Ceramide Composition in Exosomes for Characterization of Glioblastoma Stem-Like Cell Phenotypes

Abstract

Glioblastoma (GBM) is one of the most malignant central nervous system tumor types. Comparative analysis of GBM tissues has rendered four major molecular subtypes. From them, two molecular subtypes are mainly found in their glioblastoma cancer stem-like cells (GSCs) derived in vitro: proneural (PN) and mesenchymal (MES) with nodular (MES-N) and semi-nodular (MES-SN) disseminations, which exhibit different metabolic, growth, and malignancy properties. Many studies suggest that cancer cells communicate between them, and the surrounding microenvironment, via exosomes. Identifying molecular markers that allow the specific isolation of GSC-derived exosomes is key in the development of new therapies. However, the differential exosome composition produced by main GSCs remains unknown. The aim of this study was to determine ceramide (Cer) composition, one of the critical lipids in both cells and their cell-derived exosomes, from the main three GSC phenotypes using mass spectrometry-based lipidomics. GSCs from human tissue samples and their cell-derived exosomes were measured using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) in an untargeted analysis. Complete characterization of the ceramide profile, in both cells and cell-derived exosomes from GSC phenotypes, showed differential distributions among them. Results indicate that such differences of ceramide are chain-length dependent. Significant changes for the C16 Cer and C24:1 Cer and their ratio were observed among GSC phenotypes, being different for cells and their cell-derived exosomes.

Keywords: LC-MS; cancer stem cells; ceramides; exosomes; glioblastoma; mesenchymal phenotype; proneural phenotype; untargeted lipidomics.

Figure 1

Metabolic profile description of GSC phenotypes from cells and their derived exosomes. (A) Chemical entities in cells and exosomes that passed the quality filters, (B, C) Principal component analysis (PCA) models of cells and exosomes: quality control injections (QC, • dark-gray dot), MES-N (•; blue dot), MES-SN (•; green dot), and PN (•; red dot). Unit variance scaling was used for both models. (D) Pie chart showing the percentages of the number of lipid classes for cells. LPE, lysophosphatidylethanolamines; LPC, lysophosphatidylcholines; PC, phosphatidylcholines; PS, phosphatidylserines; PE, phosphatidylethanolamines; PC-ether, ether-phosphatidylcholines; PE-ether, ether-phosphatidylethanolamines; PG, phosphatidylglycerols; PI, phosphatidylinositols; SM, sphingomyelins; Cer, ceramides; HexCer, hexosylceramides; TG, triacylglycerols.

Figure 2

Characteristic ESI(+)-MS/MS fragmentation pattern for structural characterization of the dLCB composition of the three types of sphingoid backbone (d18:2, d18:1, and d18:0). The fragments ions arising from losses of H₂O are common ions seen for all ceramides. Fragment ions possessing dLCB are framed in a green dashed square, and fragment ions possessing an FA chain structure are framed in a blue dashed square. Proposed structures for main ion fragments observed based on published predictions (47).

Figure 3

Characteristic MS/MS fragmentation pattern for structural characterization of fatty acyl composition. (A) MS/MS spectra in ESI(+) and ESI(−) for Cer(d18:1/24:1); (B) MS/MS spectra in ESI(+) and ESI(−) for Cer(d18:1/24:0). The fragment ions arising from losses of H₂O, HCHO, and H₂O + CHOH are common ions seen for all Cer. Fragment ions possessing dLCB are framed in a green dashed square, and fragment ions possessing an FA chain structure are framed in a blue dashed square. Cer with saturated FA are more retained in reverse-phase chromatography than the one with unsaturated double bonds. The fragment ion of [M-H-256]⁻ arising from the combined losses of H₂O and the LCB as an aldehyde is a common ion observed for ceramides with a dLCB(18:1) (47, 49).

Figure 4

Ceramide profile in cells, exosomes (EXO), MV, and AB for the three GBM phenotypes. (A) Ceramide profile for the three phenotypes for cells and their derived exosomes (EXO). (B) Ceramide distribution between exosomes and their parent cells for each phenotype independently. (C) Ceramide distribution among MV, AB, exosomes, and their parent cells for each phenotype independently. In blue, MES-N phenotype; in green, MES-SN phenotype; and in red, PN phenotype. For all bar graphs, error bars represent the mean ± SEM. *p < 0.05; **p < 0.01; and ***p < 0.001.

Figure 5

Normalized ceramide profiles among the three phenotypes in cells and exosomes. In blue, MES-N phenotype; in green, MES-SN phenotype; and in red, PN phenotype. For all bar graphs, error bars represent mean ± SEM. *p < 0.05; **p < 0.01; and ***p < 0.001.

Figure 6

C16/C24:1 Cer ratio in GSCs and their cell-derived exosomes. In blue, MES-SN phenotype; in green, MES-N phenotype; and in red, PN phenotype. For all bar graphs, error bars represent mean ± SEM. *p < 0.05; ***p < 0.001; and ****p < 0.0001.