Mesenchymal glioblastoma-induced mature de-novo vessel formation of vascular endothelial cells in a microfluidic device.

High vascularization is a biological characteristic of glioblastoma (GBM); however, an in-vitro experimental model to verify the mechanism and physiological role of vasculogenesis in GBM is not well-established. Recently, we established a self-organizing vasculogenic model using human umbilical vein endothelial cells (HUVECs) co-cultivated with human lung fibroblasts (hLFs). Here, we exploited this system to establish a realistic model of vasculogenesis in GBM. We developed two polydimethylsiloxane (PDMS) devices, a doughnut-hole dish and a 5-lane microfluidic device to observe the contact-independent effects of glioblastoma cells on HUVECs. We tested five patient-derived and five widely used GBM cell lines. Confocal fluorescence microscopy was used to observe the morphological changes in Red Fluorescent Protein (RFP)-HUVECs and fluorescein isothiocyanate (FITC)-dextran perfusion. The genetic and expression properties of GBM cell lines were analyzed. The doughnut-hole dish assay revealed KNS1451 as the only cells to induce HUVEC transformation to vessel-like structures, similar to hLFs. The 5-lane device assay demonstrated that KNS1451 promoted the formation of a vascular network that was fully perfused, revealing the functioning luminal construction. Microarray analysis revealed that KNS1451 is a mesenchymal subtype of GBM. Using a patient-derived mesenchymal GBM cell line, mature de-novo vessel formation could be induced in HUVECs by contact-independent co-culture with GBM in a microfluidic device. These results support the development of a novel in vitro research model and provide novel insights in the neovasculogenic mechanism of GBM and may potentially facilitate the future detection of unknown molecular targets

Molecular diagnosis of diffuse glioma using a chip-based digital PCR system to analyze IDH, TERT, and H3 mutations in the cerebrospinal fluid.

PURPOSE: Conventional genetic analyzers require surgically obtained tumor tissues to confirm the molecular diagnosis of diffuse glioma. Recent technical breakthroughs have enabled increased utilization of cell-free tumor DNA (ctDNA) in body fluids as a reliable resource for molecular diagnosis in various cancers. Here, we tested the application of a chip-based digital PCR system for the less invasive diagnosis (i.e., liquid biopsy) of diffuse glioma using the cerebrospinal fluid (CSF). METHODS: CSF samples from 34 patients with diffuse glioma were collected from the surgical field during craniotomy. Preoperative lumbar CSF collection was also performed in 11 patients. Extracted ctDNA was used to analyze diagnostic point mutations in IDH1 R132H, TERT promoter (C228T and C250T), and H3F3A (K27M) on the QuantStudio® 3D Digital PCR System. These results were compared with their corresponding tumor DNA samples. RESULTS: We detected either of the diagnostic mutations in tumor DNA samples from 28 of 34 patients. Among them, we achieved precise molecular diagnoses using intracranial CSF in 20 (71%). Univariate analyses revealed that the World Health Organization (WHO) grade (p = 0.0034), radiographic enhancement (p = 0.0006), and Mib1 index (p = 0.01) were significant predictors of precise CSF-based molecular diagnosis. We precisely diagnosed WHO grade III or IV diffuse gliomas using lumbar CSF obtained from 6 (87%) of 7 patients with tumors harboring any mutation. CONCLUSION: We established a novel, non-invasive molecular diagnostic method using a chip-based digital PCR system targeting ctDNA derived from CSF with high sensitivity and specificity, especially for high-grade gliomas

HRM analysis using different amplicon lengths.

<p>Multiple amplicon lengths (90, 129, and 212 bp) were tested for mutation analyses in order to optimize the HRM analysis. First row: -<i>d</i>¹ curve; second row: -<i>d</i>² curve. The 90-bp amplicon showed the most interpretable heteroduplex-derived peaks.</p

HRM analysis for hotspot mutations of IDH2 and BRAF.

<p>First column: Sequence-wild-type DNA results, including no heteroduplex-derived peak in either derivative curve. Second column: Clear heteroduplex-derived peaks are seen in both derivative curves of the sequence-mutant DNA. C) Distribution plots of HRM-MI for 52 DNA samples analyzed for <i>BRAF</i>^<i>V600E</i>. HRM-MI values completely match the sequence results.</p

Primer sequences for high resolution melting analysis.

<p>Primer sequences for high resolution melting analysis.</p

Discrepant results between a duplicate HRM analyses.

<p>Difference plots of discriminated wild-type calls and variant (i.e., mutant) calls from the first (left upper) and second (right upper) 96 runs, which are displayed as light blue and light red curves, respectively. A representative discrepancy for a duplicate HRM analysis, i.e., one with a wild-type call in the first run and a variant call in the second run, is shown as a black curve in both difference plots. Negative derivative curves of this discrepancy were similar to those of less interpretable plots (left lower, first run; right lower, second run).</p

HRM-MI assay.

<p>A) Representation of the HRM-Mutation Index (HRM-MI), which is defined as the difference between the low-temperature melting transition and high-temperature melting transition. B) Representative -<i>d</i>² curves of mutated and wild-type DNA samples. The <i>x</i>-axis indicates the position relative to <i>Tm</i>. A single asterisk shows <i>Z</i>_HTMT, and double asterisk shows <i>Z</i>_LTMT. Mutated DNA samples presented positive values of HRM-MIs owing to heteroduplex-derived peaks (upper graph). Conversely, wild-type DNA samples showed simple ascending curves between <i>Z</i>_HTMT and <i>Z</i>_LTMT, and negative HRM-MI values (lower graph). The colors represent different samples. C) Distribution plots of HRM-MI for 192 DNA samples analyzed for <i>IDH1</i>^<i>R132</i>. All-HRM-MI values of sequence-mutant (seq-mut) DNA samples were distributed in the positive range (black plots). The corresponding values for all wild-type (seq-wt) DNA samples were negatively dispersed (red plots), except for six values (blue plots) obtained from the duplicated results of three DNA samples.</p