Comparative DIA Proteomics of Neuroblastoma: Laser-Microdissected FFPE Tumors Versus Cell Lines

Formalin-fixed, paraffin-embedded (FFPE) tumor specimens are widely available and represent a valuable resource for retrospective cancer research; however, chemical cross-linking has historically limited their application in proteomic studies. In this study, we developed a proteomic workflow for laser-microdissected FFPE neuroblastoma tissues and evaluated their biological characteristics in comparison with established neuroblastoma cell lines. Tumor regions were isolated from FFPE sections by laser microdissection, and protein profiles were analyzed using data-independent acquisition mass spectrometry. Seven high-risk neuroblastoma FFPE samples and four neuroblastoma cell lines were examined. A total of 4511 proteins were identified in FFPE tissues and 6812 proteins in cell lines, with 4311 proteins shared between both systems. After quality filtering, 2186 proteins were retained for downstream quantitative analyses. Principal component analysis and hierarchical clustering demonstrated clear separation between FFPE tissues and cell lines despite a strong overall correlation in mean protein abundance across the quantified proteome. Differential abundance and functional enrichment analyses revealed distinct proteomic features between the two systems. Proteins enriched in FFPE tissues were associated with extracellular vesicles, the extracellular space, the cornified envelope, intermediate filament organization and cell junctions. In contrast, proteins enriched in neuroblastoma cell lines were associated with RNA binding, translation, rRNA processing, oxidative phosphorylation, and glycolysis/gluconeogenesis, reflecting enhanced biosynthetic and metabolic activity. Together, these findings demonstrate that FFPE tissues and neuroblastoma cell lines capture complementary aspects of neuroblastoma biology, supporting their combined use as a comprehensive framework for biomarker discovery and translational research in pediatric oncology.