Illuminating the Role of A-to-I Editing in Gastric Cancer using EndoVIA 2.0

Background & AimsAdenosine-to-inosine (A-to-I) RNA editing is an essential post-transcriptional modification catalyzed by ADAR enzymes, and emerging evidence suggests its dysregulation can contribute to cancer. However, technical barriers have hindered spatial analysis of editing activity in formalin-fixed paraffin-embedded (FFPE) tissues--an abundant but challenging sample type. Here, we introduce EndoVIA 2.0, an optimized immunostaining assay that enables spatial detection of edited RNAs in FFPE tissues. MethodsUsing human Endonuclease V (hEndoV) as a molecular affinity reagent, we developed a calcium-dependent staining workflow compatible with crosslinked tissues. EndoVIA 2.0 was validated in cell lines with altered ADAR1 expression, fresh frozen tissues, gastric organoids, and FFPE tissue sections. Quantitative imaging was combined with machine-learning segmentation and spatial analysis. ResultsEndoVIA 2.0 successfully detected A-to-I editing changes in ADAR1 knockout and overexpression models and revealed differential editing patterns across tissues. In gastric organoids and FFPE tissues, the assay spatially resolved editing heterogeneity and distinguished ADAR1-deficient from ADAR1-sufficient cells. Application to long-archived lung and breast cancer FFPE tissues suggest EndoVIA 2.0s broad utility and potential of capturing disease-associated hyper-editing in malignant samples. ConclusionsEndoVIA 2.0 enables robust, spatial detection of A-to-I editing in FFPE tissues--circumventing the limitations of RNA extraction and unlocking access to archived clinical specimens. This platform lays the foundation for mapping RNA editing dynamics in cancer progression and may support future biomarker discovery.