The defining features of hybrid epithelial-mesenchymal state in head and neck cancer

Epithelial-mesenchymal plasticity represents a major axis of tumor cell adaptation, enabling cancer cells to dynamically and reversibly transition between epithelial and mesenchymal states through epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET). EMT is an evolutionarily conserved cellular plasticity program that allows cancer cells to adopt multiple intermediate phenotypes along an epithelial-mesenchymal spectrum. Notably, complete EMT is relatively rare in human tumors, as most cancer cells do not fully activate terminal mesenchymal markers. Instead, they adopt an intermediate or hybrid epithelial/mesenchymal (hybrid E/M) state, also referred to as partial EMT, in which epithelial traits are retained while mesenchymal features are gradually acquired. In this study, we focus on elucidating the clinical significance of the hybrid E/M state in head and neck squamous cell carcinoma (HNSCC) and investigate whether this transitional phenotype is governed by specific molecular regulators. By integrating multi-omics clinical datasets and spatial transcriptomic analyses of patient tumor tissues, we found that the tumor invasive front exhibits marked cellular heterogeneity, which strongly correlates with the hybrid epithelial/mesenchymal cell phenotype. Digital Spatial Profiling analysis revealed that epithelial markers were evenly distributed between the primary and metastatic lymph node tumor regions. In contrast, mesenchymal markers were highly expressed in outer invasive regions and metastatic tumor sites. By analyzing both TCGA datasets and our own RNA-seq cohort, we found that the hybrid E/M phenotype is significantly associated with advanced tumor stage in HNSCC patients. Through single-cell transcriptomic profiling of clinical HNSCC samples, we found that hybrid E/M cells represent a unique cell state defined by their enriched expression of adhesion-related markers. We further developed an in vitro platform to model hybrid epithelial-mesenchymal transition and performed ATAC-seq profiling. Our analysis revealed that members of the GATA transcription factor family, particularly GATA3, play a crucial role in maintaining this intermediate state. Surprisingly, depletion of GATA3 led to a disruption of the hybrid epithelial/mesenchymal phenotype. Taken together, our findings provide compelling evidence that the hybrid epithelial/mesenchymal state is not only clinically relevant in HNSCC but is also shaped by distinct molecular mechanisms. Hybrid E/M cells display a unique adhesion-related gene expression profile, suggestive of a specialized function in promoting collective invasion during tumor progression. These findings enhance our understanding of EMT plasticity and highlight hybrid E/M cells and their control mechanisms as promising targets for slowing tumor evolution and metastasis.