Spatially resolved multimodal hallmarks of response to neoadjuvant immunotherapies in the melanoma ecosystem in 2D and 3D

Neoadjuvant immunotherapy has transformed cancer treatment, yet the spatial molecular architecture governing response and resistance across distinct immune checkpoint blockade (ICB) regimens remains poorly defined. We assembled the largest neoadjuvant ICB (NICB) spatial multi-omics cohort to date, profiling over 112 million cells at single-cell resolution across three melanoma ICB regimens using MERFISH spatial transcriptomics, multiplexed immunofluorescence, and scRNA-sequencing. These analyses revealed the full multicellular spatial architecture of the NICB tumor microenvironment, including mature TLS with germinal centers, TCF7+ stem-like T cells, myeloid cells organized into spatially distinct cellular neighborhoods with unique intercellular signaling circuits, and CCL19/CCL21-expressing fibroblasts as a previously unrecognized stromal scaffold sustaining these immune hubs. We developed three purpose-built computational tools that together enabled comprehensive quantification of this microenvironment for the first time: SCIRA for whole-slide single-cell receptor-ligand quantification, GCSCAN for molecularly grounded TLS and germinal center structural delineation, and PathNet-TLS for automated TLS detection on H&E images. Applying these tools across the cohort, we defined the immune and stromal composition and cellular neighborhood organization distinguishing responders from non-responders. We also quantified cell-cell interactions and regimen-specific immune architectures, including a markedly stronger mature TLS/germinal center response with IPI-NIVO than NIVO-RELA. Importantly, GCSCAN-quantified TLS and germinal center density each stratified disease-free survival, with responders that lack germinal centers having an elevated risk of relapse. Open-top light-sheet imaging and CODA-based 3D reconstruction further uncovered interconnected germinal center-TLS tunnels invisible to standard 2D histopathology. These findings establish a discovery-to-tool paradigm linking single-cell tumor microenvironment interrogation to clinically deployable computational pathology for biomarker-driven NICB assessment across cancer types.