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

Abstract

Adenosine-to-inosine (A-to-I) RNA editing is an essential post-transcriptional modification catalyzed by adenosine deaminases acting on RNA (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 quantification and spatial mapping of edited RNAs in FFPE tissues. Using 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 having altered ADAR1 expression, fresh frozen (FF) tissues, gastric organoids, and FFPE tissue sections. Using quantitative imaging combined with machine-learning segmentation and spatial analysis, EndoVIA 2.0 successfully detected A-to-I editing changes in ADAR1 knockout (KO) and overexpression models, revealing differential editing patterns across tissues. In gastric organoids and FFPE tissues, the assay also spatially resolved editing heterogeneity and distinguished ADAR1-deficient from ADAR1-sufficient cells. Application to long-archived lung and breast cancer FFPE tissues suggest EndoVIA 2.0's broad utility and potential for capturing disease-associated changes in A-to-I editing. Finally, staining across the gastric neoplastic cascade revealed stage-dependent increases in A-to-I editing and changes in subcellular localization, further supporting the possibility that ADAR plays a critical role in the early development of gastric cancer. EndoVIA 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.