Highly Sensitive Spatial Host‐Microbiome Transcriptomics in FFPE Tissues via Iterative Hydrogel Expansion

Spatial transcriptomics (ST) of archival formalin-fixed paraffin-embedded (FFPE) tissues is fundamentally limited by a trade-off between spatial resolution and transcriptomic sensitivity due to severe molecular crowding and RNA degradation. Here, we present Ex-spRandom, a highly sensitive platform that overcomes this limitation by directly converting diverse, fragmented RNA biotypes into stably anchored cDNA through the integration of random-primed in situ cDNA synthesis chemistry with interpenetrating polymer network (IPN)-based tissue expansion chemistry. This physicochemical synergy physically decrowds the dense FFPE matrix, directly converting highly fragmented host and microbial RNAs into stably anchored cDNA. Consequently, Ex-spRandom improves spatial signal confinement while enabling the detection of nearly 10,000 median genes per 50-µm spatial bin. Leveraging this synergistic high resolution and sensitivity, the platform captures over 44,000 unique host genes, enabling the precise delineation of continuous neurodevelopmental trajectories in the complex embryonic eye. Furthermore, Ex-spRandom achieves simultaneous, spatial profiling of host epithelial architecture and microbial transcript signals in the colon, detecting over 150,000 unique microbial genes. Ultimately, this scalable platform establishes a framework for sensitive, cross-kingdom spatial profiling in archival FFPE tissues.