Background <p>Breast calcifications are frequent mammographic findings and serve as critical indicators for the early detection of breast cancer. Breast calcifications are formed by a mineralization process considered passive deposits resulting from tissue necrosis, but accumulating evidence suggests that calcifications may instead arise from active stromal remodeling within the tumor microenvironment. Among the diverse stromal components, mesenchymal stem cells are particularly implicated in mediating mineralization processes. Adipose-derived mesenchymal stem cells (ADSCs), abundant in breast tissue and possessing strong osteogenic potential, could therefore play a key role in mineralization process. However, their contribution of ADSCs to malignant calcification formation remains poorly understood.</p> Methods <p>RNA sequencing was performed on malignant and benign calcified breast tissues to identify differentially expressed genes. Candidate genes were validated using public datasets, immunohistochemistry and survival analysis. ADSCs were isolated from patient breast tissue, phenotypically characterized and genetically modified to overexpress SQLE or HGD genes. Osteogenic differentiation was assessed by alkaline phosphatase activity, Alizarin Red S staining, qPCR and Western blot. In vivo effects were evaluated using a breast cancer xenograft model. Transcriptomic profiling, Seahorse mitochondrial stress analysis, JC-1 staining, reactive oxygen species (ROS) quantification, calcium imaging and transmission electron microscopy were used to explore the underlying mechanisms.</p> Results <p>Transcriptomic screening identified SQLE as a top upregulated gene in malignant calcifications, correlating with poor patient survival. SQLE was highly expressed in stromal cells of malignant lesions. Functional assays showed that the overexpressed SQLE markedly enhanced ADSC osteogenic differentiation and matrix mineralization, with a stronger effect than HGD. In vivo, SQLE-overexpressing ADSCs promoted ectopic calcification within tumors. Mechanistically, the overexpressed SQLE altered the ADSC transcriptome, enriched cancer- and calcium-related pathways, impaired mitochondrial oxidative phosphorylation, reduced mitochondrial membrane potential and ROS levels, and induced mitochondrial calcium accumulation with mineral deposition. The upregulation of PMCA2 and MCU mediated by overexpressed SQLE occurred prior to differentiation, followed by sustained mitochondrial calcium accumulation.</p> Conclusions <p>This study identifies SQLE as a novel driver of stromal mineralization and malignant breast calcification via mitochondrial dysfunction and calcium dysregulation. These findings challenge the passive necrosis model of calcification, positioning SQLE as both a biomarker for malignant calcifications and a potential therapeutic target for microenvironment-focused breast cancer interventions.</p> Graphical Abstract <p></p>