Comprehensive surfaceome profiling reveals tumor specific targets in osteosarcoma

Osteosarcoma (OS) is an aggressive bone cancer characterized by significant genomic alterations. Despite recent efforts to study OS, the extracellular proteins driving tumorigenesis and metastasis in xenograft models remain understudied due to technical limitations in enriching low-abundance surface proteins. Here, we employed quantitative surfaceome profiling via Wheat Germ Agglutinin (WGA)-HRP mediated cell surface protein enrichment to map the dynamic surface protein landscape of primary and metastatic OS, uncovering clinically actionable targets. We applied an integrated proteomics approach combining WGA-HRP-based cell surface capture and deep offline peptide fractionation coupled with DIA-PASEF mass spectrometry to achieve deep and accurate surfaceome coverage. Primary and metastatic OS cell lines (n = 9) and PDX tumors (n = 4-5), along with controls (n = 3), were analyzed using WGA-HRP mediated surface enrichment followed by Neutravidin based pulldown. PDX tumors were mechanically dissociated to obtain single suspensions. Enriched proteins were fractionated offline, and a deep spectral library was generated using high-pH fractionation and DDA-PASEF, enabling high-resolution, quantitative mapping of the OS surfaceome across diverse samples. The optimized workflow identified ~6,000 total proteins, of which over 600 were validated as druggable surface proteins through Surfy database (FDR < 0.01). PCA revealed clear separation of samples by disease state and phenotype, demonstrating the high reproducibility and biological fidelity of the surfaceome profiles. Comparative analysis of all metastatic OS cell lines vs. controls identified numerous highly dysregulated surface proteins (log₂FC ≥ 5). The surfaceome profile revealed both reported & novel targets, including a pronounced and coordinated upregulation of proteins critically involved in enhanced nutrient transport and metabolic reprogramming, key cell-cell adhesion and structural maintenance molecules, and prominent growth signaling receptors. These upregulated functional classes are critically implicated in enhanced metastatic capacity and aggressive tumor progression. These findings provide a comprehensive surfaceome map of OS remodelling, revealing interconnected, tumor-specific dysregulation and actionable surface targets. The identified protein classes highlight potential biomarkers for early diagnosis, prognosis, and monitoring therapeutic response, and can guide the rational development of targeted biologics and immunotherapies to improve outcomes for OS patients. Acknowledgement: This project is supported by the OCE, FDA of the U.S. Dept of Health and Human Services (HHS) as part of a financial assistance award [CERSI grant to UCSF, U01FD005978] totaling $1,257,392. The contents are those of the author(s) & do not necessarily represent the official views of, nor an endorsement by FDA/HHS, or the U.S. Govt.