Epithelial Responses and Remodeling in the Submandibular Gland Post-irradiation

Radiation therapy for head and neck cancer often causes irreversible salivary gland dysfunction, resulting in chronic xerostomia. Understanding the cellular and molecular mechanisms underlying this dysfunction is essential for developing regenerative therapies. Given the cellular complexity of the salivary gland single-cell RNA sequencing (scRNA-seq) provides a powerful approach to investigate cell-type-specific injury responses to ionizing radiation (IR) and uncover transcriptional changes that may be masked in bulk analyses. This study investigates the cellular and molecular responses of the murine submandibular gland (SMG) to irradiation, with a focus on epithelial loss and remodeling, and regenerative failure. In order to effectively use scRNA-seq for transcriptomic analyses, we also sought to develop a rigorous data cleaning pipeline to improve data quality and deconvolute mixed-gland tissue samples. Our findings reveal that multiple epithelial populations adopt a conserved stress-adaptive transcriptional program post-irradiation, marked by upregulation of inflammatory, mitochondrial, and translational genes, alongside persistent downregulation of metabolic and secretory regulators. Subclustering analysis also identified distinct SMG acinar subpopulations with different stress, secretory, and proliferative profiles, though all converged on a similar injury response. Notably, a subset of acinar cells upregulated Bpifa2, which encodes parotid secretory protein (PSP), suggesting the emergence of a stress-associated, non-regenerative state. We also observed a progressive loss of SMGC⁺ intercalated duct cells, indicating that certain ductal populations are susceptible to long-term damage following radiation injury and may contribute to disease progression. These results demonstrate that radiation injury leads to lasting transcriptional and structural changes in the salivary epithelium, accompanied by chronic inflammation and fibrosis.The failure of acinar regeneration and the emergence of a fibrotic microenvironment likely underlie long-term glandular dysfunction. Understanding these responses provides a foundation for developing regenerative therapies targeting epithelial preservation, progenitor activation, and anti-fibrotic intervention.