Single-cell transcriptomics and functional validation reveal a TUBB4B-associated malignant epithelial state in nasopharyngeal carcinoma

Abstract

Intratumoral heterogeneity of malignant epithelial cells limits prognostic stratification and therapeutic development in NPC, yet transcriptionally distinct malignant epithelial states and their associated molecular programs remain poorly characterized. We aimed to identify clinically relevant malignant epithelial states and define their associated molecular programs in NPC. Fresh normal nasopharyngeal tissue, primary NPC, and nodal metastases were profiled by scRNA-seq, yielding 27,330 cells. Malignant epithelial states were characterized by transcriptomic reclustering, cell-cycle analysis, CNV inference, pseudotime trajectory analysis, and pathway enrichment analyses. An independent public scRNA-seq cohort (GSE150825) was reanalyzed to validate the identified malignant epithelial state and its associated molecular programs. TUBB4B function was examined using knockdown and overexpression assays, cell-cycle analysis, clonogenic assays, and xenograft experiments. Clinical relevance was evaluated by immunohistochemistry and immunofluorescence in NPC tissue sections and by survival analysis in an independent NPC tissue microarray cohort with long-term follow-up. We identified a malignant epithelial state occupying an early branch of the inferred pseudotime trajectory, characterized by increased inferred CNV burden and increased cell-cycle engagement, and further distinguished by coordinated microtubule- and cilium-associated transcriptional programs and selective enrichment of TUBB4B expression. Reanalysis of GSE150825 identified a phenotypically similar malignant epithelial state subpopulation with concordant microtubule- and cilium-associated pathway enrichment. TUBB4B localized to mitotic structures; its knockdown reduced the proportion of cells in G2/M phase, whereas overexpression increased PCNA expression, clonogenic growth, and xenograft tumor growth. In clinical tissue sections, TUBB4B expression was higher in NPC than in normal nasopharyngeal epithelium and was accompanied by an increased Ki-67-positive area. In the tissue microarray cohort, high cytoplasmic TUBB4B expression was associated with significantly worse overall survival and progression-free survival using a data-derived cut-off, and remained associated with overall survival after adjustment for staging in multivariable Firth penalized Cox models, although this association was attenuated using an alternative cut-off. We identify a TUBB4B-linked malignant epithelial transcriptional program in NPC associated with proliferation, inferred CNV burden, and adverse clinical outcome in this cohort. These findings provide a transcriptionally resolved framework for understanding malignant epithelial heterogeneity in NPC progression and nominate TUBB4B as a candidate prognostic biomarker and potential therapeutic vulnerability warranting further validation.