The human U-87 MG (ATCC HTB-14) cell line, which is derived from a glioblastoma of unknown origin, was purchased from the American Type Culture Collection. The U-87 MG cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM; cat. no. 11965; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; cat. no. 16000; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (cat. no. 15140; Thermo Fisher Scientific, Inc.). Human brain microvascular endothelial cells (HBMECs; passages 7–9; ACBRI 376; Cell Systems) were cultured at 37°C in Endothelial Cell Growth Medium-2 BulletKit^™-microvascular (EGM-2-MV; CC-3202; Lonza Group, Ltd.). For the experiments under hypoxic conditions, cells were incubated at 37°C in a hypoxic incubator (Forma^™; cat. no. 3131; Thermo Fisher Scientific, Inc.) filled with 1% O₂, 5% CO₂ and balance N₂. Isolinderalactone (ALB-RS-6003) was obtained from ALB Technology, Ltd. and dissolved in dimethyl sulfoxide (DMSO; Duchefa Biochemie B.V.). Isolinderalctone was used to treat U-87 cells under hypoxia (2.5 or 5 µg/ml) and HBMECs were treated with HCM (2 µg/ml). Recombinant human basic fibroblast growth factor (BFGF; F0291) was purchased from Sigma-Aldrich. Recombinant human VEGF (293-VE) was purchased from R&D Systems, Inc. and reconstituted in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (cat. no. 0332; VWR International, LLC.) according to the manufacturer's protocol.

Total RNA was isolated from U-87 cells using TRIzol^® Reagent (cat. no. 15596; Thermo Fisher Scientific, Inc.). A PrimeScript^™ 1st Strand cDNA Synthesis Kit (cat. no. 6110; Takara Bio, Inc.) was used to synthesize cDNA from 2 µg total RNA, according to the manufacturer's protocol. End-point PCR for the measurement of VEGF was performed using GAPDH as the reference gene for normalization with the following primers: VEGF (23) forward, 5′-GAGAATTCGGCCTCCGAAACCATGAACTTTCTGCT-3′ (nucleotides plus EcoRI adapter) and reverse, 5′-GAGCATGCCCTCCTGCCCGGCTCACCGC-3′ (nucleotides plus SphI adapter) with a melting temperature of 65°C for 30 cycles; and GAPDH forward, 5′-CGTGGAAGGACTCATGAC-3′ and reverse, 5′-CAAATTCGTTGTCATACCAG-3′ with a melting temperature of 55°C for 27 cycles. The PCR products were mixed with Ezstain DNA loading dye (cat. no. B006M; Enzynomics, Inc.) and analyzed using a 1.2% agarose gel.

U-87 cells were lysed using RIPA buffer (cat. no. 9806; Cell Signaling Technology, Inc.) supplemented with a protease inhibitor mixture (P3100; GenDEPOT) and a phosphatase inhibitor mixture (P3200; GenDEPOT). Total protein content was determined by Pierce^™ BCA Protein Assay Kit (cat. no. #23225; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Total protein (20–30 µg/lane) was separated on 12% gels using SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham; Cytiva). After transfer, the membrane was incubated with 5% skimmed milk (cat. no. #232100; Becton, Dickinson and Company) in PBS containing 0.1% Tween-20 (cat. no. TR1027-500-00; Biosesang) for 1 h at room temperature. The membrane was immunoblotted using an anti-VEGF antibody (1:1,000; sc-7269; Santa Cruz Biotechnology, Inc.) for 16 h at 4°C. An α-tubulin antibody (1:3,000; T5168; Sigma-Aldrich; Merck KGaA), as the internal control, was incubated for 16 h at 4°C. For the incubation with secondary antibody, HRP-conjugated anti-mouse antibody (1:10,000; #1031-05; SouthernBiotech) was applied for 1 h at room temperature. Chemiluminescence intensity was measured using an ImageQuant^™ LAS 4000 apparatus (Cytiva). The quantification of band intensity was performed using ImageJ software (V1.8.0; National Institutes of Health) and normalized to the intensity of α-tubulin.

The CM from normoxic U-87 cells (NCM) and the CM from hypoxic U-87 cells (HCM) were collected, concentrated and used in further experiments.

U-87 cells were seeded at 1.5×10⁶ cells/100 mm dish and cultured for 2 days. The medium was replaced with EGM-2-MV (without VEGF and bFGF) and incubated under normoxic or hypoxic conditions. After 16 h, CM was harvested, filtered using a Minisart^® Syringe Filter (pore size 0.45 µm; Sartorius AG), concentrated twice (for wounding migration assay) or 10 times (for tube formation assay) with an Amicon^® Ultra-15 Centrifugal Filter Unit (UFC901024; MilliporeSigma) and used to treat HBMECs.

The U-87 cell medium was replaced with DMEM containing 0.5% FBS and incubated under normoxic or hypoxic conditions for 16 h. CM was collected, filtered using a Minisart Syringe Filter (pore size 0.45 µm), concentrated 100 times with an Amicon Ultra-15 Centrifugal Filter Unit and mixed with Matrigel (50 µl concentrated CM + 500 µl Matrigel).

U-87 cells cultured in DMEM containing 10% FBS were incubated under normoxic or hypoxic conditions for 16 h. CM was collected, filtered using a Minisart Syringe Filter (pore size 0.45 µm), and mixed with EGM-2-MV (without VEGF and bFGF) at a 2:1 ratio for use as interstitial fluid.

The migration assay was performed as described previously (24). Briefly, HBMECs were seeded into 60-mm culture dishes and incubated until they reached ~90% confluence. The monolayer was wounded with a razor and washed with an endothelial basal medium to remove cell debris, and the NCM (5 ml) or HCM (5 ml) from U-87 cells and 1 mM thymidine (T1895; Sigma-Aldrich) were added. The HCM-treated cells were incubated with or without isolinderalactone (2 µg/ml), and the cells were incubated for 16 h. The cells were then fixed, stained and imaged with an Olympus TH4-200 microscope (light microscope; Olympus Corporation). The migration activity was quantified by counting the number of cells that moved beyond the reference line.

HBMECs (3×10⁴ cells/well) were seeded onto polymerized Matrigel (cat. no. 354230; BD Biosciences) in a 24-well plate, and a tube formation assay was performed as previously described (24,25). HBMECs incubated with NCM (1 ml) or HCM (1 ml), the latter with or without 2 µg/ml isolinderalactone, were imaged with a TH4-200 microscope every hour for 10 h. Capillary-like tube networks were observed and the tube length and tube branching points (>3) were analyzed using iSolution full image analysis software (Image & Microscope Technology).

3D chips (DAX01) were purchased from AIM Biotech Pte. Ltd. and an angiogenic sprouting assay was performed as previously described (10). AIM connectors (LUC-1; AIM Biotech Pte. Ltd.) and a 1-cc syringe (Jung Rim Medical Industrial Co., Ltd.) were used to apply interstitial flow. Briefly, the central gel channel was filled with 2.5 mg/ml collagen gel solution (pH 7.4; 10 µl) prepared using Collagen I, rat tail (cat. no. 354236; Corning, Inc.), 10X PBS (cat. no. 70011-044; Thermo Fisher Scientific, Inc.) and 0.5 M NaOH (cat. no. 221465; Sigma-Aldrich), following the AIM Biotech protocol. The gel-filled device was polymerized for 30 min at 37°C, the lateral media channels were hydrated using growth medium (EGM-2-MV, without VEGF or bFGF), and the devices were kept in a humidified CO₂ incubator until cell seeding. HBMECs were suspended in growth medium at a concentration of 1.5×10⁶ cells/ml, and the cell suspension (40 µl) was seeded onto one lateral channel of the 3D chip. After seeding the cells, the chips were tilted 90° to allow the cells to adhere to the gel surface and incubated at 37°C for 15 min. The chips were then returned to their original orientation and incubated for 3 h. Then, the medium was replaced with fresh growth medium to remove any unattached cells. The next day, the AIM connectors and 1-cc syringe were connected. Subsequently, 200 µl growth medium was supplied to the cell seeding channel, and 1,000 µl of a mixture of NCM or HCM with growth medium and bFGF (10 ng/ml) was supplied to the medium channel. DMSO (0.1%) or isolinderalactone (2 µg/ml) was added to the medium channel containing the HCM mixture 6 days later. The medium was replaced daily, and sprouting was monitored using a TH4-200 phase-contrast microscope.

After 8 days of culture on the chip, immunofluorescence staining was performed according to the manufacturer's protocol. Briefly, HBMECs were washed, fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with 0.1% Triton X-100 for 30 min at room temperature. The cells were incubated with blocking buffer comprising 10% normal goat serum (S-1000; Vector Laboratories, Inc.) in 1X PBS for 2 h at room temperature, and then incubated with anti-VE-cadherin antibody (1:100 in 1X PBS; sc-9989; Santa Cruz Biotechnology, Inc.) overnight at 4°C. The next day, the cells were washed with PBS and incubated with Alexa Fluor 488-conjugated secondary antibody (1:200; A-11001; Thermo Fisher Scientific, Inc.) and rhodamine-phalloidin for F-actin (5 U/ml; R415; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Nuclei were stained with DAPI (D1306; Molecular Probes; Thermo Fisher Scientific, Inc.). The z-stack images of angiogenic sprouting were captured using a Zeiss LSM 700 laser scanning confocal microscope (Carl Zeiss AG). The sprout diameter was analyzed using images captured from orthographic views of the middle of the sprouts, as previously reported (26). Sprout length was measured starting from the point at which the sprouts began to extend. All analyses were performed using iSolution full image analysis software.

A total of 16 C57BL/6 mice (6-week-old males; 20–25 g) were purchased from Nara Biotechnology. The mice were housed under a 12-h light/dark cycle at 23±1°C with 40–60% humidity and allowed ad libitum access to food and water. The mice were adapted to these conditions for at least 7 days prior to the experiments. The mice were maintained in accordance with the Pusan National University Guidelines for the Care and Use of Laboratory Animals and the experiments using the mice were approved by the Institutional Review Board of Pusan National University (PNU-2019-2361). Humane endpoints for euthanasia were considered to be the prolonged observation of a 40% reduction in food and water intake and/or unstable breathing. The feeding and condition of the mice were assessed and monitored daily post-surgery for any signs of decreased activity and a reduction in food intake. However, no signs of these conditions were observed. The following groups were established: NCM (n=4), HCM + DMSO (n=6) and HCM + iso (n=6). Growth factor-reduced Matrigel (500 µl; cat. no. 354230) containing NCM or HCM (concentrated 100 times) was injected subcutaneously under anesthesia. The mice were anesthetized with isoflurane delivered using a face mask (3% for induction and 1.5% for maintenance, in 80% N₂O and 20% O₂). The next day, the treatment of the mice in the HCM + iso group was initiated with an intraperitoneal injection of 5 mg/kg/day isolinderalactone for 6 days. The mice in the HCM + DMSO group were treated with DMSO at the same time points. On day 8, the mice were sacrificed by exposure to 70% carbon dioxide following deep anesthesia with 50 mg/kg sodium thiopental (IP), and the Matrigel plugs were harvested. The hemoglobin content of the Matrigel plugs was measured using a hemoglobin assay kit (MAK115; Sigma-Aldrich) to evaluate blood vessel formation.

All data are expressed as the mean ± standard error of the mean. The differences between groups were analyzed for statistical significance using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To examine the direct angiogenic effect of GBM, CM from U-87 tumor cells was used. The CM was obtained from U-87 cells under normoxic or hypoxic conditions. Before collecting the CM, western blotting and RT-PCR analyses confirmed that VEGF expression was stimulated in U-87 cells under hypoxic conditions (Fig. 1). Hypoxia significantly increased VEGF protein and mRNA levels in the U-87 cells compared with those in U-87 cells cultured under normoxic conditions, and these increases in VEGF levels were significantly decreased by treatment with isolinderalactone. Culture supernatants were obtained from normoxic and hypoxic U-87 cells and used to perform in vitro and in vivo assays (Fig. 2A).

First, whether HCM affects angiogenesis was investigated using in vitro angiogenesis assays, in which HCM was used to simulate the angiogenesis-stimulating effects of glioblastoma on neighboring endothelial cells. The effect of HCM on endothelial migration, a critical feature in the formation of new blood vessels, was evaluated (27). HCM significantly stimulated the migration of HBMECs, whereas isolinderalactone treatment significantly attenuated the HCM-induced endothelial migration (Fig. 2B). The effect of HCM on capillary-like tube structure stabilization was then examined. HCM clearly increased capillary-like tube formation, whereas isolinderalactone suppressed the HCM-induced formation of tube structures (Fig. 3A). Quantification of the tube length and branching points revealed the significant suppression of HCM-mediated tube formation by isolinderalactone (Fig. 3B and C).

The anti-angiogenic effect of isolinderalactone was further examined in a 3D microfluidic device after the application of interstitial flow using glioblastoma CM (Fig. 4A). Angiogenic sprout growth was observed following HCM stimulation, and the addition of isolinderalactone to the cell culture inhibited sprout formation (Fig. 4B). Immunofluorescence staining of the HBMECs with anti-VE-cadherin (green) and anti-F-actin (red) antibodies showed that sprout length was significantly increased in the HCM-treated group, and the HCM-induced increase in sprout length was significantly decreased by isolinderalactone (Fig. 4C and D). However, sprout diameter did not differ among the groups (Fig. 4E).

To evaluate whether U-87 cell CM induces in vivo angiogenesis, which is inhibited by isolinderalactone, an in vivo mouse Matrigel plug assay was performed (Fig. 5). Mice were subcutaneously injected with Matrigel containing either NCM or HCM. Isolinderalactone treatment of mice with HCM was initiated after 24 h (Fig. 5A). HCM strongly increased angiogenic activity in the Matrigel plug, which was notably suppressed by isolinderalactone (Fig. 5B). The Matrigel with HCM had a significantly increased hemoglobin concentration compared with that in the Matrigel with NCM, and the hemoglobin concentration in the isolinderalactone-injected HCM group was significantly lower compared with that in the untreated HCM group (Fig. 5C).