REZCUE: Rescuing 3D spatial omics from archived FFPE tissue

Abstract

Archived formalin-fixed, paraffin-embedded (FFPE) tissue represents a vast clinical resource, yet three-dimensional imaging and omics-wide molecular profiling remain largely incompatible because current antigen retrieval and clearing can degrade nucleic acids. We developed REZCUE, a depth-invariant antigen retrieval chemistry that enables 3D multiplexed immunofluorescence imaging of intact FFPE tissue while quantitatively preserving DNA, RNA, and protein for multi-omics analyses. REZCUE restores antigenicity in tissue archived for up to 30 years, supports 160 commercial whole-IgG antibodies across tissue types, and enables imaging to 6 mm depth. Imaging before destructive molecular profiling establishes a 3D spatial reference for registering molecular assays to intact tissue architecture. REZCUE enabled multi-modal mapping of spatial niches of the mouse brain and identified a human breast tumor compartment not distinguishable by conventional section-based spatial-omics, revealed after integrating 3D image-guided segmentation with spatial transcriptomics, mass spectrometry, miRNA sequencing, and whole-genome sequencing. In short, REZCUE transforms archived FFPE specimens into spatially registered, molecularly resolved, 3D substrates for sequential spatial multi-omic interrogation.

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