Profiling extracellular vesicles from cerebrospinal fluid for classification of intradural spinal tumors

Extracellular vesicles (EVs) transport biomolecules that could serve as biomarkers for disease diagnosis and monitoring. The clinical utility of EVs derived from cerebrospinal fluid (CSF) in patients with intradural spinal tumors (IST) has not yet been investigated. Here, we obtained EVs from CSF of adult patients with intraspinal ependymoma (n = 9), meningioma (n = 9), hemangioma (n = 4) and schwannian tumors (n = 7), as well as comparison group ('CG', normal pressure hydrocephalus, n = 7), by ultrafiltration. CSF-EVs were characterized by electron microscopy and nanoparticle tracking analysis. EV populations according to the presence of tetraspanins (CD9, CD63, CD81) were measured by imaging flow cytometry (IFCM). CD81⁺ EVs were more prevalent in the comparison group, meningioma, ependymoma WHO grade 2, and hemangioma, whereas CD9⁺ EVs were predominant in ependymoma grade 1 and Schwannian tumors. CD63⁺ EVs per milliliter/CSF differed between ependymoma WHO grades 1 and 2 (FC = 24.6, AUC = 90%, p < 0.05). Based on results from a bead-based multiplex profiling, we selected ITGB1, CD44, CD133 and HLA-DR/DQ/DP for further phenotyping in CSF-EVs using IFCM, in combination with each tetraspanin as double-positive subpopulations. Compared to CG, CD44⁺ EVs were the most relevant population in CSF from IST patients, followed by ITGB1. Notable differences in absolute (EVs/mL CSF) and relative (percentages of CSF-EVs) levels were: CD44⁺/CD81⁺ for ependymoma grade 1 (FC = 196.5 and 34.5; p < 0.01) and grade 2 (%FC = 6.1, p < 0.05); CD44⁺/CD63⁺ for meningioma (abs. and %FC > 1000, p < 0.05); ITGB1⁺/CD81⁺ for hemangioma (%FC = 4.8, p < 0.05); and ITGB1⁺/CD9⁺ for schwannian tumors (abs.FC = 19.8, p < 0.01). In conclusion, we identified distinct EV subpopulations in the CSF of IST patients, potentially facilitating tumor classification.