DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain barrier and be detected in peripheral blood of patients

Abstract

Tumor-cell-secreted extracellular vesicles (EVs) can cross the disrupted blood-brain barrier (BBB) into the bloodstream. However, in certain gliomas, the BBB remains intact, which might limit EVs release. To evaluate the ability of tumor-derived EVs to cross the BBB, we used an orthotopic xenotransplant mouse model of human glioma-cancer stem cells featuring an intact BBB. We demonstrated that all types of tumor cells-derived EVs-apoptotic bodies, shedding microvesicles and exosomes-cross the intact BBB and can be detected in the peripheral blood, which provides a minimally invasive method for their detection compared to liquid biopsies obtained from cerebrospinal fluid (CSF). Furthermore, these EVs can be readily distinguished from total murine EVs, since they carry human-specific DNA sequences relevant for GBM biology. In a small cohort of glioma patients, we finally demonstrated that peripheral blood EVs cargo can be successfully used to detect the presence of IDH1G395A, an essential biomarker in the current management of human glioma.

Keywords: biomarkers; blood-brain barrier; brain tumors; extracellular vesicles.

Figure 1. Experimental procedure flowchart

A. Isolated hCSCs from 2 GBM patients were xenotransplanted in athymic mice. After 12 weeks, the animals were transcardially perfused. B. BBB permeability was evaluated using three assays: MRI, Evans Blue staining, and albumin extravasation. C. EVs (ABs, SMVs, and EXOs) were isolated from hCSCs-enriched culture supernatants and from mouse peripheral blood. D. EVs were identified using TEM, tracking analysis, and CD63 tetraspanin quantification. E. To ensure that the analyzed DNA was confined within the EVs, supernatants and plasma were treated with DNase before gDNA isolation; after the isolation, gDNA was pre-amplified before performing PCR analysis with human-specific primers. F. Sequences detected were sequenced to confirm their human origin.

Figure 2. Evaluation of BBB leakage in two GBM models: GBM27 presents an intact BBB

A. Representative T2- and T1-weighted images of GBM27 and GBM38. The GBM27 tumor xenotransplant T2-weighted image depicts diffuse hyperintense infiltrative involvement. GBM38 xenograft tumor showed well-defined borders. T2-weighted images revealed a hyperintense mass compressing ventricular structures. GBM27 features an intact BBB, as revealed by the lack of any contrast enhancement. GBM38 shows a homogeneous enhancement, suggesting that the BBB integrity is compromised. B. Evans Blue extravasation. Examination of the brains of perfused animals previously stained with Evans Blue confirmed BBB disruption in the GBM38 model. C. Quantification of Evans Blue extravasation. *P <0.05. D-E. Immunofluorescence staining of human vimentin (yellow), mouse CD105 (green), and mouse albumin (red). Nuclei were stained with DAPI (blue). GBM27 presents no sign of albumin staining throughout the tissue, which indicates that the BBB is intact. GBM38 features a leaky BBB, as shown by albumin spreading (white asterisk) from the blood vessels (white arrows) through the tissue. Total slides with anti-human vimentin are shown in Fig S1. Scale bar: 50 μm.

Figure 3. Morphological characterisation of EVs isolated from hCSCs supernatant from GBM27 and gDNA isolation

A. Transmission electron microscopy images. ABs (500 nm to 1 μm), SMVs (500–150 nm) and EXOs (150–60 nm). B. Size distribution of EVs, as measured using Nanosizer tracking analysis. C. Quantification of the tetraspanin cell-surface glycoprotein CD63. D. Relative distribution of EVs. E. Most representative sequences analyzed are present in EVs isolated from GBM27 cells. F. Histogram showing the frequency of occurrence of target sequences after 6 consecutive experiments. G. Presence of ERBB2, CDK4, AKT3, and MDM2 sequences in all types of EVs. The remaining sequences were found randomly in ABs, SMVs, and EXOs. No sequences were detected in the supernatant. Scale bars: 1 μm (ABs), 0.2 μm (SMVs and EXOs).

Figure 4. Human gDNA sequences are confined inside EVs isolated from xenografted mice

AKT3, MDM4, PIK3CA, and EGFR sequences were detected in EVs isolated from the peripheral blood of 10 xenografted mice. The multiple sequence alignment shows complete homology among gDNA sequences from GBM27 hCSCs, EVs isolated from hCSCs-enriched culture supernatants, and EVs found in mouse peripheral blood. These results confirm the human origin of the sequences. Keyword: GBM1= GBM27.