Michael Patrick, Evelyn Metzger, Daniel McGuire, Dwayne Dunaway, Patrick Danaher, Margaret L Hoang, Andrew Grootsky, Megan Vandenberg, Shanshan He, Rachel Liu, Michael McKean, Michael Rhodes, Joseph M Beechem. bioRxiv 2024.11.27.625536; doi: https://doi.org/10.1101/2024.11.27.625536

Approachable Spatial Data Analysis

By utilizing barcoded probes and an image-based readout, the WTX workflow maps gene expression down to subcellular levels. The AtoMx® Spatial Informatics Platform (SIP) streamlines data analysis, efficiently processing billions of transcripts from millions of cells. Advanced cloud computing and machine-learning algorithms enable rapid multi-sample analyses and provide detailed insights into subcellular spatial patterns. This sophisticated approach creates whole-transcriptome maps that facilitate advanced analyses, such as identifying rare cell populations, mapping cell type-specific pathways, and visualizing ligand-receptor interactions in unique tissue niches.

High Reproducibility and Scalability

Rigorous testing demonstrated the reproducibility of the CosMx WTX assay. Three independent experiments conducted on a 36-cell-line FFPE array across varying imaging scales showed high consistency, with correlation coefficients of approximately 0.97. These results affirm that scaling up imaging does not compromise data quality. Additionally, pseudo-bulk WTX profiles closely matched Cancer Cell Line Encyclopedia (CCLE) bulk RNA-seq data, confirming the assay’s reliable gene expression quantification over a broad dynamic range.

Sub-cellular Imaging of the Entire Protein-Coding Human Transcriptome (18933-plex) on FFPE Tissue Using Spatial Molecular Imaging. Rustem Khafizov, Erin Piazza, Yi Cui, Michael Patrick, Evelyn Metzger, Daniel McGuire, Dwayne Dunaway, Patrick Danaher, Margaret L Hoang, Andrew Grootsky, Megan Vandenberg, Shanshan He, Rachel Liu, Michael McKean, Michael Rhodes, Joseph M Beechem bioRxiv 2024.11.27.625536; doi: https://doi.org/10.1101/2024.11.27.625536

Unveiling Tumor Complexity

In one notable example, the study focused on colon adenocarcinoma, integrating CosMx WTX data with pathway analyses. Results revealed heightened activity in pathways like EGFR, TGF-beta, and VEGF within tumor regions, along with reduced apoptotic signals such as TRAIL. These findings indicate an aggressive tumor phenotype driven by pro-survival and pro-angiogenesis mechanisms. The ability to map these expression patterns at a single-cell level highlights the importance of spatial context in uncovering intratumor heterogeneity, offering insights unattainable through dissociative methods. Moreover, the WTX panel’s ability to monitor all pathways simultaneously introduces a groundbreaking “pathways-first” perspective for spatial biology.

Yi Cui (2025), CosMx® SMI Spatially Resolved Whole Transcriptome in FFPE Tissue – A Paradigm Shift for Tissue Analysis, Bruker Spatial Biology, Seattle, WA

Final Thoughts

This study paves the way for whole-transcriptome spatial profiling to become a cornerstone of discovery research, uniting expansive gene coverage with intact tissue context. The CosMx WTX solution removes the need to compromise between high-plex transcriptomic analysis and subcellular resolution.

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