Spatial Proximity Sequencing Maps Developmental Dynamics in the Germinal Center

Spatial profiling of proteins and protein interactions with mRNAs is essential for studies understanding cellular signaling. However, technologies capable of integrating these modalities remain limited. Here, we present Spatial Proximity-Sequencing (Sprox-seq), a method that combines proximity ligation assay with spatial transcriptomics to simultaneously profile mRNAs, surface proteins, and their interactions in intact tissues. Applied to human tonsil tissue, Sprox-seq profiled 32 proteins and their pairwise interactions alongside thousands of mRNAs. We developed a robust computational framework for analyzing protein proximity data, enabling both the statistical identification and interaction strength measurements of protein complexes. Clustering based on protein interaction strength recapitulated RNA-defined tissue architecture. Interaction networks constructed from protein complexes revealed significantly higher interaction complexity in Light zone. Trajectory inference based on protein interaction strength uncovered a maturation path distinct from that inferred by RNA data, and spatially enriched protein complexes were associated with functional gene-expression pathways. Moreover, Sprox-seq also captured cell–cell interactions mediated by protein complex ITGA4–VCAM1 in Light zone. Overall, Sprox-seq provides a spatially resolved, multi-modal view of cell states, offering a powerful tool for studying immune responses, tissue development, and disease progression. Overall design: The assay was performed on fresh-frozen human tonsil tissue sections, including three biological replicates: two spatially distinct regions from the same donor (samples A1 and B1) and one section from a different donor (sample D1) and analyzed using Spatial Proximity-Sequencing (Sprox-seq) by combing proximity ligation assay with spatial transcriptomics to simultaneously profile mRNAs, surface proteins, and their interactions in intact tissues.