Integrating functional biosignatures and same-cell spatial multiomics to predict synergistic benefit of combination checkpoint therapy in head and neck cancer

Effective immune checkpoint blockade (ICB) requires coordinated activation, spatial organization, and functional cross-talk among immune populations within the tumor microenvironment (TME). Farcast TruTumor™ platform preserves viable tumor, stromal, and immune compartments in their near-native architecture, aligning with the FDA's New Approach Methodologies (NAM) framework for human-relevant cancer models. The model has shown that in head and neck squamous cell carcinoma (HNSCC) anti-PD-1 response is associated with cytotoxic T-cell (CTL) infiltration into tumor nests, and that moderate responders may be rescued by adding anti-CTLA-4, accompanied by increased spatial separation between CTLs and T regulatory (Treg) cells. Here, we integrate TruTumor's functional characterization with the CosMx® Spatial Molecular Imager (SMI) using same-cell spatial multiomics (64-plex IO protein panel plus whole-transcriptome RNA) to elucidate the dynamics of improved efficacy with nivolumab (N) plus ipilimumab (I) leading to predictors of ICB responses with greater accuracy. Patient-derived HNSCC explants were cultured on the TruTumor histoculture platform and treated for 72 hours with N or N+I. Treatment effects were assessed using histopathology, cleaved caspase-3 IHC, multiparameter flow cytometry, and NanoString nCounter bulk transcriptomics. Spatial profiling was performed using the CosMx SMI same-cell multiomic assay, enabling subcellular quantification of immune checkpoints, activation and exhaustion markers, costimulatory molecules, and whole-transcriptome signatures within intact tissue architecture. Analyses included spatial clustering, cell-cell neighborhood modeling, immune-tumor interface mapping, and ligand-receptor interaction inference. CosMx multiomics revealed treatment-dependent remodeling of immune states and spatial niches not captured by bulk assays. N+I responders exhibited: (i) expansion potential of cytotoxic and proliferative CTLs; (ii) depletion or spatial exclusion of suppressive Tregs from tumor nests; (iii) enhanced antigen-presentation and IFN-gamma-response programs in tumor and myeloid cells; and (iv) rewired immune checkpoint and costimulatory ligand-receptor networks at tumor-immune borders. Same-cell integration of protein and RNA markers defined discrete CTL activation and exhaustion trajectories and uncovered microenvironments uniquely sensitized to combination therapy. Combining functional response metrics with spatial features yielded a mechanism-driven biosignature predictive of treatment outcome. Integrating the TruTumor histoculture models with CosMx SMI same-cell spatial multiomics establishes a scalable, NAM-aligned translational platform that enables discovery of predictive biomarkers for next-generation immuno-oncology therapies.