Graph-Based 3-Dimensional Spatial Gene Neighborhood Networks of Single Cells in Gels and Tissues

Objective: We developed 3-dimensional spatially resolved gene neighborhood network embedding (3D-spaGNN-E) to find subcellular gene proximity relationships and identify key subcellular motifs in cell-cell communication (CCC). Impact Statement: The pipeline combines 3D imaging-based spatial transcriptomics and graph-based deep learning to identify subcellular motifs. Introduction: Advancements in imaging and experimental technology allow the study of 3D spatially resolved transcriptomics and capture better spatial context than approximating the samples as 2D. However, the third spatial dimension increases the data complexity and requires new analyses. Methods: 3D-spaGNN-E detects single transcripts in 3D cell culture samples and identifies subcellular gene proximity relationships. Then, a graph autoencoder projects the gene proximity relationships into a latent space. We then applied explainability analysis to identify subcellular CCC motifs. Results: We first applied the pipeline to mesenchymal stem cells (MSCs) cultured in hydrogel. After clustering the cells based on the RNA count, we identified cells belonging to the same cluster as homotypic and those belonging to different clusters as heterotypic. We identified changes in local gene proximity near the border between homotypic and heterotypic cells. When applying the pipeline to the MSC-peripheral blood mononuclear cell (PBMC) coculture system, we identified CD4⁺ and CD8⁺ T cells. Local gene proximity and autoencoder embedding changes can distinguish strong and weak suppression of different immune cells. Lastly, we compared astrocyte-neuron CCC in mouse hypothalamus and cortex by analyzing 3D multiplexed-error-robust fluorescence in situ hybridization (MERFISH) data and identified regional gene proximity differences. Conclusion: 3D-spaGNN-E distinguished distinct CCCs in cell culture and tissue by examining subcellular motifs.