Genome-wide methylation profiling of glioblastoma cell-derived extracellular vesicle DNA allows tumor classification

Abstract

Background Genome-wide DNA methylation profiling has recently been developed into a tool that allows tumor classification in central nervous system tumors. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioblastoma cell-derived EVs reflects genome-wide tumor methylation and mutational profiles and allows non-invasive tumor subtype classification. Methods DNA was isolated from EVs secreted by glioblastoma cells as well as from matching cultured cells and tumors. EV-DNA was localized and quantified by direct stochastic optical reconstruction microscopy. Methylation and copy number profiling was performed using 850k arrays. Mutations were identified by targeted gene panel sequencing. Proteins were differentially quantified by mass spectrometric proteomics. Results Genome-wide methylation profiling of glioblastoma-derived EVs correctly identified the methylation class of the parental cells and original tumors, including the MGMT promoter methylation status. Tumor-specific mutations and copy number variations (CNV) were detected in EV-DNA with high accuracy. Different EV isolation techniques did not affect the methylation profiling and CNV results. DNA was present inside EVs and on the EV surface. Proteome analysis did not allow specific tumor identification or classification but identified tumor-associated proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids. Conclusions This study provides proof of principle that EV-DNA reflects the genome-wide methylation, CNV and mutational status of glioblastoma cells and enables their molecular classification.

Calcitriol Promotes Differentiation of Glioma Stem-Like Cells and Increases Their Susceptibility to Temozolomide

“…The additional four primary lines are MNOF1244, which has not been described previously (see Materials and Methods), 17/01 [ 29 ], MNOF1300 [ 34 ] and the primary gliosarcoma line MNOF168 [ 35 ]. In addition, we also included the cell lines established and described in the literature, GS-73 [ 36 ], NCH421k [ 27 ] and NCH481 [ 37 ]. We analyzed the sensitivity of the cells using LDAs after treatment with 50 nM of calcitriol for 7 days and calculated the fold-change in stem-cell frequency to better compare the results from the different cell lines.…”

“…For the experiments, the following GSC lines were employed: 17/01 [ 29 ], 17/02 [ 29 ], GS-5 [ 28 ], GS-73 [ 36 ], the gliosarcoma line MNOF168 [ 35 ], MNOF1244 (see below, Section 4.1 ), MNOF1300 [ 34 ], NCH421k [ 27 ], NCH481 [ 37 ] and NCH644 [ 27 ]. All cell lines, except MNOF168, MNFO1244 and MNOF1300, were used and cultured in Neurobasal medium (Gibco, Darmstadt, Germany).…”

Calcitriol Promotes Differentiation of Glioma Stem-Like Cells and Increases their Susceptibility to Temozolomide

“…The additional 4 primary lines are MNOF1244, which has not been described previously (see materials and methods), 17/01 [29], MNOF1300 [34] and the primary gliosarcoma line MNOF168 [35]. In addition, we also included 3 cell lines established and described in the literature GS-73 [36], NCH421k [27] and NCH481 [37]. We analyzed the sensitivity of the cells using LDAs after treatment with 50 nM calcitriol for 7 days and calculated the fold-change in stem-cell frequency to better compare the results from the different cell lines.…”

“…For the experiments the following GSC lines were employed: 17/01 [29], 17/02 [29], GS-5 [28], GS-73 [36], the gliosarcoma line MNOF168 [35], MNOF1244 (see below), MNOF1300 [34], NCH421k [27], NCH481 [37] and NCH644 [27]. All cell lines except MNOF168, MNFO1244 and MNOF1300 were used and cultured in Neurobasal medium (Gibco, Darmstadt, Germany).…”

Characteristics and Clinical Application of Extracellular Vesicle-Derived DNA

“…Analysis of specific patterns of DNA methylation is attracting attention as a potential biomarker for the detection and diagnosis of diseases such as cancer [100,101]. Several studies have evaluated the methylation of EV DNA and demonstrated similarities in methylation profiles between gDNA and EV DNA in murine melanoma cells [26]; the gastric juice in patients with gastric cancer [73,102]; and the serum in patients with breast cancer and melanoma [103], metastatic castration-resistant prostate cancer [104], diffuse large B-cell lymphoma [105], and glioblastoma [106] (Table 1). These studies have revealed differences in the methylation of cancer cells and normal cells, and suggested methylation as a biomarker.…”