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

DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain... www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 1), pp: 1416-1428 Research Paper DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain barrier and be detected in peripheral blood of patients 1,2,* 1,3,* 1,3,* Noemí García-Romero , Josefa Carrión-Navarro , Susana Esteban-Rubio , 4 5 6 Elisa Lázaro-Ibáñez , María Peris-Celda , Marta M. Alonso , Juan Guzmán-De- 7 8 1 1 Villoria , Carlos Fernández-Carballal , Ana Ortiz de Mendivil , Sara García-Duque , 4 9 1,3 Carmen Escobedo-Lucea , Ricardo Prat-Acín , Cristóbal Belda-Iniesta , Angel 1,2,3 Ayuso-Sacido Fundación de Investigación HM Hospitales, HM Hospitales, Madrid, Spain IMDEA Nanoscience, Madrid, Spain Facultad de Medicina (IMMA), Universidad San Pablo-CEU, Madrid, Spain Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Clínica Universidad de Navarra, CIMA, Pamplona, Spain Servicio de Radiodiagnóstico, Hospital General Universitario Gregorio Marañón, Madrid, Spain Servicio de Neurocirugía, Hospital General Universitario Gregorio Marañón, Madrid, Spain Departamento de Neurocirugía, Hospital Universitario la Fe, Valencia, Spain These authors have contributed equally to this work Correspondence to: Angel Ayuso-Sacido, email: [email protected] Keywords: extracellular vesicles, brain tumors, blood-brain barrier, biomarkers Received: September 20, 2016 Accepted: November 07, 2016 Published: November 26, 2016 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 nally fi demonstrated that peripheral blood EVs cargo can be successfully used to detect the presence of G395A IDH1 , an essential biomarker in the current management of human glioma Gliomas are commonly detected through clinical INTRODUCTION assessment and imaging techniques. However, the final Primary malignant brain tumors account for 3% of diagnosis relies on the histological analysis of the biopsy adult cancer deaths and are the second cause of tumoral tissue, in accordance with the WHO current standard mortality in children [1]. High-grade gliomas are the most for glioma diagnostic. Recently published molecular common primary malignant brain tumors in adults. Despite pathology-based glioma classification drastically improved advances in treatment, the median patient survival rate is 12 tumor diagnostics and prognostics, essentially through the R132H to 15 months, as tumor eventually recurs in all patients [2]. detection of the IDH1 mutation [3, 4]. www.impactjournals.com/oncotarget 1416 Oncotarget With the current treatment protocol, tissue specimens whether EVs of glioma cells can cross an intact BBB into are suitable for the evaluation of tumor histopathology at blood vessels. the very beginning of the disease, but they do not allow Tumoral mutated sequences have been recently molecular evolution assessment of the tumor along the detected in circulating DNA (ctDNA) and EVs extracted course of the disease, which is critical for improving from cerebrospinal fluid (CSF) [18, 19]. However, patient survival [5]. Thus, the last few years have seen a CSF extraction through lumbar puncture is an invasive marked increase in using liquid biopsies for monitoring procedure and not recommended in patients with high cancer genetics, by analyzing circulating cells, nucleic intracraneal pressure (commonly present in brain tumors). acids, or extracellular vesicles (EVs) released from tumors In addition, bearing this in mind, additional efforts to [6]. The EVs are present in readily accessible biofluids develop a blood-based liquid biopsy method for GBM [7–9] and they carry lipids [10], proteins [11], and distinct patients are required. species of nucleic acids [12–14] originating from donor To investigate the ability of tumor-derived cells. According to the Vesiclepedia nomenclature [15], EVs to cross an intact BBB, we used an orthotopic three types of EVs can be distinguished: apoptotic bodies xenotransplant model of human cancer stem cells (ABs), shedding microvesicles (SMVs), and exosomes (hCSCs), previously described and characterized in (EXOs). All of these EVs might be useful to identify the our laboratory, which produces a disseminated brain- tumor molecular profile at any time, using minimally tumor phenotype featuring an intact BBB [20]. Using invasive procedures. this model, we showed that all three types of EVs The amount and proportion of these circulating derived from human brain tumor cells can cross the tumor-derived EVs might be determined by the integrity undisrupted BBB and reach the bloodstream. These of the BBB, as well as the tumor size and distribution. EVs carried human genomic-DNA (gDNA) sequences Recent studies have demonstrated that EXOs administered corresponding to those of the xenotransplanted cells, intranasally [16] or injected through the tail vein in mice and could be isolated and enriched from peripheral [17] can cross the BBB and deliver their cargo within blood (Figure 1). In a cohort of glioma patients with the parenchymal brain, but the barrier’s integrity was not undisrupted BBB, we demonstrated that peripheral discussed in these studies. Therefore, it remains unknown blood EV cargo can be successfully used to detect 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. www.impactjournals.com/oncotarget 1417 Oncotarget G395A the presence of specific mutations, such as IDH1 . featuring a disrupted BBB in nude mice (Supplementary This finding provides evidence that liquid biopsies can Figure S1). We performed stereotactic transplants of 1 successfully improve the current management of brain × 10 cells from GBM27 and GBM38 into the striatum tumors. of mice. Twelve weeks later we evaluated the functional competence of the BBB through 3 distinct procedures (Supplementary and Figure S2). RESULTS First, we evaluated the BBB integrity in the xenotransplant models GBM27 and GBM38 as well as GBM xenograft model with intact BBB in control mice (Figure 2A and Supplementary Figure S2A) with MRI images. On GBM38 xenograft tumor, Our first aim was to evaluate the capacity of EVs we observed an enhancement in T1-weighted images secreted by glioma cells to cross normal BBB into the with Gadolinium-based contrast agent, a sensitive marker bloodstream. For this purpose, we used an orthotopic of blood-brain barrier disruption (Gd-DTPA-BMA; xenotransplant model of hCSCs culture (GBM27), which Omniscan, Amersham Health, Oslo, Norway). Conversely, developed an infiltrative brain-tumor phenotype featuring no enhancement of Gd-DTPA was observed on the an intact BBB in nude mice. We compared this model GBM27 tumor suggesting an intact BBB. Control mice to a second orthotopic xenotransplant model of hCSCs showed no MRI abnormalities. culture (GBM38) that generated a nodular brain tumor 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. www.impactjournals.com/oncotarget 1418 Oncotarget To verify these findings, we visualised and Our results confirmed that the GBM27 orthotopic quantified the amount of Evans Blue present in the xenotransplant model of hCSCs culture displays an intact parenchymal brain after its injection through the tail BBB. This allowed us to examine whether tumor-derived vein. This test showed that Evans Blue extravasation was EVs can cross intact BBB to the bloodstream. significantly increased in the mice xenotransplanted with GBM38 cells compared to the GBM27 xenotransplanted Morphological visualization and size distribution ones (p < 0.05) (Figure 2BC), which confirmed that BBB of EVs was intact in GBM27 xenotransplants. No Evans Blue dye was detected in control brains (Supplementary Figure After demonstrating the suitability of our model, S2B). Finally, to demonstrate the functional competence we studied whether all three types of EVs could be of the BBB, we also analysed albumin extravasation using isolated and enriched from hCSCs (GBM27) culture immunofluorescence (Figure 2D and E). Considerable supernatant as well as from plasma of xenotransplanted albumin diffusion from blood vessels into the parenchymal mice. EVs were isolated and enriched using brain of GBM38 xenotransplanted mice was observed centrifugation techniques, and then visualized using (Figura 2D). Consistently, no albumin extravasation was transmission electron microscopy (TEM) (Figure 3A detected in GBM27 xenotransplants nor in control mice and Supplementary Figure S3A). TEM images revealed (Figure 2 and Supplementary Figure S2D). the typical morphology and expected diameter ranges of 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). www.impactjournals.com/oncotarget 1419 Oncotarget the ABs (>1 μm), SMVs (~200 nm), and EXOs (~100 derived EVs can carry relevant human gDNA sequences, nm). To confirm the enrichment of the different fractions cross the undisrupted BBB and reach the bloodstream. of EVs from GBM27 cells, we analyzed them using a We hypothesised that if this was the case, we should be Zetasizer: the AB fraction ranged from 2000 to 500 nm able to detect relevant human gDNA sequences within in size, the SMV fraction showed an average size of the pool of EVs (both mouse and human) isolated 600 nm, and the EXO fraction displayed a main peak at from peripheral blood. Thus, we isolated and separated 180 nm (Figure 3B). The supernatant also showed a small all three types of EVs from plasma obtained from the 10-nm peak, compatible with the presence of proteins. inferior vena cava, isolated total DNA from each type These results confirmed the utility of the protocol used to of EV and performed PCR assays to specifically amplify isolate EVs from the supernatant. Although these images human gDNA sequences. PCR-amplification products of and sizes were consistent with the presence of all three the expected sizes were obtained for AKT3 from ABs, types of EVs, we also quantified the relative expression PIK3CA from ABs and SMVs, and MDM4 and EGFR of tetraspanin CD63. The results indicated similar CD63 from EXOs. Multiple sequence alignment showed a expression in SMVs and EXOs and a considerably lower complete homology among these sequences and their expression in ABs (Figure 3C). Lastly, EVs distribution counterparts from the supernatants of hCSCs-enriched was quantified using TEM: the EXO fraction was almost cultures (Figure 4). 4 times larger than the AB and SMV fractions in the G395A hCSCs supernatant (Figure 3D). A similar trend was IDH1 gDNA sequences successfully identified observed in the case of EVs isolated from mouse plasma within EVs isolated from peripheral blood (Supplementary Figure S3B). of human glioma patients regardless of BBB integrity EVs from hCSCs contain gDNA Having detected tumor-specific gDNA sequences The presence of human gDNA in EVs was within EVs isolated from peripheral blood in a mouse verified using a collection of primers specifically model with intact BBB, we next wondered whether designed and validated to amplify human gDNA glioma-derived EVs could be detected in the bloodstream sequences (Supplementary Figure S4). We detected of patients, regardless of the BBB integrity. To accomplish all the gDNA sequences assayed, except for CDKN2a, this, we screened peripheral blood EVs from 21 patients PTEN, and TP53 sequences (Figure 3E). Then, we (20 diagnosed with low- and high-grade glioma and 1 speculated whether the lack of PCR-amplification of brain metastasis) for the presence of IDH1 mutations, these gene sequences might be related to the existence the most relevant mutation for human glioma diagnostic of chromosomal aberrations. Accordingly, we examined and prognostic [3] (Table 1). To investigate BBB the chromosomal status of GBM27 cells using integrity, we used the analysis of contrast acquisition comparative genomic hybridisation (CGH). The CGH in T1-weighted images, which takes advantage of the analysis revealed loss of heterozygosity of chromosome incapacity of gadolinium contrast agent to cross the intact 9 at p21.3, chromosome 10 at q23.1 and chromosome BBB. In addition, we used Dynamic Contrast-Enhanced 17 at position p13.1, which respectively affect the (DCE) MRI to longitudinally measure the vascular trans CDKN2a, PTEN and TP53 locations (Supplementary constant transfer K , which reflects BBB permeability Figure S5). (Supplementary Figure S7). The results revealed 3 patients Interestingly, gDNA sequences relevant for the (HGUGM002, HGUGM003 and HGUGM007) with an GBM biology were detected in all three types of EVs. intact BBB, in contrast to the remaining 18 patients that Certain sequences appeared in all EVs, such as ERBB2, showed a disrupted BBB (Table 1). Next, we assayed for R132H EGFR, CDK4, AKT3, and MDM2, whereas IDH1 was the presence of IDH1 on surgical solid samples by IHC detected only in ABs and EXOs, and a few sequences (the Standard of Gold technique) and conventional PCR. appeared exclusively in one type of EV: PIK3CA, IHC gave positive results for 3 low-grade (HGUGM003, MDM4, IDH2, and ASCL1 in ABs, AKT1 in SMVs, and HGUGM007 and HM001) and 1 high-grade (HGUGM008) G395A MGMT and RB1 in EXOs (Figure 3E-G). Nevertheless, glioma (Table 1). Consistent with these results, IDH1 we observed a 70,4% frequency of bias in this assay, was also detected by conventional PCR on these 3 low- which was independent of our experimental procedure grade glioma samples. Furthermore, this technique detected G395A (Supplementary Figure S6). IDH1 in an additional low-grade glioma sample (HGUGM002), while it was negative for the high-grade glioma HGUGM008. Finally, we extracted total DNA from hCSC-derived EVs cross the intact BBB into the EVs from peripheral blood obtained immediately before bloodstream surgery. As a first approach, we used conventional PCR to G395A amplify IDH1 gDNA sequences, however, IDH1 was The GBM27 orthotopic xenotransplant model observed in none of the low-grade gliomas, and only in 4 was used to analyze whether all three types of GBM27- www.impactjournals.com/oncotarget 1420 Oncotarget G395A of the high-grade glioma samples, including HGUGM008, which, in our hands, is able to enrich and detect IDH1 R132H for which the presence of IDH1 was detected on a when its relative representation is at least as low as 10% surgical solid sample by IHC (Table 1). Considering these of total IDH1 sequences (Supplementary Figure S8). We G395A results, we hypothesized that IDH1 sequences might used fast ColdPCR on solid samples, for which the results wt be underrepresented within EVs compared to IDH1 . To were similar to those obtained by conventional PCR. solve this problem, we tested the fast ColdPCR technique, Notably, when we used fast ColdPCR on DNA isolated 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. www.impactjournals.com/oncotarget 1421 Oncotarget from peripheral blood EVs, we detected the presence of questionable. Further investigation is therefore required to G395A IDH1 in 47.6% of the samples included in the cohort. elucidate the mechanisms underlying nucleic acid uptake G395A Within the low-grade gliomas, IDH1 was identified in by EVs. 80% of the samples, matching our previous results from Another critical factor that must be addressed in IHC and conventional PCR on solid samples. Interestingly, order to assess the use of EVs as biomarkers is the ability G395A within high-grade gliomas, we also detected IDH1 in to separate EVs secreted by tumor cells from the total EVs 40% of the samples, including those previously detected pool. Here, we have shown that orthotopic xenotransplant by conventional PCR. The same technique identified only animal models of human tumor cells are suitable models wt IDH1 on DNA isolated from peripheral blood EVs of a for providing ‘proof of concepts’ related to the utility of patient diagnosed with adenocarcinoma brain metastasis, specific molecular biomarkers present within EVs. used as a negative control. The presence of circulating EVs secreted from These results support the idea that EVs secreted glioma cells has been strongly correlated with tumor size by brain tumor cells can cross the BBB, whether intact and the BBB status [27]. Here, we demonstrated for the or disrupted, and enters the bloodstream. Therefore, the first time that human gDNA sequences relevant for the analysis of their cargo might be useful as a biomarker GBM biology can be detected within all three types of not only for high-grade gliomas but also for low-grade EVs isolated from the bloodstream of tumor-bearing mice. gliomas, most of which conserve an intact BBB. In this context, some groups have recently postulated the use of CSF as a source of tumor-derived DNA sequences [19, 21]. However, the lumbar puncture is often unfeasible DISCUSSION in glioma patients mostly due to the high intracranial pressure [28]. The BBB maintains the brain environment and Notably, our findings confirm that not only tumor- protects it against external factors. In glioma-diagnosed secreted EXOs but also ABs and SMVs can cross the intact patients and xenografted mice, the BBB dysfunction BBB into the bloodstream. This is essential to standardize is partially due to the impairment of tight junctions, the use of circulating EVs in serum as glioma biomarkers which explains the fluid leakage and cerebral oedema regardless of the BBB status, providing a minimally associated with these tumors [21, 22]. However, a invasive method compared to CSF. However, the exact completely functional BBB is occasionally found in mechanism used by EVs to cross the BBB remains elusive orthotopic xenograft mouse models [23], in certain low- and might differ depending on the EV type. grade human gliomas and a few high-grade gliomas Finally, the new molecular pathology-based [24]. In agreement with these reports, we demonstrated classification of gliomas expanded current molecular that the GBM27 orthotopic xenotransplant mouse knowledge about prognostic and diagnostic implications model of hCSCs culture displays a functional BBB, of a variety of biomarkers used to characterize glioma using three approaches: Gd-DTPA MRI, external tracer patients. For example, IDH1-mutated patients are Evans Blue, and evaluation of albumin extravasation by expected to have longer survival rates than their wild- immunofluorescence. Thus, using the GBM27 model, type counterparts despite similar histopathologic features we are able to assess the potential presence of brain [29]. Moreover, immunotherapy is a field of growing tumor EVs in peripheral blood after crossing the intact interest in brain oncology and specific glioma hallmarks BBB. are considered as immuno-targets. In this regard, a few We have shown that gDNA sequences are present IDH1 peptide vaccines trials are currently going on [30]. inside all three types of EVs isolated from the hCSCs Unfortunately, there are obvious limitations to obtain a supernatant. Other groups have also demonstrated the representative glioma tissue sample. This restrains the presence of gDNA sequences within total and fractioned access to new therapies and clinical trials and impairs EVs [13, 25]. monitoring of drug resistance and/or clonal dynamics Interestingly, we noted a key variability in the during treatment. As a consequence, almost all clinical detection of specific gDNA sequences in the EVs, and R132H trials accept first biopsies as a tissue to assess IDH1 based also on previous reports [26], we hypothesised that or any other target status. In this context, we addressed this variability might be more related to the representation the potential use of tumor-derived EVs cargo in serum of such sequences within the cell of origin than to the isolated from high or low grade brain tumor patients by isolation and pre-amplification procedures. Although G395A looking for the presence of IDH1 , currently the most analysing the gDNA sequences contained within all three valuable molecular marker for human glioma diagnostics types of EVs might be useful for obtaining clinically and prognostics [3]. In our hands, conventional PCR was relevant information, our results raise at least two useful to detect the wild type form, but unable to reveal the technical issues. Firstly, the amount of tumor-derived EVs mutated form of IDH1 in DNA sequences from peripheral within the total pool of circulating EVs is relatively low. blood EVs, which might explain why previous works also Secondly, the relative presence of mutated gDNA versus reported negative results [19, 21]. Here, we demonstrated wild type sequences within tumor-derived EVs cargo is www.impactjournals.com/oncotarget 1422 Oncotarget G395A Table 1: IDH1 gDNA sequence identification in liquid biopsies from human glioma patients with or without BBB disruption Patient ID Patient Pathological anatomy BBB integrity evaluation by Surgical sample analysis Liquid biopsy analysis variables MRI-based techniques Sex Age Pathologic Grade BBB Contrast Ktrans IHQ FFPE FFPE Fast EVs EVs Fast diagnosis integrity acquisition (10-3/ Conventional COLDPCR Conventional ColdPCR min) PCR PCR HGUGM002 F 63 Oligodendro II No No 0 WT G395A G395A N/D G395A glioma disrupted HGUGM003 M 33 Oligodendro II No No 10 R132H G395A G395A WT G395A glioma disrupted HGUGM007 F 48 Oligoastro II No No N/D R132H G395A G395A N/D G395A cytoma disrupted HM001 M 31 Astrocytoma II Disrupted Yes N/D R132H G395A G395A WT G395A HM012 M 56 Astrocytoma II Disrupted Yes N/D WT WT WT N/D WT HM009 M 56 Astrocytoma III Disrupted Yes N/D WT WT WT WT WT HGUGM004 M 47 Grade III IV Disrupted Yes 184- WT WT WT N/D WT Astrocytoma 427 with grade IV areas HGUGM005 F 43 Giant-Cells IV Disrupted Yes N/D WT WT WT G395A G395A GBM HGUGM006 M 53 GBM IV Disrupted Yes N/D WT WT WT WT G395A HGUGM008 M 39 GBM, recurrence IV Disrupted Yes N/D R132H WT WT G395A G395A HGUGM009 F 64 GBM, recurrence IV Disrupted Yes N/D WT WT WT N/D WT HM002 M 42 GBM IV Disrupted Yes N/D WT WT WT WT WT HM003 F 61 GBM IV Disrupted Yes N/D WT WT WT G395A G395A HM004 M 65 GBM IV Disrupted Yes N/D WT WT WT WT WT HM005 F 36 GBM IV Disrupted Yes N/D WT WT WT WT WT HM006 M 48 GBM IV Disrupted Yes N/D WT WT WT G395A G395A HM007 F 66 GBM IV Disrupted Yes N/D WT WT WT N/D G395A HM008 F 65 GBM IV Disrupted Yes N/D WT WT WT N/D WT HM010 M 43 GBM IV Disrupted Yes N/D WT WT WT N/D WT HM011 F 61 GBM IV Disrupted Yes N/D WT WT WT WT WT HGUGM001 F 61 Adenocarcinoma O Disrupted Yes 638 WT WT WT WT WT Brain metastases trans Samples are organized by tumor grade. BBB integrity is evaluated mainly by contrast acquisition, but also by K when possible. Surgical tumor tissues were analyzed by IHQ, conventional PCR and Fast Cold PCR. Liquid biopsies were analyzed mainly by Fast Cold PCR. Keywords: BBB, blood brain barrier and N/D, not determined. for the first time, that fast Cold-PCR can be successfully are needed to consolidate and validate the use of DNA used to enrich the mutated form of IDH1 in DNA sequences isolated from the peripheral blood EVs of brain sequences from peripheral blood EVs isolated from brain tumor patients. tumor patients. In our cohort, the results obtained from In conclusion, we have demonstrated that all liquid biopsies were consistent with those observed from three types of EVs secreted by human glioma cells can solid samples of low-grade gliomas, but not with high- cross the intact BBB. Furthermore, we prove that DNA G395A grade gliomas. The higher number of positive IDH1 sequences from peripheral blood EVs isolated from observed in high-grade gliomas, as compared with their brain tumor patients can be successfully used to detect corresponding solid samples, might be explained by the the presence or absence of specific molecular alterations G395A intra-tumor heterogeneity, for the small specimen analyzed such as IDH1 . This finding supports, for the first time, by pathological anatomy barely represents the whole the utility of tumor-derived DNA within all three types tumor. However, additional studies with larger cohorts of circulating EVs as potential biomarkers to improve www.impactjournals.com/oncotarget 1423 Oncotarget diagnostics, prognostics and follow-up for both low- and T2-weighted images were acquired by using the high-grade gliomas. rapid acquisition with refocused echo (RARE) sequence with the following parameters: TR/TE (repetition time/ echo time) = 2500/44 ms, field of view = 2.3 cm, 6 averages, MATERIALS AND METHODS matrix size = 256 × 256, number of slices = 14, and slice thickness = 1 mm without a gap. The total scan time required Human samples and derivation of glioblastoma- to concurrently acquire T2-weighted images was 6 min. cancer stem cells-enriched cultures Subsequently, we modified certain parameters for T1-weighted images; for example, we used the multi- Two tumor samples from GBM patients (GBM27 slice multi-echo (MSME) sequence, TR/TE = 3500/10.6 and GBM38) were processed within 12 h after extraction ms, 3 averages, and total time of acquisition = 3 min 2 s. according to the protocol described previously [31]. 2+ The contrast agent used, 0.3 M Gd-DTPA, was injected Briefly, the samples were minced and washed in Ca / 2+ intraperitoneally. Mg -free HBSS (Hanks balanced salt solution). Enzymatic digestion was sequentially performed with -1 Solution I (papain (14 U ml , Sigma-Aldrich) and DNase Evans blue injection and brain extraction -1 I (10 U ml , Sigma) in PIPES solution) for 90 min at -1 A 2% Evans Blue dye solution (Sigma-Aldrich) 37°C and Solution II (papain (7 U ml ) and DNase I (15 -1 -1 was administered (2 ml kg ) through the tail vein and, U ml ) in 1:1 PIPES: proliferation medium) for 30 min 1 h later, the mice were transcardially perfused and the at 37°C. The cells were then dissociated using diameter- brains were extracted, weighed, and homogenised using tapering polished Pasteur pipettes, filtered through a a TissueLyser LT (Qiagen) in twice their volume of 70-μm mesh, and resuspended in defined proliferative N,N-dimethylformamide (Sigma-Aldrich). The tissues media. These CSCs lines were previously described and were incubated overnight at 55°C and then centrifuged characterized in detail by our laboratory [20]. for 20 min at 9300 × g. The optical density (OD) of the Solid surgical tissue samples and peripheralblood supernatant was measured at 610–635 nm, and the amount were obtained from patients operated at HM Hospitales of Evans Blue extravasation was quantified as nanograms (HM), Madrid, Spain; Hospital Universitario la Fe per milligram of tissue. (HUlaFe), Valencia Sapin and Hospital General Universitario Gregorio Marañón (HGUGM), Madrid, Immunofluorescence staining Spain. Peripheralblood samples from patients were collected prior to surgery. These blood samples were left Immunofluorescence staining for mouse albumin to clot for 30 min at room temperature and serum was was performed to determine the vascular integrity of the isolated and stored at -80°C until use. brain. Brain sections obtained from GBM27 and GBM38 hCSCs and PBS control xenotransplants were incubated Xenotransplants with a 5% blocking solution of the specific serum, and then incubated (overnight, 4°C) in solutions containing Approximately 10 cells from 2 cancer-stem cell the following primary antibodies: goat anti-mouse CD105 cultures (GBM27 and GBM38) were injected into the right (R&D Systems), goat anti-mouse albumin (Santa Cruz striatum of 10 immunosuppressed athymic nude female Biotechnology), and mouse anti-human vimentin (Santa mice by using a stereotactic device. As a negative control, Cruz Biotechnology). Then, Alexa Fluor-conjugated PBS was injected into 2 mice following the same protocol. secondary antibodies were used for 1 h (donkey anti-goat Twelve weeks post-injection, mice were anaesthetised 568, rabbit anti-goat 488, and goat anti-mouse 660; Life and then transcardially perfused with saline and prefixed Technologies, USA), and then nuclei were counterstained with 4% paraformaldehyde. The brains were post-fixed with DAPI and coverslips were mounted using FluorSave™ for 48 h after the infusion and embedded in paraffin, after reagent (Millipore). Fluorescence was examined under a which 3-μm coronal sections were obtained. Leica TCS SP5 inverted confocal microscope. Magnetic resonance imaging (MRI) in mice Immunohistochemistry Magnetic resonance experiments were performed Formalin-fixed paraffin-embedded sections were on a 7.0-T Bruker Pharmascan (Bruker Medical Gmbh, stained (as per the manufacturer’s staining protocol) with Ettlingen, Germany) superconducting magnet, with the Bond Polymer Refine Detection Kit on a Bond-max™ Paravision 5.1 software. T2-weighted MRI and T1- fully automated staining system (Leica Microsystems weighted MRI after paramagnetic contrast-agent GmbH, Germany), using a mouse monoclonal antibody administration were used to assess tumor implantation and R132H against human IDH1 (Clon H09, Dianova) for the BBB integrity, at time 0 and at 90 days. Prior to scanning, R132H detection of mutant IDH1 . mice were anaesthetised with isofluorane. www.impactjournals.com/oncotarget 1424 Oncotarget Mice plasma samples DNA isolation and pre-amplification and PCR Twelve weeks after the xenotransplantation, mouse Formalin-fixed paraffin-embedded (FFPE) samples peripheral blood was collected from the inferior vena were deparaffinized and extracted using the DNeasy Blood cava, and centrifuged at 1500 × g for 10 min to obtain the & Tissue Kit spin columns according to the manufacter´s plasma. protocol. (Qiagen, Germany). Before DNA isolation, supernatant and plasma samples were treated with 27 -1 Kunitz U ml of DNase I (Qiagen) for 30 min at 37°C Extracellular vesicles isolation to remove potential free-DNA contaminants. Total DNA EVs from plasma and serum samples and cell was extracted from EVs and cells by using a DNeasy medium supernatants were isolated through differential Blood and Tissue Kit (Qiagen), as per manufacturer ultracentrifugation as previously described [32], with recommendations. Next, a working solution of the certain modifications. Briefly, to remove cellular debris, extracted DNA was pre-amplified using the GenomePlex samples were centrifuged at 1200 × g for 5 min. The Complete WGA2 Kit (Sigma-Aldrich), according to the supernatant was carefully aspirated off without disturbing manufacturer’s instructions. All PCRs were performed the pellet and centrifuged at 8000 × g for 20 min to obtain following the protocol of the Paq5000 enzyme (Agilent ABs. Next, this supernatant was centrifuged at 25,000 Technologies) (Supplementary Figure S8B). The final × g for 20 min to obtain SMVs, and then the remaining PCR products were electrophoretically separated on 1.8% supernatant was ultracentrifuged at 117,000 × g for 90 agarose gels. min to obtain EXOs (Optima-LE 80K ultracentrifuge, 50.2 Ti rotor; Beckman Coulter). All centrifugations were Fast Cold-PCR performed at 4°C. A sample of the EVs-free supernatant was collected after the ultracentrifugation and used as a For the enrichment and detection of IDH1 mutated negative control. In samples from human, total EVs were gDNA sequences within EVs isolated from peripheral blood analyzed. Samples were immediately used or stored at of human patients, DNA was amplified using the reported -20°C. primers: forward 5’- CGGTCTTCAGAGAAGCCATT-3’ and reverse 5’-GCAAAATCACATTATTGCCAAC-3’ [33] (Supplementary Figure S8A). Cycling conditions Transmission electron microscopy were as follows: a first denaturation step of 10 min at 96ºC, EVs samples were individually added onto glow- followed by a set of 20 cycles of 96ºC for 15 sec and 60ºC discharged 150-mesh formvar copper grids (EMS™), for 15 sec, and a second set of 30 cycles of 15 sec at T subjected to the glow-discharge procedure (2 min, 2.4 (critical temperature) and 60ºC for 15 sec (Supplementary MA), and then incubated for 2 min at 4°C. The grids Figure S8C). were washed, negatively stained with 2% aqueous uranyl acetate solution, dried, and analyzed by performing TEM Sequencing and assembly (FEI Tecnai Spirit G2 and Tecnai 12) at 80 kV. EVs were DNA sequencing of the PCR-amplified products of classified based on their size, photographed, and counted AKT3, EGFR, MDM4, PIK3CA and IDH1 was performed using a Soft Image System Morada camera. by the sequencing service at Spanish National Cancer Research Center (CNIO). Sequences were compared Tracking analysis and aligned using the BLAST algorithm and CLUSTAL Mean droplet sizes of EVs were measured using the Omega, respectively. dynamic light scattering method and a Zetasizer Nano ZS (Malvern Instruments, UK). Dynamic contrast enhanced (DCE)-MRI data acquisition and analysis Flow-cytometry analysis of CD63 expression Preoperative MR images were obtained using a 1.5 AB, SMV, or EXO samples were adsorbed onto T MRI scanner (Achieva of Intera, Philips Healthcar, Best, 4-mm aldehyde/sulphate latex beads (Invitrogen, Paisley, The Netherlands) and 8-channel SENSE head coil. For UK) overnight at 4°C. The reaction was stopped by adding DCE-MRI, baseline 3D T1-weighted images were obtained glycine 100 mM. Membrane-bound beads were washed in with the following parameters: TR 76 ms, TE 3ms, slice PBS/1% BSA, incubated with mouse anti-CD63 (Abcam, thickness 5mm, Field of View (FOV) 230 mm, matrix size Cambridge, UK) or appropriate isotype control for 30 min of 116 x128, 35 volumes, temporal resolution 5,4 s and flip- at 4°C, stained with FITC-conjugated secondary antibody angles of 5º and 15º to create two precontrast datasets. Then, (R&D Systems) for 30 min at 4°C, and resuspended in 0.5 ml a DCE perfusion imaging dynamic series was performed of PBS. Samples were analyzed using a FACS Calibur flow using T1-weigthed sequences with the same MR parameters cytometer (BD Biosciences, San Jose, CA, USA). except for an increased flip angle of 15º. At the end of the www.impactjournals.com/oncotarget 1425 Oncotarget second volume acquisition, a bolus of 14 ml of gadobenate and PI14/00077) and the ‘Miguel Servet Program’ dimeglumine (Multihance, Bracco Imaging, Spain) was (CP11/00147) from the ‘Instituto de Salud Carlos III’ injected intravenously at a rate of 3-4 ml/s. (AAS), RTC-2015-3846-1 from Ministerio de Economía Structural contrast 3D T1 fast field echo (FFE) y Competitividad and FEDER funds. sequence was performed and the detail parameters were as follows: TR/TE= 4,6/9,4 ms, flip angle 8º, FOV 256 Author contributions × 256 mm, matrix size 256x 256 and reconstructed voxel N.G.R., J.C.N., S.E.R. and A.A.S. conceived, size of 1 × 1 × 1mm. designed and performed research with assistance from To observe BBB permeability, vascular constant trans -1 E.L.I., M.P.C. and C.B.I.. M.M.A. analyzed CD63 transfer (K min ) values were calculated using expression in EVs supernatants. A.O.M. and J.G.V. Philips IntelliSpace Portal v.6 Software by simultaneous provided MRI assistance. All authors contributed to observation of axial postcontrast T1-weigthed images and trans the general discussion and comment on the manuscript. corresponding colour parametric K maps. One region of N.G.R., J.C.N., S.E.R. and A.A.S. wrote the manuscript interest was manually positioned on the solid tumoral area. with inputs from C.E.L., C.F.C., S.G.C., R.P.A. and C.B.I. Statistics REFERENCES Statistical analysis were performed using a 2-tailed Student t test. Data are presented as means ± standard 1. Siegel RL, Miller KD, Jemal A. Cancer statistics. 2016; 66: deviation and were calculated using the software package 7–30. doi: 10.3322/caac.21332. GraphPad Prism v. 5.0. Statistical values of p > 0.05 were 2. Chinot OL, Wick W, Mason W, Henriksson R, Saran F, not considered significant. Nishikawa R, Carpentier AF, Hoang-Xuan K, Kavan P, Cernea D, Brandes AA, Hilton M, Abrey L, et al. 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DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain barrier and be detected in peripheral blood of patients

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www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 1), pp: 1416-1428 Research Paper DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain barrier and be detected in peripheral blood of patients 1,2,* 1,3,* 1,3,* Noemí García-Romero , Josefa Carrión-Navarro , Susana Esteban-Rubio , 4 5 6 Elisa Lázaro-Ibáñez , María Peris-Celda , Marta M. Alonso , Juan Guzmán-De- 7 8 1 1 Villoria , Carlos Fernández-Carballal , Ana Ortiz de Mendivil , Sara García-Duque , 4 9 1,3 Carmen Escobedo-Lucea , Ricardo Prat-Acín , Cristóbal Belda-Iniesta , Angel 1,2,3 Ayuso-Sacido Fundación de Investigación HM Hospitales, HM Hospitales, Madrid, Spain IMDEA Nanoscience, Madrid, Spain Facultad de Medicina (IMMA), Universidad San Pablo-CEU, Madrid, Spain Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Clínica Universidad de Navarra, CIMA, Pamplona, Spain Servicio de Radiodiagnóstico, Hospital General Universitario Gregorio Marañón, Madrid, Spain Servicio de Neurocirugía, Hospital General Universitario Gregorio Marañón, Madrid, Spain Departamento de Neurocirugía, Hospital Universitario la Fe, Valencia, Spain These authors have contributed equally to this work Correspondence to: Angel Ayuso-Sacido, email: [email protected] Keywords: extracellular vesicles, brain tumors, blood-brain barrier, biomarkers Received: September 20, 2016 Accepted: November 07, 2016 Published: November 26, 2016 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 nally fi demonstrated that peripheral blood EVs cargo can be successfully used to detect the presence of G395A IDH1 , an essential biomarker in the current management of human glioma Gliomas are commonly detected through clinical INTRODUCTION assessment and imaging techniques. However, the final Primary malignant brain tumors account for 3% of diagnosis relies on the histological analysis of the biopsy adult cancer deaths and are the second cause of tumoral tissue, in accordance with the WHO current standard mortality in children [1]. High-grade gliomas are the most for glioma diagnostic. Recently published molecular common primary malignant brain tumors in adults. Despite pathology-based glioma classification drastically improved advances in treatment, the median patient survival rate is 12 tumor diagnostics and prognostics, essentially through the R132H to 15 months, as tumor eventually recurs in all patients [2]. detection of the IDH1 mutation [3, 4]. www.impactjournals.com/oncotarget 1416 Oncotarget With the current treatment protocol, tissue specimens whether EVs of glioma cells can cross an intact BBB into are suitable for the evaluation of tumor histopathology at blood vessels. the very beginning of the disease, but they do not allow Tumoral mutated sequences have been recently molecular evolution assessment of the tumor along the detected in circulating DNA (ctDNA) and EVs extracted course of the disease, which is critical for improving from cerebrospinal fluid (CSF) [18, 19]. However, patient survival [5]. Thus, the last few years have seen a CSF extraction through lumbar puncture is an invasive marked increase in using liquid biopsies for monitoring procedure and not recommended in patients with high cancer genetics, by analyzing circulating cells, nucleic intracraneal pressure (commonly present in brain tumors). acids, or extracellular vesicles (EVs) released from tumors In addition, bearing this in mind, additional efforts to [6]. The EVs are present in readily accessible biofluids develop a blood-based liquid biopsy method for GBM [7–9] and they carry lipids [10], proteins [11], and distinct patients are required. species of nucleic acids [12–14] originating from donor To investigate the ability of tumor-derived cells. According to the Vesiclepedia nomenclature [15], EVs to cross an intact BBB, we used an orthotopic three types of EVs can be distinguished: apoptotic bodies xenotransplant model of human cancer stem cells (ABs), shedding microvesicles (SMVs), and exosomes (hCSCs), previously described and characterized in (EXOs). All of these EVs might be useful to identify the our laboratory, which produces a disseminated brain- tumor molecular profile at any time, using minimally tumor phenotype featuring an intact BBB [20]. Using invasive procedures. this model, we showed that all three types of EVs The amount and proportion of these circulating derived from human brain tumor cells can cross the tumor-derived EVs might be determined by the integrity undisrupted BBB and reach the bloodstream. These of the BBB, as well as the tumor size and distribution. EVs carried human genomic-DNA (gDNA) sequences Recent studies have demonstrated that EXOs administered corresponding to those of the xenotransplanted cells, intranasally [16] or injected through the tail vein in mice and could be isolated and enriched from peripheral [17] can cross the BBB and deliver their cargo within blood (Figure 1). In a cohort of glioma patients with the parenchymal brain, but the barrier’s integrity was not undisrupted BBB, we demonstrated that peripheral discussed in these studies. Therefore, it remains unknown blood EV cargo can be successfully used to detect 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. www.impactjournals.com/oncotarget 1417 Oncotarget G395A the presence of specific mutations, such as IDH1 . featuring a disrupted BBB in nude mice (Supplementary This finding provides evidence that liquid biopsies can Figure S1). We performed stereotactic transplants of 1 successfully improve the current management of brain × 10 cells from GBM27 and GBM38 into the striatum tumors. of mice. Twelve weeks later we evaluated the functional competence of the BBB through 3 distinct procedures (Supplementary and Figure S2). RESULTS First, we evaluated the BBB integrity in the xenotransplant models GBM27 and GBM38 as well as GBM xenograft model with intact BBB in control mice (Figure 2A and Supplementary Figure S2A) with MRI images. On GBM38 xenograft tumor, Our first aim was to evaluate the capacity of EVs we observed an enhancement in T1-weighted images secreted by glioma cells to cross normal BBB into the with Gadolinium-based contrast agent, a sensitive marker bloodstream. For this purpose, we used an orthotopic of blood-brain barrier disruption (Gd-DTPA-BMA; xenotransplant model of hCSCs culture (GBM27), which Omniscan, Amersham Health, Oslo, Norway). Conversely, developed an infiltrative brain-tumor phenotype featuring no enhancement of Gd-DTPA was observed on the an intact BBB in nude mice. We compared this model GBM27 tumor suggesting an intact BBB. Control mice to a second orthotopic xenotransplant model of hCSCs showed no MRI abnormalities. culture (GBM38) that generated a nodular brain tumor 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. www.impactjournals.com/oncotarget 1418 Oncotarget To verify these findings, we visualised and Our results confirmed that the GBM27 orthotopic quantified the amount of Evans Blue present in the xenotransplant model of hCSCs culture displays an intact parenchymal brain after its injection through the tail BBB. This allowed us to examine whether tumor-derived vein. This test showed that Evans Blue extravasation was EVs can cross intact BBB to the bloodstream. significantly increased in the mice xenotransplanted with GBM38 cells compared to the GBM27 xenotransplanted Morphological visualization and size distribution ones (p < 0.05) (Figure 2BC), which confirmed that BBB of EVs was intact in GBM27 xenotransplants. No Evans Blue dye was detected in control brains (Supplementary Figure After demonstrating the suitability of our model, S2B). Finally, to demonstrate the functional competence we studied whether all three types of EVs could be of the BBB, we also analysed albumin extravasation using isolated and enriched from hCSCs (GBM27) culture immunofluorescence (Figure 2D and E). Considerable supernatant as well as from plasma of xenotransplanted albumin diffusion from blood vessels into the parenchymal mice. EVs were isolated and enriched using brain of GBM38 xenotransplanted mice was observed centrifugation techniques, and then visualized using (Figura 2D). Consistently, no albumin extravasation was transmission electron microscopy (TEM) (Figure 3A detected in GBM27 xenotransplants nor in control mice and Supplementary Figure S3A). TEM images revealed (Figure 2 and Supplementary Figure S2D). the typical morphology and expected diameter ranges of 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). www.impactjournals.com/oncotarget 1419 Oncotarget the ABs (>1 μm), SMVs (~200 nm), and EXOs (~100 derived EVs can carry relevant human gDNA sequences, nm). To confirm the enrichment of the different fractions cross the undisrupted BBB and reach the bloodstream. of EVs from GBM27 cells, we analyzed them using a We hypothesised that if this was the case, we should be Zetasizer: the AB fraction ranged from 2000 to 500 nm able to detect relevant human gDNA sequences within in size, the SMV fraction showed an average size of the pool of EVs (both mouse and human) isolated 600 nm, and the EXO fraction displayed a main peak at from peripheral blood. Thus, we isolated and separated 180 nm (Figure 3B). The supernatant also showed a small all three types of EVs from plasma obtained from the 10-nm peak, compatible with the presence of proteins. inferior vena cava, isolated total DNA from each type These results confirmed the utility of the protocol used to of EV and performed PCR assays to specifically amplify isolate EVs from the supernatant. Although these images human gDNA sequences. PCR-amplification products of and sizes were consistent with the presence of all three the expected sizes were obtained for AKT3 from ABs, types of EVs, we also quantified the relative expression PIK3CA from ABs and SMVs, and MDM4 and EGFR of tetraspanin CD63. The results indicated similar CD63 from EXOs. Multiple sequence alignment showed a expression in SMVs and EXOs and a considerably lower complete homology among these sequences and their expression in ABs (Figure 3C). Lastly, EVs distribution counterparts from the supernatants of hCSCs-enriched was quantified using TEM: the EXO fraction was almost cultures (Figure 4). 4 times larger than the AB and SMV fractions in the G395A hCSCs supernatant (Figure 3D). A similar trend was IDH1 gDNA sequences successfully identified observed in the case of EVs isolated from mouse plasma within EVs isolated from peripheral blood (Supplementary Figure S3B). of human glioma patients regardless of BBB integrity EVs from hCSCs contain gDNA Having detected tumor-specific gDNA sequences The presence of human gDNA in EVs was within EVs isolated from peripheral blood in a mouse verified using a collection of primers specifically model with intact BBB, we next wondered whether designed and validated to amplify human gDNA glioma-derived EVs could be detected in the bloodstream sequences (Supplementary Figure S4). We detected of patients, regardless of the BBB integrity. To accomplish all the gDNA sequences assayed, except for CDKN2a, this, we screened peripheral blood EVs from 21 patients PTEN, and TP53 sequences (Figure 3E). Then, we (20 diagnosed with low- and high-grade glioma and 1 speculated whether the lack of PCR-amplification of brain metastasis) for the presence of IDH1 mutations, these gene sequences might be related to the existence the most relevant mutation for human glioma diagnostic of chromosomal aberrations. Accordingly, we examined and prognostic [3] (Table 1). To investigate BBB the chromosomal status of GBM27 cells using integrity, we used the analysis of contrast acquisition comparative genomic hybridisation (CGH). The CGH in T1-weighted images, which takes advantage of the analysis revealed loss of heterozygosity of chromosome incapacity of gadolinium contrast agent to cross the intact 9 at p21.3, chromosome 10 at q23.1 and chromosome BBB. In addition, we used Dynamic Contrast-Enhanced 17 at position p13.1, which respectively affect the (DCE) MRI to longitudinally measure the vascular trans CDKN2a, PTEN and TP53 locations (Supplementary constant transfer K , which reflects BBB permeability Figure S5). (Supplementary Figure S7). The results revealed 3 patients Interestingly, gDNA sequences relevant for the (HGUGM002, HGUGM003 and HGUGM007) with an GBM biology were detected in all three types of EVs. intact BBB, in contrast to the remaining 18 patients that Certain sequences appeared in all EVs, such as ERBB2, showed a disrupted BBB (Table 1). Next, we assayed for R132H EGFR, CDK4, AKT3, and MDM2, whereas IDH1 was the presence of IDH1 on surgical solid samples by IHC detected only in ABs and EXOs, and a few sequences (the Standard of Gold technique) and conventional PCR. appeared exclusively in one type of EV: PIK3CA, IHC gave positive results for 3 low-grade (HGUGM003, MDM4, IDH2, and ASCL1 in ABs, AKT1 in SMVs, and HGUGM007 and HM001) and 1 high-grade (HGUGM008) G395A MGMT and RB1 in EXOs (Figure 3E-G). Nevertheless, glioma (Table 1). Consistent with these results, IDH1 we observed a 70,4% frequency of bias in this assay, was also detected by conventional PCR on these 3 low- which was independent of our experimental procedure grade glioma samples. Furthermore, this technique detected G395A (Supplementary Figure S6). IDH1 in an additional low-grade glioma sample (HGUGM002), while it was negative for the high-grade glioma HGUGM008. Finally, we extracted total DNA from hCSC-derived EVs cross the intact BBB into the EVs from peripheral blood obtained immediately before bloodstream surgery. As a first approach, we used conventional PCR to G395A amplify IDH1 gDNA sequences, however, IDH1 was The GBM27 orthotopic xenotransplant model observed in none of the low-grade gliomas, and only in 4 was used to analyze whether all three types of GBM27- www.impactjournals.com/oncotarget 1420 Oncotarget G395A of the high-grade glioma samples, including HGUGM008, which, in our hands, is able to enrich and detect IDH1 R132H for which the presence of IDH1 was detected on a when its relative representation is at least as low as 10% surgical solid sample by IHC (Table 1). Considering these of total IDH1 sequences (Supplementary Figure S8). We G395A results, we hypothesized that IDH1 sequences might used fast ColdPCR on solid samples, for which the results wt be underrepresented within EVs compared to IDH1 . To were similar to those obtained by conventional PCR. solve this problem, we tested the fast ColdPCR technique, Notably, when we used fast ColdPCR on DNA isolated 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. www.impactjournals.com/oncotarget 1421 Oncotarget from peripheral blood EVs, we detected the presence of questionable. Further investigation is therefore required to G395A IDH1 in 47.6% of the samples included in the cohort. elucidate the mechanisms underlying nucleic acid uptake G395A Within the low-grade gliomas, IDH1 was identified in by EVs. 80% of the samples, matching our previous results from Another critical factor that must be addressed in IHC and conventional PCR on solid samples. Interestingly, order to assess the use of EVs as biomarkers is the ability G395A within high-grade gliomas, we also detected IDH1 in to separate EVs secreted by tumor cells from the total EVs 40% of the samples, including those previously detected pool. Here, we have shown that orthotopic xenotransplant by conventional PCR. The same technique identified only animal models of human tumor cells are suitable models wt IDH1 on DNA isolated from peripheral blood EVs of a for providing ‘proof of concepts’ related to the utility of patient diagnosed with adenocarcinoma brain metastasis, specific molecular biomarkers present within EVs. used as a negative control. The presence of circulating EVs secreted from These results support the idea that EVs secreted glioma cells has been strongly correlated with tumor size by brain tumor cells can cross the BBB, whether intact and the BBB status [27]. Here, we demonstrated for the or disrupted, and enters the bloodstream. Therefore, the first time that human gDNA sequences relevant for the analysis of their cargo might be useful as a biomarker GBM biology can be detected within all three types of not only for high-grade gliomas but also for low-grade EVs isolated from the bloodstream of tumor-bearing mice. gliomas, most of which conserve an intact BBB. In this context, some groups have recently postulated the use of CSF as a source of tumor-derived DNA sequences [19, 21]. However, the lumbar puncture is often unfeasible DISCUSSION in glioma patients mostly due to the high intracranial pressure [28]. The BBB maintains the brain environment and Notably, our findings confirm that not only tumor- protects it against external factors. In glioma-diagnosed secreted EXOs but also ABs and SMVs can cross the intact patients and xenografted mice, the BBB dysfunction BBB into the bloodstream. This is essential to standardize is partially due to the impairment of tight junctions, the use of circulating EVs in serum as glioma biomarkers which explains the fluid leakage and cerebral oedema regardless of the BBB status, providing a minimally associated with these tumors [21, 22]. However, a invasive method compared to CSF. However, the exact completely functional BBB is occasionally found in mechanism used by EVs to cross the BBB remains elusive orthotopic xenograft mouse models [23], in certain low- and might differ depending on the EV type. grade human gliomas and a few high-grade gliomas Finally, the new molecular pathology-based [24]. In agreement with these reports, we demonstrated classification of gliomas expanded current molecular that the GBM27 orthotopic xenotransplant mouse knowledge about prognostic and diagnostic implications model of hCSCs culture displays a functional BBB, of a variety of biomarkers used to characterize glioma using three approaches: Gd-DTPA MRI, external tracer patients. For example, IDH1-mutated patients are Evans Blue, and evaluation of albumin extravasation by expected to have longer survival rates than their wild- immunofluorescence. Thus, using the GBM27 model, type counterparts despite similar histopathologic features we are able to assess the potential presence of brain [29]. Moreover, immunotherapy is a field of growing tumor EVs in peripheral blood after crossing the intact interest in brain oncology and specific glioma hallmarks BBB. are considered as immuno-targets. In this regard, a few We have shown that gDNA sequences are present IDH1 peptide vaccines trials are currently going on [30]. inside all three types of EVs isolated from the hCSCs Unfortunately, there are obvious limitations to obtain a supernatant. Other groups have also demonstrated the representative glioma tissue sample. This restrains the presence of gDNA sequences within total and fractioned access to new therapies and clinical trials and impairs EVs [13, 25]. monitoring of drug resistance and/or clonal dynamics Interestingly, we noted a key variability in the during treatment. As a consequence, almost all clinical detection of specific gDNA sequences in the EVs, and R132H trials accept first biopsies as a tissue to assess IDH1 based also on previous reports [26], we hypothesised that or any other target status. In this context, we addressed this variability might be more related to the representation the potential use of tumor-derived EVs cargo in serum of such sequences within the cell of origin than to the isolated from high or low grade brain tumor patients by isolation and pre-amplification procedures. Although G395A looking for the presence of IDH1 , currently the most analysing the gDNA sequences contained within all three valuable molecular marker for human glioma diagnostics types of EVs might be useful for obtaining clinically and prognostics [3]. In our hands, conventional PCR was relevant information, our results raise at least two useful to detect the wild type form, but unable to reveal the technical issues. Firstly, the amount of tumor-derived EVs mutated form of IDH1 in DNA sequences from peripheral within the total pool of circulating EVs is relatively low. blood EVs, which might explain why previous works also Secondly, the relative presence of mutated gDNA versus reported negative results [19, 21]. Here, we demonstrated wild type sequences within tumor-derived EVs cargo is www.impactjournals.com/oncotarget 1422 Oncotarget G395A Table 1: IDH1 gDNA sequence identification in liquid biopsies from human glioma patients with or without BBB disruption Patient ID Patient Pathological anatomy BBB integrity evaluation by Surgical sample analysis Liquid biopsy analysis variables MRI-based techniques Sex Age Pathologic Grade BBB Contrast Ktrans IHQ FFPE FFPE Fast EVs EVs Fast diagnosis integrity acquisition (10-3/ Conventional COLDPCR Conventional ColdPCR min) PCR PCR HGUGM002 F 63 Oligodendro II No No 0 WT G395A G395A N/D G395A glioma disrupted HGUGM003 M 33 Oligodendro II No No 10 R132H G395A G395A WT G395A glioma disrupted HGUGM007 F 48 Oligoastro II No No N/D R132H G395A G395A N/D G395A cytoma disrupted HM001 M 31 Astrocytoma II Disrupted Yes N/D R132H G395A G395A WT G395A HM012 M 56 Astrocytoma II Disrupted Yes N/D WT WT WT N/D WT HM009 M 56 Astrocytoma III Disrupted Yes N/D WT WT WT WT WT HGUGM004 M 47 Grade III IV Disrupted Yes 184- WT WT WT N/D WT Astrocytoma 427 with grade IV areas HGUGM005 F 43 Giant-Cells IV Disrupted Yes N/D WT WT WT G395A G395A GBM HGUGM006 M 53 GBM IV Disrupted Yes N/D WT WT WT WT G395A HGUGM008 M 39 GBM, recurrence IV Disrupted Yes N/D R132H WT WT G395A G395A HGUGM009 F 64 GBM, recurrence IV Disrupted Yes N/D WT WT WT N/D WT HM002 M 42 GBM IV Disrupted Yes N/D WT WT WT WT WT HM003 F 61 GBM IV Disrupted Yes N/D WT WT WT G395A G395A HM004 M 65 GBM IV Disrupted Yes N/D WT WT WT WT WT HM005 F 36 GBM IV Disrupted Yes N/D WT WT WT WT WT HM006 M 48 GBM IV Disrupted Yes N/D WT WT WT G395A G395A HM007 F 66 GBM IV Disrupted Yes N/D WT WT WT N/D G395A HM008 F 65 GBM IV Disrupted Yes N/D WT WT WT N/D WT HM010 M 43 GBM IV Disrupted Yes N/D WT WT WT N/D WT HM011 F 61 GBM IV Disrupted Yes N/D WT WT WT WT WT HGUGM001 F 61 Adenocarcinoma O Disrupted Yes 638 WT WT WT WT WT Brain metastases trans Samples are organized by tumor grade. BBB integrity is evaluated mainly by contrast acquisition, but also by K when possible. Surgical tumor tissues were analyzed by IHQ, conventional PCR and Fast Cold PCR. Liquid biopsies were analyzed mainly by Fast Cold PCR. Keywords: BBB, blood brain barrier and N/D, not determined. for the first time, that fast Cold-PCR can be successfully are needed to consolidate and validate the use of DNA used to enrich the mutated form of IDH1 in DNA sequences isolated from the peripheral blood EVs of brain sequences from peripheral blood EVs isolated from brain tumor patients. tumor patients. In our cohort, the results obtained from In conclusion, we have demonstrated that all liquid biopsies were consistent with those observed from three types of EVs secreted by human glioma cells can solid samples of low-grade gliomas, but not with high- cross the intact BBB. Furthermore, we prove that DNA G395A grade gliomas. The higher number of positive IDH1 sequences from peripheral blood EVs isolated from observed in high-grade gliomas, as compared with their brain tumor patients can be successfully used to detect corresponding solid samples, might be explained by the the presence or absence of specific molecular alterations G395A intra-tumor heterogeneity, for the small specimen analyzed such as IDH1 . This finding supports, for the first time, by pathological anatomy barely represents the whole the utility of tumor-derived DNA within all three types tumor. However, additional studies with larger cohorts of circulating EVs as potential biomarkers to improve www.impactjournals.com/oncotarget 1423 Oncotarget diagnostics, prognostics and follow-up for both low- and T2-weighted images were acquired by using the high-grade gliomas. rapid acquisition with refocused echo (RARE) sequence with the following parameters: TR/TE (repetition time/ echo time) = 2500/44 ms, field of view = 2.3 cm, 6 averages, MATERIALS AND METHODS matrix size = 256 × 256, number of slices = 14, and slice thickness = 1 mm without a gap. The total scan time required Human samples and derivation of glioblastoma- to concurrently acquire T2-weighted images was 6 min. cancer stem cells-enriched cultures Subsequently, we modified certain parameters for T1-weighted images; for example, we used the multi- Two tumor samples from GBM patients (GBM27 slice multi-echo (MSME) sequence, TR/TE = 3500/10.6 and GBM38) were processed within 12 h after extraction ms, 3 averages, and total time of acquisition = 3 min 2 s. according to the protocol described previously [31]. 2+ The contrast agent used, 0.3 M Gd-DTPA, was injected Briefly, the samples were minced and washed in Ca / 2+ intraperitoneally. Mg -free HBSS (Hanks balanced salt solution). Enzymatic digestion was sequentially performed with -1 Solution I (papain (14 U ml , Sigma-Aldrich) and DNase Evans blue injection and brain extraction -1 I (10 U ml , Sigma) in PIPES solution) for 90 min at -1 A 2% Evans Blue dye solution (Sigma-Aldrich) 37°C and Solution II (papain (7 U ml ) and DNase I (15 -1 -1 was administered (2 ml kg ) through the tail vein and, U ml ) in 1:1 PIPES: proliferation medium) for 30 min 1 h later, the mice were transcardially perfused and the at 37°C. The cells were then dissociated using diameter- brains were extracted, weighed, and homogenised using tapering polished Pasteur pipettes, filtered through a a TissueLyser LT (Qiagen) in twice their volume of 70-μm mesh, and resuspended in defined proliferative N,N-dimethylformamide (Sigma-Aldrich). The tissues media. These CSCs lines were previously described and were incubated overnight at 55°C and then centrifuged characterized in detail by our laboratory [20]. for 20 min at 9300 × g. The optical density (OD) of the Solid surgical tissue samples and peripheralblood supernatant was measured at 610–635 nm, and the amount were obtained from patients operated at HM Hospitales of Evans Blue extravasation was quantified as nanograms (HM), Madrid, Spain; Hospital Universitario la Fe per milligram of tissue. (HUlaFe), Valencia Sapin and Hospital General Universitario Gregorio Marañón (HGUGM), Madrid, Immunofluorescence staining Spain. Peripheralblood samples from patients were collected prior to surgery. These blood samples were left Immunofluorescence staining for mouse albumin to clot for 30 min at room temperature and serum was was performed to determine the vascular integrity of the isolated and stored at -80°C until use. brain. Brain sections obtained from GBM27 and GBM38 hCSCs and PBS control xenotransplants were incubated Xenotransplants with a 5% blocking solution of the specific serum, and then incubated (overnight, 4°C) in solutions containing Approximately 10 cells from 2 cancer-stem cell the following primary antibodies: goat anti-mouse CD105 cultures (GBM27 and GBM38) were injected into the right (R&D Systems), goat anti-mouse albumin (Santa Cruz striatum of 10 immunosuppressed athymic nude female Biotechnology), and mouse anti-human vimentin (Santa mice by using a stereotactic device. As a negative control, Cruz Biotechnology). Then, Alexa Fluor-conjugated PBS was injected into 2 mice following the same protocol. secondary antibodies were used for 1 h (donkey anti-goat Twelve weeks post-injection, mice were anaesthetised 568, rabbit anti-goat 488, and goat anti-mouse 660; Life and then transcardially perfused with saline and prefixed Technologies, USA), and then nuclei were counterstained with 4% paraformaldehyde. The brains were post-fixed with DAPI and coverslips were mounted using FluorSave™ for 48 h after the infusion and embedded in paraffin, after reagent (Millipore). Fluorescence was examined under a which 3-μm coronal sections were obtained. Leica TCS SP5 inverted confocal microscope. Magnetic resonance imaging (MRI) in mice Immunohistochemistry Magnetic resonance experiments were performed Formalin-fixed paraffin-embedded sections were on a 7.0-T Bruker Pharmascan (Bruker Medical Gmbh, stained (as per the manufacturer’s staining protocol) with Ettlingen, Germany) superconducting magnet, with the Bond Polymer Refine Detection Kit on a Bond-max™ Paravision 5.1 software. T2-weighted MRI and T1- fully automated staining system (Leica Microsystems weighted MRI after paramagnetic contrast-agent GmbH, Germany), using a mouse monoclonal antibody administration were used to assess tumor implantation and R132H against human IDH1 (Clon H09, Dianova) for the BBB integrity, at time 0 and at 90 days. Prior to scanning, R132H detection of mutant IDH1 . mice were anaesthetised with isofluorane. www.impactjournals.com/oncotarget 1424 Oncotarget Mice plasma samples DNA isolation and pre-amplification and PCR Twelve weeks after the xenotransplantation, mouse Formalin-fixed paraffin-embedded (FFPE) samples peripheral blood was collected from the inferior vena were deparaffinized and extracted using the DNeasy Blood cava, and centrifuged at 1500 × g for 10 min to obtain the & Tissue Kit spin columns according to the manufacter´s plasma. protocol. (Qiagen, Germany). Before DNA isolation, supernatant and plasma samples were treated with 27 -1 Kunitz U ml of DNase I (Qiagen) for 30 min at 37°C Extracellular vesicles isolation to remove potential free-DNA contaminants. Total DNA EVs from plasma and serum samples and cell was extracted from EVs and cells by using a DNeasy medium supernatants were isolated through differential Blood and Tissue Kit (Qiagen), as per manufacturer ultracentrifugation as previously described [32], with recommendations. Next, a working solution of the certain modifications. Briefly, to remove cellular debris, extracted DNA was pre-amplified using the GenomePlex samples were centrifuged at 1200 × g for 5 min. The Complete WGA2 Kit (Sigma-Aldrich), according to the supernatant was carefully aspirated off without disturbing manufacturer’s instructions. All PCRs were performed the pellet and centrifuged at 8000 × g for 20 min to obtain following the protocol of the Paq5000 enzyme (Agilent ABs. Next, this supernatant was centrifuged at 25,000 Technologies) (Supplementary Figure S8B). The final × g for 20 min to obtain SMVs, and then the remaining PCR products were electrophoretically separated on 1.8% supernatant was ultracentrifuged at 117,000 × g for 90 agarose gels. min to obtain EXOs (Optima-LE 80K ultracentrifuge, 50.2 Ti rotor; Beckman Coulter). All centrifugations were Fast Cold-PCR performed at 4°C. A sample of the EVs-free supernatant was collected after the ultracentrifugation and used as a For the enrichment and detection of IDH1 mutated negative control. In samples from human, total EVs were gDNA sequences within EVs isolated from peripheral blood analyzed. Samples were immediately used or stored at of human patients, DNA was amplified using the reported -20°C. primers: forward 5’- CGGTCTTCAGAGAAGCCATT-3’ and reverse 5’-GCAAAATCACATTATTGCCAAC-3’ [33] (Supplementary Figure S8A). Cycling conditions Transmission electron microscopy were as follows: a first denaturation step of 10 min at 96ºC, EVs samples were individually added onto glow- followed by a set of 20 cycles of 96ºC for 15 sec and 60ºC discharged 150-mesh formvar copper grids (EMS™), for 15 sec, and a second set of 30 cycles of 15 sec at T subjected to the glow-discharge procedure (2 min, 2.4 (critical temperature) and 60ºC for 15 sec (Supplementary MA), and then incubated for 2 min at 4°C. The grids Figure S8C). were washed, negatively stained with 2% aqueous uranyl acetate solution, dried, and analyzed by performing TEM Sequencing and assembly (FEI Tecnai Spirit G2 and Tecnai 12) at 80 kV. EVs were DNA sequencing of the PCR-amplified products of classified based on their size, photographed, and counted AKT3, EGFR, MDM4, PIK3CA and IDH1 was performed using a Soft Image System Morada camera. by the sequencing service at Spanish National Cancer Research Center (CNIO). Sequences were compared Tracking analysis and aligned using the BLAST algorithm and CLUSTAL Mean droplet sizes of EVs were measured using the Omega, respectively. dynamic light scattering method and a Zetasizer Nano ZS (Malvern Instruments, UK). Dynamic contrast enhanced (DCE)-MRI data acquisition and analysis Flow-cytometry analysis of CD63 expression Preoperative MR images were obtained using a 1.5 AB, SMV, or EXO samples were adsorbed onto T MRI scanner (Achieva of Intera, Philips Healthcar, Best, 4-mm aldehyde/sulphate latex beads (Invitrogen, Paisley, The Netherlands) and 8-channel SENSE head coil. For UK) overnight at 4°C. The reaction was stopped by adding DCE-MRI, baseline 3D T1-weighted images were obtained glycine 100 mM. Membrane-bound beads were washed in with the following parameters: TR 76 ms, TE 3ms, slice PBS/1% BSA, incubated with mouse anti-CD63 (Abcam, thickness 5mm, Field of View (FOV) 230 mm, matrix size Cambridge, UK) or appropriate isotype control for 30 min of 116 x128, 35 volumes, temporal resolution 5,4 s and flip- at 4°C, stained with FITC-conjugated secondary antibody angles of 5º and 15º to create two precontrast datasets. Then, (R&D Systems) for 30 min at 4°C, and resuspended in 0.5 ml a DCE perfusion imaging dynamic series was performed of PBS. Samples were analyzed using a FACS Calibur flow using T1-weigthed sequences with the same MR parameters cytometer (BD Biosciences, San Jose, CA, USA). except for an increased flip angle of 15º. At the end of the www.impactjournals.com/oncotarget 1425 Oncotarget second volume acquisition, a bolus of 14 ml of gadobenate and PI14/00077) and the ‘Miguel Servet Program’ dimeglumine (Multihance, Bracco Imaging, Spain) was (CP11/00147) from the ‘Instituto de Salud Carlos III’ injected intravenously at a rate of 3-4 ml/s. (AAS), RTC-2015-3846-1 from Ministerio de Economía Structural contrast 3D T1 fast field echo (FFE) y Competitividad and FEDER funds. sequence was performed and the detail parameters were as follows: TR/TE= 4,6/9,4 ms, flip angle 8º, FOV 256 Author contributions × 256 mm, matrix size 256x 256 and reconstructed voxel N.G.R., J.C.N., S.E.R. and A.A.S. conceived, size of 1 × 1 × 1mm. designed and performed research with assistance from To observe BBB permeability, vascular constant trans -1 E.L.I., M.P.C. and C.B.I.. M.M.A. analyzed CD63 transfer (K min ) values were calculated using expression in EVs supernatants. A.O.M. and J.G.V. 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Published: Nov 26, 2016

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