Intratumoral bacteria are now recognized as biologically relevant members of the tumor microenvironment, but the spatial relationships between microbial communities and host gene expression remain incompletely characterized. A major methodological limitation is that many spatial transcriptomics approaches for microbial detection require fresh frozen tissue, which excludes formalin-fixed paraffin-embedded (FFPE) specimens that constitute the majority of clinical archives. The source study addresses this gap by developing a probe-based approach compatible with the chemistry used by commercial platforms, enabling spatially resolved microbial profiling in FFPE samples.
The authors implemented a custom probe design pipeline that targets variable regions of bacterial 16S rRNA. These probes were formulated to work with the probe-based chemistry of the 10X Genomics Visium CytAssist platform, allowing microbial detection to be integrated into the same experimental workflow used for host transcriptomics in FFPE tissue. The source reports that this integration was achieved by adding the custom microbial probes into the standard Visium workflow.
To demonstrate feasibility, the method was applied to a pilot cohort comprising six FFPE colorectal cancer tumor specimens and matched normal adjacent tissue. The study focused on whether adding microbial probes would permit bacterial detection while preserving the expected structure of host gene expression and cell-type organization in the sections.
According to the source, integration of custom microbial probes into the Visium workflow preserved host transcriptomic structure. Clustering analysis of host gene expression recapitulated expected colonic cell type architecture, indicating that the addition of microbial probes did not disrupt the host transcriptome profiling. This preservation supports the approach as a dual-purpose assay for simultaneous host and microbial spatial profiling in FFPE samples.
Bacterial signal was detected across all six samples in the pilot cohort. The observed signals were spatially patterned and focal, rather than homogeneous, demonstrating the method’s capacity to resolve localized microbial distributions within tissue architecture. Spatially resolved detection thus enabled assessment of where bacterial communities locate relative to host tissue features in FFPE colorectal specimens.
One tumor sample in the pilot cohort exhibited markedly elevated bacterial signal intensity with a distinct invasive distribution pattern. The source attributes this pattern to spatially structured signals from Bacteroides-Phocaeicola and Porphyromonas, which showed divergent intratumoral trajectories. This example illustrates how probe-based spatial metatranscriptomics in FFPE tissue can reveal taxon-specific spatial behavior within tumors.
The work reported is a preprint and has not been certified by peer review. The authors declared no competing interests. The source provided links for data and code access: a GEO accession (GSE334323) and a GitHub repository for the project, indicating that raw data and analysis code are available as reported in the original article.
The source concludes that probe-based spatial metatranscriptomics is feasible in FFPE tissue using custom 16S rRNA probes compatible with the Visium CytAssist platform. The approach preserves host transcriptomic profiling and detects spatially patterned, focal bacterial signals in colorectal cancer and adjacent normal tissue. By enabling microbial detection in FFPE specimens, this method provides a generalizable framework for studying host–microbiome interactions in clinically archived materials where fresh frozen tissue is unavailable. The preprint presents a pilot demonstration and supplies data and code resources; details beyond those reported in the source (for example, specific performance metrics, sensitivity, or broader validation) were not provided in the article text.