Abstract
Tumor-associated macrophages (TAMs) are pivotal effector cells in angiogenesis. Here, we tested whether CYP4X1 inhibition in TAMs by flavonoid CH625 prolongs survival and normalizes glioma vasculature. CH625 was selected against the CYP4X1 3D model by virtual screening and showed inhibitory activity on the CYP4X1 catalytic production of 14,15-EET-EA in the M2-polarized human peripheral blood mononuclear cells (IC50 = 16.5 μM). CH625 improved survival and reduced tumor burden in the C6 and GL261 glioma intracranial and subcutaneous model. In addition, CH625 normalized vasculature (evidenced by a decrease in microvessel density and HIF-1α expression and an increase in tumor perfusion, pericyte coverage, and efficacy of temozolomide therapy) accompanied with the decreased secretion of 14,15-EET-EA, VEGF, and TGF-β in the TAMs. Furthermore, CH625 attenuated vascular abnormalization and immunosuppression induced by coimplantation of GL261 cells with CYP4X1high macrophages. In vitro TAM polarization away from the M2 phenotype by CH625 inhibited proliferation and migration of endothelial cells, enhanced pericyte migration and T cell proliferation, and decreased VEGF and TGF-β production accompanied with the downregulation of CB2 and EGFR-dependent downstream STAT3 expression. These effects were reversed by overexpression of CYP4X1 and STAT3 or exogenous addition of 14,15-EET-EA, VEGF, TGF-β, EGF, and CB2 inhibitor AM630. These results suggest that CYP4X1 inhibition in TAMs by CH625 prolongs survival and normalizes tumor vasculature in glioma via CB2 and EGFR-STAT3 axis and may serve as a novel therapeutic strategy for human glioma.
Introduction
Glioblastoma (GBM) is the most common primary malignant brain tumor (Wick et al., 2017). Angiogenesis is integral to the pathology of GBM (Turkowski et al., 2018). Inhibitors of vascular endothelial growth factor (VEGF) are commonly used to block GBM angiogenesis in the clinic but fail to improve overall survival (Wick et al., 2016). Moreover, antiangiogenic treatment with sunitinib, a multitargeted receptor tyrosine kinase inhibitor, increases PD-L1 expression and fosters an immunosuppressive microenvironment (Ramjiawan et al., 2017). The challenge moving forward will be to overcome these limitations and expand the benefits of antiangiogenic therapy. Vascular normalization induced by antiangiogenic treatment modestly enhances T cell infiltration and improves the therapeutic efficacy of anticancer drugs and may represent a potential therapeutic strategy for human glioma (Goel et al., 2011; Peterson et al., 2016).
Tumor-associated macrophages (TAMs) constitute the majority of the stromal cells within a GBM (Huijbers et al., 2016). TAMs are correlated with poor survival of patients with recurrent glioblastoma (Huijbers et al., 2016; Kloepper et al., 2016). TAMs contribute to angiogenesis by indirect paracrine secretion of proangiogenic growth factors, including VEGF, transforming growth factor (TGF-β) and hypoxia-inducible factor-1α (HIF-1α) (Huijbers et al., 2016). Conversely, depletion of TAMs inhibits tumor growth and angiogenesis and improves the efficacy of chemotherapy as a result of vascular normalization (Guerriero et al., 2017; Wang et al., 2017). Under different stimuli, macrophages can polarize into different phenotypes: the two extremes of the phenotypic spectrum of TAMs are defined as the alternatively activated protumor (M2) versus classically activated antitumor (M1) states (Peterson et al., 2016; Wang et al., 2017). Polarization of TAMs is a novel strategy for vascular normalization and antitumor immunity (Huang et al., 2011). Our previous study showed that reprogramming of M2-polarized macrophages results in tumor growth inhibition and vascular normalization in rat C6 glioma (Wang et al., 2017). Therefore, inhibiting tumor angiogenesis by altering TAMs to an antitumoral phenotype could represent a promising therapeutic strategy for glioblastoma.
The endocannabinoid system comprises endogenous ligands such as anandamide (AEA), cannabinoid receptors (CB) 1/2, and the proteins responsible for their synthesis and degradation (Snider et al., 2010). AEA markedly inhibits glioma growth and angiogenesis through decreasing production of VEGF and TGF-β (Picardi et al., 2014; Ma et al., 2016). Cytochrome P450 (CYP) 4X1, an orphan P450 protein, is expressed in the brain and metabolizes AEA to 14,15-epoxyeicosatrienoic acid-ethanolamide (14,15-EET-EA) (Snider et al., 2010). Rhythmic regulation of Cyp4x1 gene expression in the rat brain and vasculature may contribute to circadian changes in blood flow (Carver et al., 2014). CYP4X1 is overexpressed in breast cancer and shows correlations with tumor grade (Murray et al., 2010). Computational identification and binding analysis of orphan human cytochrome P450 4X1 enzyme with substrates provide useful information on structure-based drug design (Kumar, 2015). However, the effects of CYP4X1 on glioma angiogenesis and its mechanism of action have not been elucidated. Importantly, there is an increasing interest in exploring dietary flavonoids as antiangiogenic agents (Ravishankar et al., 2013). In this regard, we showed that CYP4A inhibition by flavonoid FLA-16 prolongs survival and normalizes tumor vasculature in glioma (Wang et al., 2017). The success prompted us to evaluate a common inhibitor of CYP4X1 from flavonoids in effectively normalizing glioma vasculature.
In this study, a novel flavonoid, 3-sulfanyl-1-triazene (CH625), was selected from the nearly 36,043 compounds by virtual database screening against the CYP4X1 3D model. We then investigated whether CYP4X1 inhibition in TAMs by CH625 normalizes glioma vasculature with the improved survival and antitumor immunity. Mechanistically, we investigated whether CYP4X1 inhibition by CH625 contributes to vascular normalization through downregulating M2 macrophage-derived VEGF and TGF-β via CB2 and EGFR-STAT3 axis. Our findings could lead to a novel strategy for the treatment of human glioma.
Materials and Methods
Reagents.
3-Sulfanyl-1-triazene (CH625, CAS: 123316-64-3) was purchased from MOLBASE (Shanghai, China), and its purity was ≥98% as determined by high-performance liquid chromatography analysis. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was purchased from Sigma Chemical Co. (St. Louis, MO). 14,15-EET-EA was purchased from Cayman Chemicals (Ann Arbor, MI). Antibodies against CD31, α-SMA, HIF-1α, and F4/80 were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). IgG/horseradish peroxidase was purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD). Antibodies against iNOS, CD206, arginase-1, p-VEGFR2, VEGFR2, VEGF, p-Smad2, Smad2, TGF-β, p-EGFR, EGFR, CB2, caspase-3, Bcl-xL, Ki67, and β-actin were purchased from Abcam, Inc. (Cambridge, MA). For the flow cytometric analysis, anti-CD8 was purchased from Abcam, Inc. Mouse recombinant IL-4 and IL-13 were purchased from PeproTech (Rocky Hill, NJ). For in vivo experiment, liposomal CH625 was prepared by mixing CH625, 1,2-dimyristoyl-sn-glycero-3-phosphocholine and cholesterol at a mole ratio of 1:5:5 according to a modification of Lim et al. (2000). The final products were stored at −20°C and warmed to room temperature just before use. For the in vitro experiment, CH625 was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (80 mM), stored at −20°C, and serial dilutions were made in DMSO. All culture reagents contained less than 0.125 endotoxin unit per milliliter as checked by Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).
Homology Modeling.
The homology model of human CYP4X1 was automatically generated by the SWISS-MODEL program using the crystal structure of human CYP4B1 (PDB id: 5T6Q) as a template (Bordoli et al., 2009). The heme was manually merged into the protein to occupy the same position as the heme of the template protein (CYP4B1) using the Coot 0.8.2 program (Emsley et al., 2010). Subsequently, a 5-ns dynamic simulation was performed using GROMACS 4.5.4 software (http://www.gromacs.org) (Pronk et al., 2013). The quality of the final model was validated by two programs, Procheck and Verify_3D, both of which belong to the structure analysis-validation online server sponsored by the UCLA-DOE Institute for Genomics and Proteomics (LaGier, 2017).
Virtual Screening.
Molecular docking studies were performed using the Surflex-Dock26 of the SYBYL suite (SYBYL-X 2.0, Tripos) (Joshi et al., 2016). For this purpose the homology model of human CYP4X1 was subjected to induced-fit docking studies. The active pocket of CYP4X1 for binding the inhibitors was generated above the heme-group of the homology model of CYP4X1 in an automatic mode. All of the molecules from the traditional Chinese medicine database (TCM Database@Taiwan) (Chen et al., 2011) were prepared and filtered by Lipinski’s Rule of five using SYBYL-X 2.0. Subsequently, structure-based virtual screening was then performed using the Surflex-Dock (with default parameters).
Cell Cultures.
The rat C6 glioma cell line, human umbilical vein endothelial cells (HUVEC), 10T1/2 cell line (mouse pericyte-like cell) and mouse endothelial cell line MS1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Human brain vascular pericytes (HBVP) were purchased from ScienCell Research Laboratories (Carlsbad, CA). The mouse glioma cell line GL261 was obtained from the National Cancer Institute (Bethesda, MD). The cells were grown in DMEM containing 10% fetal bovine serum in a humidified atmosphere of 5% CO2/95% air at 37°C.
Human peripheral blood mononuclear cells (PBMCs) and T cells were isolated from the blood of healthy volunteers according to the Helsinki Declaration. All donors gave informed consent and were healthy nonsmoking men. Our study was approved by the ethics boards of the Medical School of Wuhan University. Human PBMCs were isolated as previously described (Tannheimer et al., 2014) and then incubated with macrophage-colony stimulating factor (50 ng/ml) for 7 days to be M0 macrophages and treated with tumor supernatants containing IL-4/IL-13 (20 ng/ml) for 12 hours to be M2 macrophages. Isolation, culturing, and characterization of mouse bone marrow-derived macrophages (BMDMs) were performed from male C57BL/6 mice as previously described (Mao et al., 2016; Dong et al., 2017) and treated with tumor supernatants containing IL-4/IL-13 (20 ng/ml) for 12 hours to be M2 macrophages. TAMs were isolated from C6 or GL261 glioma tissues using immunomagnetic selection as described previously (Chen et al., 2017).
Tumor Models and Treatment Regimes.
Wistar rats (male, 6–8 weeks old) and C57BL/6 mice (male, 6–8 weeks old) were provided by the Experimental Animal Center of Wuhan University, housed on a 12-hour light/12-hour dark cycle in a pathogen-free environment, and allowed ad libitum access to food and water. All animal studies were approved by the Animal Research Committee of Wuhan University and maintained in accordance with the guidelines by the Association for Assessment and Accreditation of Laboratory Animal Care International. For the C6 subcutaneous tumor model, rat C6 glioma cells (5 × 106) or mouse GL261 glioma cells (2 × 106) were injected subcutaneously into the right flank. When palpable tumors formed approximately 0.5 cm in diameter, CH625 was administered intraperitoneally at the dose of 10 and 20 mg/kg in rats and 25 and 50 mg/kg in mice once daily, which demonstrated no drug-related toxicity. The in vivo doses were selected on the basis of preliminary experiments. After euthanizing the rats or mice on day 8, tumors were collected and analyzed. The C6 or GL261 orthotopic tumor model was established as described previously (Qin et al., 2015). Briefly, the rats or mice were anesthetized with an intraperitoneal dose 3% or 0.6% pentobarbital, secured in a stereotaxic apparatus, and implanted 10 μl of cell suspension containing 1 × 106 rat C6 glioma cells or 3 μl of cell suspension containing 5 × 105 mouse GL261 glioma cells into the right caudatum. The animals were then returned to the cages after 2 hours and received standard diet and water ad libitum. Animal body weight was measured every day throughout the study periods. CH625 was administered intraperitoneally at the dose of at the dose of 10 and 20 mg/kg in rats and 25 and 50 mg/kg in mice once daily after tumor implantation. The survival time for each animal after inoculation was measured. To evaluate the effects of CH625 on chemotherapy, temozolomide (TMZ) was injected intraperitoneally either as a single dose of 20 mg/kg after treatment with CH625 (20 mg/kg) for 7 days to determine the concentration of TMZ in tumor tissues or at a dose of 20 mg/kg once daily for 7 consecutive days to evaluate the tumor growth and TMZ concentration.
TAM depletion was achieved by using anti-CSF-1 antibody as described previously (Guerriero et al., 2017). The anti-CSF-1 antibody (50 mg/kg) was administrated intraperitoneally before tumor implantation for 24 hours, followed by repeated injections of 25 mg/kg every 5 days, and then CH625 (20 mg/kg) was injected intraperitoneally once daily for a week from day 7 of treatment. The efficiency of macrophage depletion was assessed by immunohistochemical analysis of tumor sections for F4/80. In co-injection model, GL261 cells (9 × 105) mixed with parental or CYP4X1high (BMDMs) (3 × 105) were injected subcutaneously into the right flank of C57BL/6 mice in a ratio of 3:1. When palpable tumors formed approximately 0.5 cm in diameter, CH625 was administered intraperitoneally at the dose of 25 and 50 mg/kg once daily. After the mice were euthanized on day 8 of treatment, tumors were collected and analyzed.
Preparation of Conditioned Media.
The conditioned media (CM) was prepared as previously described (Chen et al., 2017). In brief, BMDM and PBMC at 1 × 106/well in six-well plates were stimulated with tumor supernatants containing IL-4/IL-13 (20 ng/ml) for 12 hours followed by treatment with CH625 (10 and 20 μM) or vehicle (dimethyl sulfoxide, DMSO) for 24 hours. The culture supernatants were harvested, and then subjected to centrifugation through an Amicon Ultra-4 filter to remove any traces of CH625. The retentate was collected as CM.
Laser Doppler Analysis of Tumor Perfusion.
The tumor perfusion was measured by laser Doppler analysis as previously described (Qin et al., 2015; Wang et al., 2017). Briefly, the C6-bearing rats and GL261-bearing mice as described above were anesthetized with 3% or 0.6% pentobarbital and placed on a heating pad (37°C). The cutaneous envelope over each tumor was carefully excised, protecting the vascular network on the tumor mass. Tumor perfusion in the animals treated with CH625 (10 and 20 mg/kg in rats and 25 and 50 mg/kg in mice) or vehicle at days 0, 2, 4, 6, and 8 was blindly measured using a laser Doppler analyzer (moorFLPI-2, Moor Instruments, Devon, UK). The tumor perfusion in arbitrary perfusion units was monitored graphically.
14, 15-EET-EA Measurement.
CYP4X1 enzymes (0.1 nmol) were reconstituted with 0.2 nmol of reductase individually at 4°C for 45 minutes. The reconstituted enzyme were supplemented with catalase (500 U) and 50 mM KPO4 buffer (pH 7.4) and diluted with water to a volume of 500 μl. The substrate anandamide was dissolved in ethanol and added to a final concentration of 2 μM, and the samples were equilibrated at 37°C for 3 minutes. The reactions were initiated by the addition of 1.2 mM NADPH and allowed to proceed for 45 minutes in a shaking water bath. The reactions were stopped by the addition of 3 ml of ethyl acetate, vortexed, and centrifuged at 1000 rpm for 5 minutes. The organic layer (top) was recovered and dried under nitrogen gas. The samples were then suspended in 100 μl of methanol.
14,15-EET-EA was analyzed by liquid chromatographic-tandem mass spectrometry as previously described (Snider et al., 2007). Briefly, samples (10 μl of each) were injected onto a Hypersil ODS column (5 μm, 4.6 × 100 mm; Thermo Fisher Scientific, Waltham, MA) that had been equilibrated with 75% solvent B (0.1% acetic acid in methanol) and 25% solvent A (0.1% acetic acid in water). The metabolites were resolved using the following gradient: 0–5 minutes, 75% B; 5–20 minutes, 75%–100% B; 20–25 minutes, 100% B; 25–26 minutes, 100%–75% B; and 26–30 minutes, 75% B. The flow rate was 0.3 ml/min. The column effluent was directed into the Agilent LSD ion trap mass spectrometer 1100 (Agilent Technologies, Palo Alto, CA) using negative electrospray ionization-MS/MS, and the peaks eluting with a mass/charge ratio (m/z) of 364 (14,15-EET-EA) were isolated and monitored.
12-Hydroxylauric Acid Measurement.
12-Hydroxylauric acid was analyzed by GC-MS as previously described (Wang et al., 2017). Briefly, the derivatized internal standard, 15-hydroxypentadecanoic acid, was monitored at m/z 387, and the derivatives of hydroxy lauric acid standards monitored at m/z 345 for 12-hydroxy lauric acid. The IC50 of CH625 for CYP4V2-dependent lauric acid hydroxylation was determined using GraphPad Prism (GraphPad Software Inc., San Diego, CA).
Flow Cytometry.
T cells were labeled with APC-labeled anti-CD3 and fluorescein isothiocyanate-labeled anti-CD8 antibodies. FACS data were acquired using a FACS Aria III flow cytometer (BD Biosciences, San Jose, CA) and then analyzed using FlowJo software (Tree Star, Ashland, OR). CD8+ T cell proliferation was measured by flow cytometry according to manufacturer’s protocols (Yang et al., 2016). T cells were stimulated with 2 μg/ml plate-bound anti-CD3 and anti-CD28 antibodies for 24 hours and incubated with CM from CH625 (10 and 20 μM)-treated PBMC for 24, 48, and 72 hours. CD8+ T cells proliferation was measured by CFSE dilution.
To investigate the macrophage polarization, macrophages were immunostained PE-Cy7 conjugated rat anti mouse CD86 monoclonal antibody (560582; BD Biosciences) and APC conjugated rat anti-mouse CD206 antibody (141708; Biolegend, San Diego, CA). FACS data were acquired using a FACS Aria III flow cytometer and then analyzed using FlowJo software (Tree Star). Results were expressed as the percentage of positive cells over the total number of cells.
Cell Proliferation and Migration Assay.
The proliferation assay was performed as previously described (Wang et al., 2015). Human umbilical vein endothelial cells (HUVEC) or mouse endothelial cells (MS1) (1 × 104/well) seeded in 96-well plates were treated with the indicated concentration of CH625 for 24 hours. Cell viability was measured according to the protocol of 3-(4,5-dimethylthiazol-2yl)-5- (3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (CellTiter 96 AQueous Assay reagent; Promega, Madison, WI).
The migration assay was performed as previously described (Wang et al., 2017). The CM from the BMDM or PBMC was added to the lower chamber. MS1, HUVEC, 10T1/2, or HBVP were added to the upper chamber. The cells were incubated over night for ability to migrate across the 5 μm transwell insert (Corning, NY) toward lower chamber. The cells that did not migrate and remained in the upper chamber were removed. The cells that migrated to the lower chamber were counted.
Statistical Analysis.
Normal distribution of the data were tested by the Kolmogorov-Smirnov test. Normal distribution was considered if P values were above 0.05. Normal distributed data were then evaluated using a one-way ANOVA followed by the Student-Newman-Keul’s test or two-tailed Student’s t test. The survival rate was estimated using the Kaplan-Meier method and compared using the log-rank test. All values were expressed as mean ± S.E.M., P values of 0.05 or less were considered significant.
Results
Discovery of Flavonoid CH625 as a Potential Inhibitor of CYP4X1.
Given that the crystal structure of CYP4X1 is not included in the Protein Data Bank, and CYP4X1 had the highest sequence identity (56%) with CYP4B1 (Fig. 1A), the CYP4B1 (PDB id: 5T6Q) was used as a template to build the 3D structure of CYP4X1. After 1.5 ns of simulation, the root mean square deviation value of the CYP4X1 tended to be convergent with fluctuations around 4.5 Å (Fig. 1B). Furthermore, PROCHECK showed that 99.5% of the residues were located in the allowed regions (90.2% most favored) and only 0.5% (2 residues) outside the allowed regions (Fig. 1C). Verify 3D also showed that 94.1% of the residues had an averaged 3D-1D score >0.2. These results indicate that the refined CYP4X1 3D model is reliable.
Homology modeling of CYP4X1 and structure-based virtual screening. (A) Sequence alignment results between CYP4X1 and template CYP4B1. The amino acid residues were colored by the Clustal method in Geneious. The dark blue color indicates conserved residues, and light blue color indicates a semiconserved substitution. (B) The backbone root mean square deviation (RMSD) values of the CYP4X1 during the dynamic simulation. (C) Ramachandran plot of the CYP4X1 model showing the distribution of residues in favored (red), allowed (yellow), and outlier (white) regions. (D) A structural view of the interaction of CH625 with CYP4X1. Heme is drawn as a red line representation, and CH625 is shown as a yellow stick representation. (E) The structure of 3-sulfanyl-1-triazene (CH625). (F) Details on the binding site interactions are shown. Nonpolar hydrogen atoms are hidden for clarity. Potential intermolecular hydrogen bonds are shown as red dashed lines.
Next, by virtual screening against the 3D structure of CYP4X1, we narrowed our interests from a total of 36,043 compounds (TCM Database) to 10 top flavonoids (Fig. 1D; Supplemental Table 1). A preliminary study was undertaken to evaluate the inhibitory activity of the 10 flavonoids on the human CYP4X1 enzyme. We observed the most promising compound CH625 (ZINC95910725, 3-sulfanyl-1-triazene) with an IC50 value of 23.1 μM (Fig. 1E; Supplemental Table 1). Next, we measured 14,15-EET-EA production in the M2-polarized PBMCs and found that CH625 significantly inhibited CYP4X1 catalytic production of 14, 15-EET-EA (IC50 = 16.5 μM). Enzyme kinetic analysis was performed to measure the type of inhibition. We found that CH625 presented a competitive type of inhibition, since the Km value increased, whereas the Vmax remained unchanged (Supplemental Fig. 1, A–C). Furthermore, the binding mode of CH625 to CYP4X1 was predicted by the Surflex-Dock program implemented in SYBYL software. We found that CH625 interacted with GLN114 and THR312 of CYP4X1 through a hydrogen bond, and formed several hydrophobic interactions with key catalytic site residues such as LEU121, ALA126, LEU315, ALA316, THR320, VAL381, and PHE491 of CYP4X1 (Fig. 1F). We also examined the specificity of CH625 for CYP4X1 and found that CH625 was approximately 4.15- and 2.88-fold selective for CYP4X1 over COX and its homolog CYP4V2, respectively. These results suggest that flavonoid CH625 is a potential inhibitor of CYP4X1.
CH625 Prolongs Survival and Delays Growth in the C6 and GL261 Gliomas.
We first treated rats bearing intracranial C6 with CH625, sunitinib, or vehicle. As shown in Fig. 2A, CH625 (20 mg/kg) significantly increased the survival (median 18.5 days) in C6-bearing rats compared with the sunitinib (13.5 days) or control-treated rats (11.5 days), accompanied by the decreased intratumoral level of 14,15-EET-EA. Conversely, sunitinib (80 mg/kg) significantly increased the intratumoral level of 14,15-EET-EA compared with the control (Fig. 2A). Similarly, CH625 (50 mg/kg) significantly increased survival of GL261-bearing mice (median 25 days) compared with the control (19 days) (Fig. 2B). The intratumoral level of 14,15-EET-EA was decreased in the CH625 (50 mg/kg)-treated group, whereas increased in the sunitinib-treated group compared with the control (Fig. 2B). Given the significant improvement in survival in animals treated with CH625 over sunitinib, we examined the impact of treatment on growth in the subcutaneous C6 and GL261 gliomas. As shown in Fig. 2, C and D, CH625 significantly decreased tumor weight and the intratumoral level of 14,15-EET-EA. In contrast, sunitinib increased the intratumoral level of 14,15-EET-EA without influencing tumor weight. These results suggest that CH625 prolongs survival and delays growth in the C6 and GL261 gliomas.
CH625 prolongs survival and delays growth in C6 and GL261 gliomas with the decreased intratumoral level of 14,15-EET-EA. (A) In the C6 intracranial glioma model, the survival time of the rats (n = 10) injected intraperitoneally with CH625 (10 and 20 mg/kg), sunitinib (80 mg/kg), or vehicle was measured, and 14,15-EET-EA in the tumor tissues from each group was determined at day 12 by LC-MS/MS. (B) In the GL261 intracranial glioma model, the survival time of the mice (n = 10) injected intraperitoneally with CH625 (25 and 50 mg/kg), sunitinib (80 mg/kg), or vehicle was measured, and 14,15-EET-EA in the tumor tissues from each group was determined at day 20 by LC-MS/MS. (C) In the C6 subcutaneous glioma model, rat C6 glioma cells (5 × 106) were injected subcutaneously into the right flank of Wistar rats. When tumors reached a size of about 100 mm3, the rats (n = 8) received CH625 (10 and 20 mg/kg), sunitinib (80 mg/kg), or vehicle by intraperitoneal injection once daily for a week. Tumor weight was measured, and 14,15-EET-EA was determined by LC-MS/MS. (D) In the GL261 subcutaneous glioma model, mouse GL261 glioma cells (2 × 106) were injected subcutaneously into the right flank of C57BL/6 mice. When palpable tumors formed approximately 0.5 cm in diameter, the mice (n = 10) received CH625 (25 and 50 mg/kg), sunitinib (80 mg/kg), or vehicle by intraperitoneal injection once daily for a week. Tumor weight was measured, and 14,15-EET-EA was determined by LC-MS/MS. The values are presented as the mean ± S.E.M., *P < 0.05; **P < 0.01 vs. control, #P < 0.05; ##P < 0.01 vs. sunitinib (80 mg/kg)-treated group.
CH625 Normalizes Tumor Vasculature in the C6 and GL261 Gliomas.
Normalization of tumor vasculature prolongs survival in glioma (Wang et al., 2017). To determine whether CH625 prolongs survival in glioma through normalizing the tumor vasculature, we assessed time course effects on tumor perfusion using laser Doppler analysis. In the subcutaneous C6 model, CH625 (10 and 20 mg/kg) improved tumor perfusion at day 2 of treatment by 19.4% and 21.3%, respectively. A steady increase in tumor perfusion until day 4 by 23.1% and 30.2% in the CH625 (10 and 20 mg/kg)-treated groups was observed, followed by a sharp decrease until day 8 by 21.7% and 21.1%, respectively. In contrast, tumor perfusion in the control group increased after 2 days by 5.8% and increased higher by 7.9% at day 4, followed by a decrease by 7.1% until day 8 (Fig. 3, A and B). We then determined tumor hypoxia by analysis for hypoxia-inducible factor (HIF)-1α. As expected, HIF-1α expression in C6 glioma was decreased in response to CH625 treatment compared with the control (Fig. 3C). Because pericyte coverage improves vessel maturation (Gilles et al., 2016), we double stained for the endothelial cell marker CD31 and the pericyte marker α-smooth muscle actin (α-SMA), and observed the decreased microvessel density and the increased pericyte coverage of tumor vessels in the CH625 (10 and 20 mg/kg)-treated groups compared with the control (Fig. 3D).
CH625 induces tumor vessel normalization in rat C6 glioma. (A–D) Rat C6 glioma cells (5 × 106) were injected subcutaneously into the right flank of Wistar rats. When tumors reached a size of about 100 mm3, the rats (n = 8) received CH625 (10 and 20 mg/kg) or vehicle by intraperitoneal injection once daily for a week. Tumor perfusion at days 0, 2, 4, 6, and 8 was measured using a laser Doppler analyzer. Scale bar, 2 mm (A). The quantitative analysis showed the relative level of tumor perfusion (B). After rats were euthanized at day 8, hypoxia induced factor (HIF)-1α in the tumor tissues was measured by Western blot (C). Double staining for CD31 (green) and α-SMA (red) in the tumor tissues was shown. Scale bar, 50 μm (D). (E) In the C6 intracranial glioma model, the survival time of the rats (n = 10) injected intraperitoneally with CH625 (20 mg/kg), temozolomide (TMZ, 20 mg/kg), CH625 (20 mg/kg) plus TMZ (20 mg/kg), or vehicle was measured, and TMZ uptake into tumor tissues was determined at day 12 by high-performance liquid chromatography (HPLC). (F) In another experiment, rat C6 glioma cells (5 × 106) were injected subcutaneously into the right flank of Wistar rats. When tumors reached a size of about 100 mm3, the rats (n = 8) received CH625 (20 mg/kg), TMZ (20 mg/kg), CH625 (20 mg/kg) plus TMZ (20 mg/kg), or vehicle by intraperitoneal injection once daily for a week. Tumor weight was measured, and TMZ uptake into tumor tissues was determined by HPLC. The values are presented as the mean ± S.E.M., *P < 0.05; **P < 0.01 vs. control, #P < 0.05; ##P < 0.01 vs. TMZ (20 mg/kg)-treated group.
Normalization of tumor blood vessels enhances the efficacy of chemotherapeutic drug temozolomide (TMZ) through increasing its concentration in the glioma tissues (Goel et al., 2011). We found that the CH625 (20 mg/kg) plus TMZ therapy significantly increased rat survival (median 22 days) compared with the TMZ (17 days) or control (12 days) in the intracranial C6 model (Fig. 3E). In addition, the dual therapy significantly increased TMZ concentration in the glioma tissues compared with the TMZ alone (Fig. 3E). Previous study showed that TMZ exerts anticancer effect by mostly affecting proliferation and apoptosis of tumor cells (Roos et al., 2007). Therefore, the proliferation marker (Ki-67) and the apoptotic markers (caspase-3 and Bcl-xL) were measured in the C6 glioma tissues. We found that the dual therapy significantly decreased Ki67 and Bcl-xL expression but increased caspase-3 expression compared with the TMZ monotherapy, although CH625 alone did not affect these parameters (Supplemental Fig. 2, A and B). Similar results were obtained in the subcutaneous C6 model (Fig. 3F; Supplemental Fig. 2, C and D). These data indicate that CH625 induces vascular normalization and thereby enhances the efficacy of TMZ.
In the subcutaneous GL261 model, CH625 (50 mg/kg) significantly decreased microvessel density and HIF-1α expression and increased pericyte coverage and tumor perfusion (Supplemental Fig. 3, A–D). Importantly, the CH625 (50 mg/kg) plus TMZ therapy significantly increased the survival in GL261-bearing mice (median 39 days) compared with the TMZ (30.5 days) or control-treated mice (19.5 days), accompanied by the increased intratumoral level of TMZ (Supplemental Fig. 3E). Additionally, the dual therapy significantly decreased tumor weight but increased the intratumoral level of TMZ in the subcutaneous GL261 glioma (Supplemental Fig. 3F). These results suggest that CH625 normalizes tumor vasculature in the C6 and GL261 gliomas.
CYP4X1 Inhibition by CH625 Reprograms TAMs, thus Decreasing VEGF and TGF-β Production and Enhancing T Cell Infiltration.
Polarization of TAMs is a novel strategy for vascular normalization and antitumor immunity (Huang et al., 2011). Therefore, we determined the alteration of the M1/2 phenotype in the subcutaneous C6 model and found that M2 (F4/80+CD206+) macrophages in the CH625 (10 and 20 mg/kg)-treated tumors were significantly decreased compared with the control (Fig. 4, A and B), though there was no difference in infiltration of F4/80+ cells among the groups. Furthermore, CH625 upregulated expression of M1 genes (iNOS and CXCL9), whereas downregulated expression of M2 genes (arginase-1 and CD206) (Fig. 4, C and D). We also observed the increased levels of Th1 (TNF-α and IL-12) and the decreased levels of Th2 (CCL2 and IL-10) cytokines in the CH625-treated groups compared with the control (Fig. 4, E and F). Importantly, CH625 (20 mg/kg) decreased 14,15-EET-EA, VEGF, and TGF-β production in the TAMs (Fig. 4G) and increased the number of CD8+ T cells in the tumor tissues (Fig. 4H).
CH625 repolarizes tumor-associated macrophages (TAMs) to the M1 phenotype through CYP4X1 inhibition. (A–H) Rat C6 glioma cells (5 × 106) were injected subcutaneously into the right flank of Wistar rats. When tumors reached a size of about 100 mm3, the rats (n = 8) received CH625 (10 and 20 mg/kg) or vehicle by intraperitoneal injection once daily for a week. Immunostaining analysis of F4/80+CD206+ macrophages in the tumor tissues from the groups treated with CH625 (10 and 20 mg/kg) or vehicle. Scale bar, 20 μm (A and B). M1 markers (iNOS and CXCL9) and M2 markers (arginase-1 and CD206) in the TAMs were measured by qPCR (C and D). Production of Th1 cytokines (TNF-α and IL-12) and Th2 cytokines (IL-10 and CCL2) in the TAMs was determined by ELISA (E and F). Production of 14,15-EET-EA, VEGF and TGF-β in the TAMs was determined by LC-MS/MS or ELISA (G). The number of CD8+ T cells was determined by flow cytometry (H). The values are presented as the mean ± S.E.M., *P < 0.05; **P < 0.01 vs. control. (I–L) Parental and CYP4X1high human peripheral blood mononuclear cells (PBMCs) stimulated with tumor supernatants containing IL-4/IL-13 (20 ng/ml) for 12 hours, and then treated with CH625 (10 and 20 μM) or vehicle for 12 hours, followed by treatment with or without 14,15-EET-EA (1 μM) for another 12 hours. Representative flow cytometry analysis of M1 and M2 macrophages. The macrophages were stained with PE-Cy7 conjugated anti-CD86 and APC-conjugated anti-CD206 monoclonal antibodies (I and J). 14,15-EET-EA, VEGF and TGF-β in culture supernatants was determined by LC-MS/MS or ELISA (K and L). T cells were stimulated with 2 μg/ml plate-bound anti-CD3 and anti-CD28 antibodies for 24 hours and incubated with CM from CH625 (10 and 20 μM)-treated PBMCs for the indicated time. The proliferation of CD8+ T cells was measured by carboxyfluorescein succinimidyl ester (CFSE) dilution (O and N). Each value represents the mean ± S.E.M. of five independent triplicate experiments. *P < 0.05; **P < 0.01 vs. control, #P < 0.05; ##P < 0.01 vs. model, ^P < 0.05; ^^P < 0.01 vs. CH625 (20 μM)-treated group. The effects of CYP4X1 overexpression on CYP4X1 protein level in PBMCs were measured by Western blot (M). Each value represents the mean ± S.E.M. of three independent triplicate experiments. *P < 0.05; **P < 0.01 vs. LV-NC.
Next, we measured the effects of CH625 on in vitro macrophage polarization, and found that treatment of IL-4/IL-13 (classic M2 macrophage activator)-stimulated human PBMCs with CH625 (10 and 20 μM) increased the number of CD86+ cells but decreased the number of CD206+ cells (Fig. 4, I and J), inhibited production of 14,15-EET-EA, VEGF, and TGF-β (Fig. 4K), without affecting the proliferation of glioma cells and macrophages (Supplemental Fig. 4, A–D). The CM from M2-polarized PBMCs treated with CH625 (10 and 20 μM) improved T cell proliferation (Fig. 4M). Conversely, overexpression of CYP4X1 by the CYP4X1 lentiviral activation particle in the M2-polarized PBMCs (Fig. 4O) or exogenous addition of 14,15-EET-EA reversed these effects of CH625 (Fig. 4, I, J, L, and N). Treatment of IL-4/IL-13-stimulated mouse BMDMs with CH625 (10 and 20 μM) increased the M1/M2 ratio; production of 14,15-EET-EA, VEGF, and TGF-β; and the number of T cells. In contrast, overexpression of CYP4X1 or exogenous addition of 14,15-EET-EA reversed these effects (Supplemental Fig. 5, A–F).
Inhibition of CYP4X1 in TAMs by CH625 Normalizes Glioma Vasculature with the Improved Antitumor Immunity.
To investigate whether the repolarization of TAMs is involved in the antitumor effect of CH625, we treated C6 glioma-bearing rats with anti-CSF-1 antibody to deplete TAMs (using lgG as the control) and compared their effects on the control tumors (containing more M2-polarized TAMs) with the CH625-treated tumors (containing more M1-polarized TAMs). The immunohistochemical analysis showed depletion of F4/80+ macrophages by 81.2% in the anti-CSF-1-treated rats. Elimination of TAMs decreased the growth of control tumors, indicating that TAMs were predominantly of the M2-like, tumor-promoting phenotype in these conditions. The CH625 (20 mg/kg)-treated tumors were smaller, but depletion of TAMs by anti-CSF-1 enhanced their growth (Fig. 5A), suggesting that CH625 skews TAMs to a more tumor-inhibitory M1-like phenotype. Elimination of TAMs by anti-CSF-1 improved tumor perfusion and pericyte coverage (Fig. 5, B–D) and decreased VEGF and TGF-β production (Fig. 5E). Importantly, CH625 (20 mg/kg) increased tumor perfusion and pericyte coverage and decreased VEGF and TGF-β expression, but TAM depletion failed to further affect these parameters, suggesting that M1-polarized TAMs do not prominently regulate vascular abnormalization in glioma (Fig. 5, B–E).
CYP4X1 inhibition in TAMs by CH625 normalizes glioma vasculature with the improved antitumor immunity. (A–E) Rat C6 glioma cells (5 × 106) were injected subcutaneously into the right flank of Wistar rats. The anti-CSF-1 antibody (50 mg/kg) was administrated intraperitoneally before tumor implantation for 24 hours, followed by repeated injections of 25 mg/kg every 5 days. CH625 (20 mg/kg) was injected intraperitoneally once daily for a week from day 7 of treatment. Tumor weight was measured (A). Tumor perfusion was evaluated by laser Doppler analysis (B). Double staining for CD31 (green) and α-SMA (red) in the tumor tissues was performed. Scale bar, 50 μm (C and D). VEGF and TGF-β in the tumor tissues were determined by ELISA (E). The values are presented as the mean ± S.E.M., n = 8. *P < 0.05; **P < 0.01 vs. control, ^P < 0.05; ^^P < 0.01 vs. CH625 (20 mg/kg)-treated group. The effects of CYP4X1 overexpression on CYP4X1 protein level in BMDMs were measured by Western blot (F). Each value represents the mean ± S.E.M. of three independent triplicate experiments. *P < 0.05 vs. LV-NC. (G-M) GL261 cells (9 × 105) mixed with parental or CYP4X1high cells (mouse bone marrow-derived macrophages, BMDMs) (3 × 105) were injected into the right flank of C57BL/6 mice in a ratio of 3:1. When palpable tumors formed approximately 0.5 cm in diameter, the mice were treated with CH625 (50 mg/kg) or vehicle by intraperitoneal injection once daily. After mice were euthanized on day 8, tumor weight was measured (G). Immunofluorescence for CD31+α-SMA+ vessels was performed (H). Tumor perfusion was evaluated by laser Doppler analysis (I). 14,15-EET-EA, VEGF and TGF-β in the tumor tissues were determined by LC-MS/MS or ELISA (J). PD-L1 expression in the tumor tissues was determined by Western blot (K). The number of CD8+ T cells was determined by flow cytometry (L). GzmB, IFN-γ, and IL-2 in CD8+ T cells were determined by ELISA (M). The values are presented as the mean ± S.E.M., n = 10. *P < 0.05; **P < 0.01 vs. WT control, ^P < 0.05; ^^P < 0.01 vs. CH625 (50 mg/kg)-treated WT group.
To clarify whether inhibition of CYP4X1 in TAMs by CH625 promotes vascular normalization with the improved antitumor immunity, GL261 glioma cells were co-implanted with wild-type or CYP4X1high BMDMs (Fig. 5F) into the right flank of C57BL/6 mice. We found that CYP4X1 overexpression in BMDMs decreased pericyte coverage, tumor perfusion, the number of CD8+ T cells and their production of GzmB, IFN-γ and IL-2, accompanied by the increased tumor burden, PD-L1 expression and 14, 15-EET-EA, VEGF and TGF-β production (Fig. 5, G–M). Conversely, CH625 (50 mg/kg) significantly increased pericyte coverage, tumor perfusion, the number of CD8+ T cells and their production of GzmB, IFN-γ and IL-2, whereas decreased tumor burden, PD-L1 expression and 14,15-EET-EA, VEGF, and TGF-β production, which were partially reversed by CYP4X1 overexpression (Fig. 5, G–M).
Inhibition of CYP4X1 in TAMs by CH625 Decreases the Proliferation and Migration of Endothelial Cells and Increases the Migration of Pericyte Cells.
TAMs-derived VEGF and TGF-β promote pericyte migration, which is the crucial step for vascular normalization (Liu et al., 2012; Wang et al., 2017). We found that the CM from CH625 (10 and 20 μM)-treated PBMCs and BMDMs decreased the proliferation and migration of endothelial cells and increased the migration of pericyte cells, which were partially reversed by overexpression of CYP4X1 or exogenous addition of 14,15-EET-EA, VEGF or TGF-β (Fig. 6, A–D). To investigate whether VEGFR2 or Smad2 plays an essential role in CH625-induced pericyte migration, we measured p-VEGFR2 and p-Smad2 and found that the CM from CH625 (10 and 20 μM)-treated PBMCs decreased p-VEGFR2 and p-Smad2 in the HBVPs and HUVECs, although there were no significant differences of VEGFR2 and Smad2 levels among the groups (Fig. 6, E and F).
CYP4X1 inhibition in TAMs by CH625 stimulates migration of pericyte cells and attenuates proliferation and migration of endothelial cells. Parental and CYP4X1high mouse bone marrow-derived macrophages (BMDMs) and human peripheral blood mononuclear cells (PBMCs) stimulated with tumor supernatants containing IL-4/IL-13 (20 ng/ml) for 12 hours, and then treated with CH625 (10 and 20 μM) or vehicle for 12 hours, followed by treatment with or without 14,15-EET-EA (1 μM), VEGF (20 ng/ml), or TGF-β (10 ng/ml) for another 12 hours. The condition medium (CM) from the above treated groups was added to the lower chamber. Pericyte cells (mouse pericyte-like cells, 10T1/2 and human brain vascular pericytes, HBVPs) (A and C) or endothelial cells (mouse endothelial cells, MS1s and human umbilical vein endothelial cells, HUVECs) (B and D) were added to the upper chamber and then incubated overnight. The cells migrated to the lower chamber were counted. MS1s or HUVECs were plated at 1 × 104 per well in 96-well culture plates and then treated with the CM-treated as above. Cell proliferation was measured using 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (MTS) assays (B and D). The levels of p-VEGFR2, VEGFR2, p-Smad2, and Smad2 in the HBVPs and HUVECs (E and F) were determined by Western blot. Each value represents the mean ± S.E.M. of five independent triplicate experiments. *P < 0.05; **P < 0.01 vs. control, #P < 0.05; ##P < 0.01 vs. model, ^P < 0.05; ^^P < 0.01 vs. CH625 (20 μM)-treated group.
CB2 and EGFR-STAT3 Axis Contributes to CH625-Induced Downregulation of VEGF and TGF-β.
We found that CB2 was decreased, whereas p-EGFR and p-STAT3 were increased in M2 macrophages stimulated by tumor supernatants containing IL-4/IL-13 (Fig. 7, A and B). In contrast, CH625 (10 and 20 μM) significantly increased CB2 and decreased p-EGFR and p-STAT3 without influencing EGFR and STAT3 (Fig. 7, A and B). To investigate whether CH625 blocks VEGF and TGF-β via STAT3, STAT3 was overexpressed by transfection with STAT3 lentiviral activation particle in PBMCs (Fig. 7C). We found that STAT3 overexpression or exogenous addition of EGF and CB2 inhibitor AM630 reversed CH625 (20 μM)-induced decrease in mRNA expression of M2 markers (arginase-1 and CD206), levels of Th2 cytokines (CCL2 and IL-10), and production of TGF-β and VEGF (Fig. 7, D–F). Importantly, overexpression of STAT3 or exogenous addition of EGF and AM630 reversed the enhanced migration of pericyte cells and the decreased proliferation and migration of endothelial cells induced by the CM from CH625 (20 μM)-treated PBMCs (Fig. 7, G–I).
CYP4X1 inhibition in TAMs by CH625 increases pericyte migration and decreases endothelial proliferation and migration via CB2 and EGFR-STAT3 axis. Parental and STAT3high human peripheral blood mononuclear cells (PBMCs) stimulated with tumor supernatants containing IL-4/IL-13 (20 ng/ml) for 12 hours, and then treated with CH625 (10 and 20 μM) or vehicle for 12 hours, followed by treatment with or without EGF (20 ng/ml) or AM630 (10 μM) for another 12 hours. p-EGFR, EGFR, CB2, p-STAT3, and STAT3 were determined in the PBMCs by Western blot (A and B). The effects of STAT3 overexpression on STAT3 protein level in the PBMCs were measured by Western blot (C). mRNA expression of M2 markers (arginase-1 and CD206) and production of Th2 cytokines (IL-10 and CCL2) in the PBMCs were measured by qPCR or ELISA (D and E). VEGF and TGF-β in the supernatants were determined by ELISA (F). The condition medium (CM) from the above treated groups was added to the lower chamber. Human brain vascular pericytes (HBVPs) (G) or human umbilical vein endothelial cells (HUVECs) (I) were added to the upper chamber and then incubated overnight. The cells migrated to the lower chamber were counted. HUVECs cells were plated at 1 × 104 per well in 96-well culture plates and then incubated with the CM treated as above. Cell proliferation was measured using the MTS assays (H). Each value represents the mean ± S.E.M. of five independent triplicate experiments. *P < 0.05; **P < 0.01 vs. control, #P < 0.05; ##P < 0.01 vs. model, ^P < 0.05; ^^P < 0.01 vs. CH625 (20 μM)-treated group. A proposed mechanism to explain the effect of flavonoid CH625 on vascular normalization by targeting TAMs in tumor microenvironment (H). TAMs: tumor-associated macrophages; CYP4X1: cytochrome P450 4X1; 14,15-EET-EA: 14,15-epoxyeicosatrienoic acid ethanolamide; CB2, cannabinoid receptors 2; ECs, endothelial cells; EGFR, epidermal growth factor receptor; PCs, pericyte cells; TGF-β, transforming growth factor β; VEGF, vascular endothelial growth factor.
Discussion
In the present study, two novel observations have been made. First, we provided direct evidence that CYP4X1 inhibition by a novel flavonoid CH625 strongly normalizes glioma vasculature with the improved survival and antitumor immunity. To our knowledge, this is the first study that directly demonstrated the vasculature normalization effect of flavonoid CH625. Second, we have demonstrated for the first time that CYP4X1 inhibition by CH625 normalizes glioma vasculature through blunting production of TAMs-derived VEGF and TGF-β via CB2 and EGFR-STAT3 signaling, and may represent a potential therapeutic strategy for human glioma. However, subcutaneous glioma models lack the local central nervous system tumor microenvironment (Lenting et al., 2017). Thus, the effects of CH625 and its mechanism of action should be further studied in intracranial glioma models.
We demonstrated that CYP4X1 inhibition by CH625 prolonged survival and delayed growth in the C6 and GL261 gliomas. Previous study showed that the stimulated proliferation and migration of endothelial cell produces immature tumor vessels characterized by the decreased perfusion and oxygenation, and thereby hampers survival in glioblastoma (Carmeliet and Jain, 2011). Conversely, vessel normalization induced by anti-VEGF therapy improves tumor perfusion and oxygenation by pruning immature vessels and increasing vascular maturation (Jain, 2005). Here we observed the decreased microvessel density and the increased pericyte coverage of tumor vessels in the CH625 (10 and 20 mg/kg)-treated groups compared with the control. Furthermore, CH625 was likely to produce a short-lived time window of about 4 days when tumor vessels were transiently normalized, while sustained antiangiogenic regimens (up to 8 days) resulted in the decreased perfusion by disrupting tumor vessels, similar to previous study (Goel et al., 2011). These data indicate that CYP4X1 inhibition by flavonoid CH625 prolongs survival through normalizing glioma vasculature.
The tumor microenvironment often polarizes macrophage to a M2 phenotype, which promotes vascular abnormalization (Rolny et al., 2011; Qin et al., 2015), whereas repolarization of TAMs away from the M2 phenotype contributes to tumor growth inhibition and vascular normalization (Rolny et al., 2011; Qin et al., 2015). The clinical benefit of angiostatic drugs remains limited due to the induction of drug resistance by stromal cells, including TAMs (Huijbers et al., 2016). Two proangiogenic growth factors, VEGF and TGF-β, are highly expressed in TAMs (Li et al., 2016; Wang et al., 2018) and play critical roles during glioma angiogenesis (Qin et al., 2015; Mangani et al., 2016). Our previous study showed that the decreased VEGF or TGF-β production in TAMs inhibits angiogenesis and thus normalizes glioma vasculature (Wang et al., 2017). Here, we demonstrated that CYP4X1 inhibition by a novel flavonoid CH625 normalized glioma vasculature and prolonged survival through blunting production of TAMs-derived VEGF and TGF-β. Thus, downregulation of VEGF and TGF-β by targeting CYP4X1 in perivascular TAMs may represent a potential therapeutic strategy for human glioma.
PD-L1, an immune checkpoint regulator, is highly expressed in TAMs and plays critical roles during immune escape (Gordon et al., 2017; Noguchi et al., 2017). Angiogenic inhibitors induce PD-L1 expression in tumors to overcome growth restrictions and escape immunosurveillance (Ramjiawan et al., 2017). A small phase I study showed that combining the immune checkpoint antibody ipilimumab with bevacizumab increases overall survival in advanced melanoma patients for more than 6 months (Ott et al., 2015). Concurrent blockade of angiogenesis and PD-L1 improves efficacy, prolongs survival, and enhances T cell infiltration and activation (Allen et al., 2017). Here, we demonstrated that CYP4X1 inhibition by CH625 decreased PD-L1 expression in TAMs and increased number of CD8+T cells and their production of GzmB, IFN-γ, and IL-2 during normalization of GL261 glioma vasculature. A previous study showed that flavonoid apigenin inhibits PD-L1 expression in human and mouse mammary carcinoma cells (Coombs et al., 2016).These data suggest that CYP4X1 inhibition in TAMs by flavonoid CH625 could stimulate antitumor immunity, thus overcoming the limitations and expanding the benefits of antiangiogenic therapy. Therefore, further experiments are being carried out to investigate this possibility.
EGFR is altered in almost 50% of glioblastomas (Zahonero and Sánchez-Gómez, 2014). However, although EGFR kinase inhibitors have proven to be useful in treating other types of tumors, they offer poor outcomes in GBM patients, with no clear explanation for the tumor resistance observed (Zahonero and Sánchez-Gómez, 2014; Weller et al., 2017). Additionally, activation of cannabinoid receptor type 2 has been suggested as a new strategy to modulate angiogenesis in vitro and in vivo (Staiano et al., 2016). A previous study showed that STAT3 tyrosine phosphorylation is induced by EGFR but inhibited by upstream receptors including CB2 (Ravi et al., 2016). Here, we demonstrated that CYP4X1 inhibition in TAMs by CH625 downregulated STAT3 signaling via both CB2 and EGFR and thus normalized tumor vasculature in glioma. Given that STAT3 mediates the angiogenic signals triggered by multiple of upstream receptors, including EGFR and CB2, novel targeted therapies founded on CYP4X1 may represent a potential strategy for prevention of glioma angiogenesis.
In summary, CYP4X1 inhibition in TAMs by flavonoid CH625 normalizes glioma vasculature through downregulation of VEGF and TGF-β via both CB2 and EGFR-dependent downstream STAT3 signaling (Fig. 7J). Our results provide evidence in support of flavonoid CH625 as a potential inhibitor of CYP4X1, thus normalizing glioma vasculature with the improved survival and antitumor immunity. These results lay a solid foundation for the development of the novel CYP4X1 inhibitors to overcome the limitations/refractoriness to anti-VEGF treatment of human glioma.
Conflict of interest statement
Authorship Contributions
Participated in research design: Wang, Y. Yang, J. Yang.
Conducted experiments: Wang, Li, Huang, X. Liu, Qiu, Y. Liu.
Performed data analysis: Wang, Li, Chen, Y. Liu.
Wrote or contributed to the writing of the manuscript: Wang, Y. Yang, J. Yang.
Conflict of interest statement
The authors declare that they have no conflict of interests.
Footnotes
- Received December 11, 2017.
- Accepted January 29, 2018.
This work was partly supported by the National Natural Science Foundation of China [Grant 81173089 (to J.Y.)] and Hubei Key Laboratory of Medical Information Analysis and Tumor Diagnosis & Treatment [Grants PJS140011508 (to C.W.) and PJS140011707 (to Y.L.)].
↵
This article has supplemental material available at jpet.aspetjournals.org.The authors declare that they have no conflict of interests.
Abbreviations
- AEA
- anandamide
- BMDM
- bone marrow-derived macrophages
- CB
- cannabinoid receptors
- CM
- conditioned media
- CYP
- cytochrome P450
- DMSO
- dimethyl sulfoxide
- EGFR
- epidermal growth factor receptor
- 14,15-EET-EA
- 14,15-epoxyeicosatrienoic acid-ethanolamide
- GBM
- glioblastoma
- HBVP
- human brain vascular pericytes
- HIF-1α
- hypoxia-inducible factor-1α
- HUVEC
- human umbilical vein endothelial cells
- IL
- interleukin
- PBMC
- human peripheral blood mononuclear cells
- α-SMA
- α-smooth muscle actin
- TAMs
- tumor-associated macrophages
- TGF
- transforming growth factor
- TMZ
- temozolomide
- VEGF
- vascular endothelial growth factor
- Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics
References
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CYP4X1 Inhibition Normalizes Glioma Vasculature
