Single-cell transcriptomics reveals cellular evolution underlying pleomorphic adenoma recurrence and malignant transformation.

Pleomorphic adenoma (PA), the most common benign salivary gland tumor, harbors unpredictable risks of recurrence and malignant transformation into carcinoma ex pleomorphic adenoma (CXPA), posing significant clinical challenges. To better delineate the tumor transformation trajectory, we performed single-cell RNA sequencing of normal salivary gland, primary PA, recurrent PA (rPA), and CXPA. Cell trajectory reconstruction, differential expression gene identification, and key gene network analysis were integrated to characterize molecular transitions and intercellular crosstalk driving PA recurrence and malignant transformation. Immunohistochemistry was used to validate key findings. GALNT13 + myoepithelial cells were identified as CXPA-specific malignant progenitors, delineating early malignant conversion. Concurrently, MIF + myoepithelial cells exhibited enhanced tissue-destructive capabilities. Fibroblasts enforced fibrotic restraint in primary PA and drove extracellular matrix degradation in CXPA. The tumor microenvironment exhibited stage-specific adaptations, with CXPA favoring pro-inflammatory MIF-CD74/CD44 signaling and rPA adopting immunosuppressive traits. Stromal reprogramming and immune-editing dynamics collectively orchestrated microenvironmental adaptation, linking cellular heterogeneity to clinical aggressiveness. This study provides the first comprehensive molecular atlas of PA-to-CXPA transformation, revealing malignant specialization of the myoepithelial subpopulation, fibroblast-mediated stromal reprogramming, and immune-editing driven microenvironmental adaptation. These findings provide a framework for precision stratification of the malignant potential of PA, while positioning microenvironmental intervention as a cornerstone of future clinical strategies.