CellWHISPER disentangles direct cell–cell communication from structural proximity

Abstract Direct, contact-dependent cell–cell communication shapes tissue physiology and disease. Spatial transcriptomics offers a unique opportunity to infer such communication mechanisms, but the few existing tools for such inference are prone to high error rates due to spatial structure in distribution of cell types and gene expression. We present CellWHISPER, a statistical framework for inferring contact-mediated cell–cell communication, including gap junctions and ligand-receptor mechanisms, from single-cell resolution spatial data. CellWHISPER achieves strict error control via a specialized hypothesis-testing procedure that accounts for the confounding effects of cell- type-specific expression and spatial organization. Its use of an exact closed-form significance score obviates expensive permutation testing and enables scalable analysis of large tissues an extensive signaling compendia. To uncover salient communication patterns, CellWHISPER also includes a latent variable model that distills recurrent patterns into maps of mutual preferences among cell types and signaling genes. Applied to mouse brain STEREO-seq and Xenium datasets, CellWHISPER identifies cell type-specific gap-junction coupling and yields a comprehensive connexin-interaction map. We experimentally confirmed Connexin 43-mediated interfaces among astrocytes, microglia and endothelial cells, which were among the highest- ranked predictions; in contrast, existing approaches fail to prioritize these confirmed interactions. Differential analysis of an Alzheimer’s disease model versus wild-type identifies preserved astroglial coupling but increased microglia-associated gap-junction communication. In summary, CellWHISPER disentangles direct cell–cell communication from structural proximity, enabling statistically robust and computationally scalable inference from spatial transcriptomics, leading to the discovery of tissue- and disease-associated contact-mediated signaling programs.