Integrated Single-Cell and Spatial Transcriptome Analysis Reveals the Biological Characteristics and Regulatory Mechanisms of Atypical Epithelium in Solid-Type Adenoid Cystic Carcinoma

(1) Background: Solid-type adenoid cystic carcinoma (ACC) is a highly invasive and heterogeneous tumor, but the molecular heterogeneity and malignant transformation mechanisms have not been clearly investigated. (2) Methods: Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) were employed to identify characteristic tumor cell subpopulations in solid-type ACC, which were then validated in tissue samples. Functional experiments were conducted to explore the regulatory mechanisms of malignant transformation. (3) Results: ACC exhibited two non-classical epithelial cell subpopulations with rapid proliferation and dedifferentiation characteristics. The non-classical epithelial marker keratin 81 (KRT81) can serve as a molecular typing marker for solid-type ACC, with its protein expression significantly higher in solid-type tissues compared to non-solid types. In vitro and in vivo experiments demonstrated that non-classical epithelial cells promoted the formation of malignant phenotype by activating epithelial-mesenchymal transition (EMT) and cell cycle progression through the MESP1/MACC1 axis. Moreover, non-classical epithelial cells can also induce tumor immune evasion by secreting migration inhibitory factor (MIF) to promote M2 macrophage polarization. (4) Conclusion: Non-classical epithelial cells represent a characteristic cellular subpopulation in solid-type ACC. MESP1/MACC1 and MIF serve as core regulatory factors in tumor cell dedifferentiation and immune evasion. These findings provide new molecular targets to overcome the current treatment challenges in solid-type ACC.