A proteomic roadmap of SaOS-2 osteoblast-to-osteocyte-like differentiation

Mineralized bone tissue is essential for skeletal integrity, mechanical stability, and mineral homeostasis. Osteocytes, the most abundant bone cell type, play central roles in bone remodeling and endocrine regulation but remain difficult to investigate because of their embedded localization within the mineralized matrix. Consequently, the molecular mechanisms underlying osteoblast-to-osteocyte differentiation remain incompletely understood. Here, we performed a comprehensive quantitative proteomic analysis of the widely used human SaOS-2 cell line during osteoblast-to-osteocyte-like differentiation. More than 5000 proteins were quantified, providing a comprehensive proteomic roadmap of this differentiation process. Proteomic profiling demonstrated extensive molecular remodeling accompanied by extracellular matrix reorganization and dynamic changes in collagen abundance. Although differentiated SaOS-2 cells retained characteristics of late osteoblasts and did not fully recapitulate the phenotype of mature osteocytes, they acquired a distinct osteocyte-like proteomic signature. Furthermore, we identified and orthogonally validated several candidate protein markers associated with the osteoblast and osteocyte-like states. Together, our study provides the first comprehensive protein-level characterization of SaOS-2 osteoblast-to-osteocyte-like differentiation. Beyond improving the molecular characterization of this widely used model, it provides a valuable resource for future studies investigating bone biology, extracellular matrix remodeling, and skeletal disease.