Same-Slide Spatial Multi-Omics Integration with IN-DEPTH Reveals Tumor Virus-Linked Spatial Reorganization of the Tumor Microenvironment.

Spatial transcriptomics and proteomics have enabled profound insights into tissue organization, yet these technologies remain largely disparate, and emerging same-slide multi-omics approaches are limited in plex, spatial resolution, signal retention, and integrative analytics. We introduce IN-situ DEtailed Phenotyping To High-resolution transcriptomics (IN-DEPTH), a streamlined, resource-efficient, commercially compatible workflow using single-cell spatial proteomics-derived imaging to guide transcriptomic capture on the same slide without RNA signal loss. To integrate modalities beyond niche-level mapping, we developed Spectral Graph Cross-Correlation (SGCC), a proteomic-transcriptomic framework resolving spatially coordinated functional state changes across interacting cell populations. Applied to diffuse large B-cell lymphoma (DLBCL), IN-DEPTH and SGCC enabled stepwise discovery from EBV-positive and EBV-negative tumor comparisons to single-cell resolution, revealing coordinated tumor-macrophage-CD4 T-cell remodeling, immunosuppressive C1Q macrophage enrichment, CD4 T-cell dysfunction, and a candidate IL-27-STAT3 signaling axis. Collectively, IN-DEPTH enables scalable spatial multi-omics to uncover clinically relevant microenvironmental mechanisms and towards robust spatial multi-modal AI models.