Abstract LB163: Spatial analysis of head and neck cancer reveals two ecosystems with distinct modes of epithelial-to-mesenchymal transition

Abstract Background: Single-cell RNA sequencing has revealed diverse malignant programs in head and neck squamous cell carcinoma (HNSCC), including partial epithelial-to-mesenchymal transition (p-EMT), a state linked to invasion and poor outcome. However, how p-EMT is spatially organized within tumors and shaped by the tumor microenvironment (TME) remains unclear. Here, we used spatial transcriptomics to define tumor-wide spatial architectures of p-EMT in HPV-positive and HPV-negative HNSCC and to identify their microenvironmental drivers. Methods: We profiled 26 primary HNSCC tumors (18 HPV-negative, 8 HPV-positive) using 10x Visium spatial transcriptomics, with paired single-cell RNA-seq for 11 tumors. Cell states were defined using consensus expression meta-programs, and malignant regions were identified via inferred copy number aberrations. Spatial interactions were quantified using distance-based neighborhood analyses, and malignant regions were stratified into tumor edge and core compartments. Key findings were validated using high-resolution Visium HD on paired sections, 25-plex CODEX spatial proteomics in an independent cohort of 40 laryngeal squamous cell carcinomas, and cytokine perturbation assays in vitro. Results: HPV-positive tumors exhibited increased cellular density, an immune-enriched microenvironment, and malignant programs consistent with hypoxia, alongside relative depletion of p-EMT. In contrast, HPV-negative tumors showed prominent p-EMT that segregated into two distinct spatial architectures. The canonical p-EMT edge pattern localized to the invasive front, where p-EMT malignant cells spatially coupled to fibroblasts through TGFβ-associated signaling. Notably, we identified an alternative p-EMT core architecture characterized by diffuse p-EMT throughout tumor cores and co-localization with immunosuppressive macrophages and neutrophils. This spatial niche-related transcriptional program was associated with worse overall survival in large external cohorts. Ligand-receptor analyses and in vitro validations implicated oncostatin M (OSM) as a key upstream driver of p-EMT in tumor cores. Both Visium HD and CODEX independently confirmed the spatial organization of p-EMT states and the corresponding TME interactions. Notably, both p-EMT architectures were consistent across multiple samples from the same tumor, indicating tumor-wide organizational states rather than region-specific variability. Conclusions: We identify two tumor-wide p-EMT architectures in HNSCC driven by distinct microenvironmental programs: fibroblast-derived TGFβ at tumor edges and myeloid-derived OSM in tumor cores. These findings establish OSM as a novel regulator of p-EMT and suggest spatially organized EMT programs as clinically relevant determinants of tumor aggressiveness, with potential implications across cancer types. Citation Format: Dor Simkin, Thomas F. Barrett, Lingling Zhang, Michael J. Moore, Alissa R. Greenwald, Noam Darnell, Michael Mints, Salma Ramadan, Victoria Yu, Riley DZ Mullins, Jesse Zaretsky, Porter Bischoff, Douglas Adkins, Wade L. Thorstad, Sana D. Karam, Randall C. Paniello, Jason T. Rich, Ryan S. Jackson, Patrik Pipkorn, Paul A. Zolkind, Guangyong Peng, R. Alex Harbison, Rebecca Chernock, Anuraag S. Parikh, Sidharth V. Puram, Itay Tirosh. Spatial analysis of head and neck cancer reveals two ecosystems with distinct modes of epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB163.