Abstract 7666: Comprehensive surfaceome profiling reveals tumor specific targets in osteosarcoma.

Abstract 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 Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7666.