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Abstract

Head and neck squamous cell carcinoma (HNSCC) is a biologically heterogeneous disease with marked differences in incidence and prognosis between sexes. While clinical sex differences have been increasingly recognized in HNSCC, the molecular consequences of sex chromosome dosage alterations, such as Loss of Y chromosome (LoY) and Extreme Downregulation of Y-linked genes (EDY), remain poorly understood. In this thesis, I investigate how such alterations contribute to tumor biology and shape the tumor microenvironment (TME), with particular focus on fibroblast and immune cell dynamics. By integrating bulk and single-cell transcriptomic datasets, I systematically explore the prevalence and implications of sex chromosome dosage variations across HNSCC patients. Using TCGA bulk RNA-seq data, I identify widespread LoY and EDY events in male patients, with a notable enrichment in HPV-negative tumors. These alterations are tightly linked, with LoY emerging as a key driver of EDY. Importantly, patients stratified by sex chromosome dosage, XX, XY, and XØ (EDY/LoY males), display distinct transcriptomic and cellular profiles. To further dissect these differences, I constructed a harmonized single-cell HNSCC atlas, enabling the analysis of gene expression, chromosomal instability, and cell-cell interactions at single-cell resolution. Tumor cells emerged as the primary site of Y chromosome downregulation, and their classification enabled refined stratification of the cohort. I show that the TME composition, particularly the abundance and activity of fibroblast subtypes (matrix and inflammatory cancer-associated fibroblasts (mCAFs and iCAFs, respectively)), varies significantly by sex chromosome dosage. Cell-cell communication analysis revealed that fibroblasts act as major signaling mediators, especially in XY tumors, where they exhibit enhanced outgoing interactions to tumor cells. Furthermore, XY iCAFs uniquely co-express COX2 and AR, suggesting a possible interaction between inflammatory and hormonal pathways that may support tumor progression. These findings were further validated by deconvolution of bulk transcriptomic data, confirming higher fibroblast abundance in XY tumors. Collectively, this work reveals that sex chromosome dosage is a biologically meaningful variable in HNSCC, driving differences in tumor–stroma communication and shaping the microenvironment. These insights highlight the importance of moving beyond binary sex classifications to uncover nuanced mechanisms of sex-biased tumor biology, with potential implications for therapeutic stratification and biomarker development.