Global Proteomic Profiling of Extracellular Vesicles derived from Oncolytic Virus (OV)-infected, patient-derived glioblastoma stem-like cells (GSCs) using Ultra Deep DIA MS

ABSTRACT: Authors: Klaudia Kiel, Jakub Godlewski, Agnieszka Bronisz Immunopeptide-based therapies promise to improve patient outcomes compared to the standard of care. Despite substantial progress in mass spectrometry applications in preclinical research, identifying cancer-specific peptide antigens remains a significant challenge in personalized medicine, as the limited presence of such antigens on the cell surface requires high sensitivity and selectivity. The limited quantity of patient brain tissue further compounds this challenge, thus requiring cell culture approaches to expand the availability of antigen-presenting material. EVs secreted by cells of interest have a larger ratio of the surface (as MHCs-presenting area) to the volume (as protein cargo devoid of ribosomal or structural proteins) than their cell-of-origin. We conducted a randomized study to identify peptides using EV derived from glioblastoma stem-like cells (GSCs) from brain cancer patients. To reflect patient biospecimen heterogeneity, a critical factor, cellular material was derived from as many as six glioblastoma patients. To transform an immunosuppressive tumor into an immunostimulatory one, these cultured ex vivo cells were infected with a clinically relevant immune modulating HSV1 oncolytic virus (OV, MOI 0.1, 12h) – rQNestin34.5 (NCT03152318). EVs were harvested from the GSCs and separated from EV-free secretome by ultracentrifugation, providing the material for proteomic and immunopeptidomic analyses by Ultra Deep DIA and DirectDIA+ HRM™ mass spectrometry technology. Protein samples were quantitatively and qualitatively characterized by TEM, NTA, and Western blotting and subjected to mass spectrometry analysis. The data enabled unbiased quantification of all detectable peptides and proteins using the sample-specific spectral library, thus proving the EV/MHCs enrichment concept. A total of 5,203 proteins represented by 45,963 peptide ion variants were detected across all samples, with notable enrichment of MHC I that covered as much as 0.1% of all EV proteins.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Human Alphaherpesvirus 1

DISEASE(S): Glioblastoma

SUBMITTER: Klaudia Kiel  

LAB HEAD: Agnieszka Bronisz

PROVIDER: PXD054707 | Pride | 2026-10-22

REPOSITORIES: Pride