Adherent and Spheroid Glioblastoma Proteomics
Dendritic cell (DC)-based immunotherapy against glioblastoma multiforme is a novel treatment hope. Glioblastoma stem-like cells are, however, potentially causing immunoresistance. Glioblastoma cells cultured as gliomaspheres show a stem-like phenotype as opposed to classical adherent culture. They are thus a promising antigen source to specifically target glioblastoma stem-like cells via DC therapy and so overcome immunoresistance. Here we study the importance of gliomasphere-specific. Methodologically, we used 7 gliomaspheres, 3 of them patient-derived, as model system. Gliomasphere-specific protein expression was explored via quantitative proteomics.
Sample Processing Protocol
Patient samples: The components were all gained from a total of three patients (n=3) that took part in a phase II clinical trial testing a novel dendritic cell-based immunotherapy (GBM-Vax, NCT01213407). All patients had given their written informed consent that their cell material can be processed for further immunological studies in addition to the clinical trial. Glioblastoma cells were harvested from the excised brain tissue that was removed as a part of the initial surgical treatment. Established gliomaspheres: the lines U87MG (ATCC, Manassas, VA), U251MG (ATCC, Manassas, VA), NCH421K (CSL Cell Lines Service, Eppendorf, Germany) and NCH644 (CSL Cell Lines Service, Eppendorf, Germany) were used. Adherent glioblastoma cells were cultured in DMEM/Glutamax (Gibco/ThermoFisher, Waltham, MA, USA) +10% FCS (Sigma-Aldrich, St. Louis, MI, USA). Gliomaspheres were generated via harvesting adherent cells and bringing them into gliomasphere medium that we defined based on work by Campos et al. and Goidts et al. as follows: DMEM/F12 (Gibco/ThermoFisher, Waltham, MA, USA) +20% BIT (Provitro, Berlin, Germany) +20ng/ml bFGF + 20ng/ml EGF (both Stemcell Technologies, Vancouver, Canada). To compare the proteome of adherent versus spheroid glioblastoma cells, we created lysates from 7 different cell lines. In 5 of them (U87MG, U251MG, Linz1, Linz2, Gli16), both phenotypes were available. In 2 cell lines with a known glioblastoma stem cell-like nature (NCH421K, NCH644), we only used the spheroid phenotype. As soon as mature spheroids had formed, cells were harvested and lysed via snap-freezing them. Then, all spheroid samples (n=7) and all adherent cell samples (n=5) were pooled to account for biological variation. To enrich the integral membrane proteins and to minimize the aqueous-soluble proteins associated with the cultivation media (e.g., bovine albumin) a Triton-X114 detergent phase separation was implemented. The membrane-tailored proteomic workflow that combines a detergent depletion step followed by filter-aided tryptic digestion was implemented as previously described. Recontituted peptides were measured using the Fluorometric Peptide Assay (Pierce, USA) and 1Âµg of the peptides was analysed by LCMS with two technical replicates. Analysis was performed using a single-shot LCMS approach with 4-h gradient using a Dionex Ultimate 3000 system (Thermo Fisher Scientific) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Germany) with LCMS parameters as described previously.
Data Processing Protocol
All MS-MS2 spectra were matched against UniProtKB/Swiss-Prot human protein database version 2016.04.14 (42,097 sequences, including isoforms) using the MaxQuant 184.108.40.206 (24) with search parameters as described previously (23). Protein identifications and intensities from MaxQuant were analysed using Perseus statistical package (version 220.127.116.11) (25). The intensity values were log-transformed and zero-intensities were imputed-replaced by normal distribution. Statistical significance of differences in protein levels between spheroid and adherent glioblastoma cells was evaluated using a two-sided t-test with p < 0.05 (applying permutation-based FDR for truncation) and a three-fold protein expression difference.
Erhart F, Weiss T, Klingenbrunner S, Fischhuber K, Reitermaier R, Halfmann A, Blauensteiner B, Lötsch D, Spiegl-Kreinecker S, Berger W, Sialana FJ, Lubec G, Felzmann T, Dohnal A, Visus C. Spheroid glioblastoma culture conditions as antigen source for dendritic cell-based immunotherapy: spheroid proteins are survival-relevant targets but can impair immunogenic interferon γ production. Cytotherapy. 2019 PubMed: 30975602