Background
Vascular endothelial growth factor receptor-1 (VEGFR-1) is a high-affinity tyrosine kinase receptor for VEGF-A, VEGF-B, and placental growth factor (PlGF) ligands [
1,
2]. VEGFR-1 is composed of seven extracellular immunoglobulin homology domains, a single transmembrane region, and an intracellular tyrosine kinase domain. The interaction of VEGFR-1 with its ligands induces receptor dimerization, tyrosine auto-phosphorylation, transphosphorylation, and docking of signaling proteins [
1‐
3]. VEGFR-1 also exists as soluble form that acts as decoy receptor preventing VEGF-A and PlGF interaction with transmembrane receptors [
3]. While VEGFR-1 does not play a relevant role in physiological angiogenesis in the adult, this receptor is indeed important in tumor angiogenesis and directly activates signaling pathways crucial for tumor growth, progression, and metastasis in cancer cells [
4,
5].
VEGF-A binds to both VEGFR-1 and VEGFR-2, while VEGF-B and PlGF interact exclusively with VEGFR-1. VEGF-A is the most widely studied angiogenic factor, and its role in tumor angiogenesis via stimulation of VEGFRs expressed on tumor endothelium is well established [
6]. It also directly interacts with VEGFRs expressed on cancer cells stimulating disease progression. In its homo- or heterodimeric form with PlGF, VEGF-A may activate VEGFR-1 and VEGFR-2 homo- or heterodimers [
7,
8]. PlGF binds to VEGFR-1 with higher or lower affinity compared with VEGF-B or VEGF-A, respectively [
4,
9]. VEGF-B role in tumor biology appears limited [
10], while PlGF seems to have an important disease-associated role because its expression, which is low or undetectable in most adult healthy tissues, is significantly up-regulated in a number of pathological conditions including cancer [
5]. Interestingly, PlGF is produced by tumor, endothelial, and other cells of the tumor stroma including inflammatory cells promoting migration, proliferation, and survival [
11,
12]. Moreover, high tumor expression levels are associated with a poor prognosis [
11,
12].
VEGFR-1 is expressed in endothelial cells during vessel formation and remodeling, macrophages, myoepithelial cells, and a variety of human cancer cells, favoring cell migration and survival [
1,
2]. In tumors, VEGFR-1 signaling inhibits apoptosis, induces chemoresistance, and predicts poor prognosis and recurrence [
1,
13,
14]. Moreover, it is involved in the mobilization of myeloid bone marrow-derived cells that generate tumor-associated macrophages [
1,
15]. VEGF-A and PlGF binding to VEGFR-1 can induce phosphorylation and activation of mitogen-activated protein kinases (MAPKs) Erk1/2 and p38 [
16], and through VEGFR-1 activation, PlGF also stimulates the trans-phosphorylation of specific VEGFR-2 tyrosine residues [
17]. Interestingly, it has been proposed that PlGF may enhance tumor cell invasiveness by augmenting matrix metalloproteinase (MMP) secretion via Erk1/2 signaling [
18].
A number of studies have been designed to disrupt tumor angiogenesis and growth by anti-VEGF-A and anti-VEGFR-2 monoclonal antibodies (mAbs) or VEGFRs small molecule tyrosine kinase inhibitors. We hypothesize that molecules selectively targeting VEGFR-1 may inhibit tumor vascularization and invasion/metastasis while producing lower systemic toxicity than agents directed against VEGF-A or VEGFR-2, which cause adverse effects due to inhibition of physiological angiogenesis [
19]. Therefore, we have generated an anti-VEGFR-1 mAb (D16F7) by immunizing mice with a peptide corresponding to amino acids 149–161 of human VEGFR-1 [
15]. While not affecting binding of VEGF-A and PlGF, D16F7 reduces VEGFR-1 homodimerization and activation by both ligands. This mAb inhibits chemotaxis of human endothelial, myelomonocytic, and melanoma cells in response to VEGFR-1 ligands. In an in vivo murine model, D16F7 is well tolerated, inhibits angiogenesis in response to inflammatory stimuli and markedly affects melanoma growth. The antitumor effect is associated with tumor cell apoptosis, vascular abnormalities, reduced monocyte/macrophage infiltration and impaired myeloid progenitor mobilization [
15].
In the present study we investigated whether D16F7 exerts inhibitory activity against human glioblastoma (GBM), which is a highly aggressive brain tumor that relies on angiogenesis for growth and histological progression [
20,
21]. The standard care of newly diagnosed GBM includes surgical tumor resection followed by radiation therapy and chemotherapy with the alkylating agent temozolomide [
22,
23]. GBM exhibits a highly abnormal blood supply, which leads to swelling and reduced blood perfusion within the tumor and causes it to become resistant to chemo- and radiotherapy. VEGF-A and PlGF expression by glioma cells additionally induces accumulation of VEGFR-1–positive bone marrow-derived myeloid cells in the tumor tissue [
24]. While anti-VEGF-A treatment has become part of standard post-surgical treatment for recurrent GBM, its beneficial effects are temporary and it does not effectively extend patient overall survival [
25].
In this context, our results demonstrate that D16F7 markedly inhibits chemotaxis and invasiveness of GBM cells and patient-derived GBM stem cells (GSCs) in response to VEGF-A and PlGF, suggesting that VEGFR-1 might represent a suitable target that deserves further investigation for GBM treatment.
Methods
Immunohistochemical analysis of VEGFR-1 in tissue samples from GBM patients
We enrolled 42 adults [mean age 60.51 (34–79), 27 males/15 females], who underwent surgery for primary GBM at the Institute of Neurosurgery, “Università Cattolica del Sacro Cuore” (Rome, Italy), from March 2005 to September 2011. Diagnosis of GBM was established on histological examination according to the WHO classification (grade IV) of tumors of the nervous system. All patients provided written consent to use their specimens for research and the research proposal was approved by the university Ethical Committee. Tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.6 at 4 °C overnight. Tissues were rehydrated in graded ethanol solutions, xylene and finally embedded in Paraplast Plus (Tyco/Healthcare, Mansfield, MA). Sections, 3–4 μm thick, were deparaffinized and incubated in 10 mM citrate buffer, pH 6.0, dry heated for 10 min each to unmask antigen sites, cooled and washed in phosphate-buffered saline (PBS). Endogenous peroxidase activity was inhibited by rinsing the slides in 3% hydrogen peroxide for 5 min. Nonspecific binding was blocked by 5 min incubation with the Super Block Solution (ScyTek Laboratories, UT). After washing in PBS, sections were incubated for 10 min at room temperature with rabbit anti-Human Flt-1/VEGFR-1 polyclonal antibody (1:50; Spring Bioscience, Pleasanton, CA). The immunostaining conditions were standardized using human placenta as positive control (data not shown). Sections were washed extensively with PBS and subsequently processed using the Ultra Tek Anti-Polyvalent kit (ScyTek Laboratories). Finally sections were treated with 3,3′-diaminobenzidine as chromogen, contrasted with hematoxylin and mounted [
26]. Two blinded examiners evaluated staining of human tumor specimens. For each specimen, the number of VEGFR-1 positive cells in total 50 cells was counted.
Cell lines and culture conditions
The human GBM cell lines A172, U87, LN18, T98G and U373 were from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate, at 37 °C in a 5% CO2 humidified atmosphere.
GSCs were isolated from 18 surgical samples of adult patients who had undergone craniotomy at the Institute of Neurosurgery, “Università Cattolica del Sacro Cuore” (Rome, Italy). Prior to surgery all patients provided written informed consent according to the Declaration of Helsinki and the research proposal was approved by the university Ethical Committee. In regard to GSCs origin, the diagnosis of GBM was established on histological examination according to the WHO classification (grade IV) of tumors of the nervous system. Tumor samples were subjected to mechanical dissociation. The resulting cell suspension was cultured in a serum-free medium supplemented with 20 ng/ml epidermal growth factor (EGF) and 10 ng/ml FGF-2 (PeproTech, Rocky Hill, NJ). Generation of GSCs was defined by the following criteria: in vitro formation of primary neurospheres expressing stem cell markers such as CD133, SOX2, Musashi-1 and nestin, capacity of self-renew, ability to co-express astrocytic as well as neuronal phenotypic markers after serum-induced differentiation in vitro [
27‐
29]. GSCs were characterized by immunofluorescence analysis as previously described [
30]. All the GSC lines tested in this study were positive for SOX2, Musashi-1 and nestin, whereas they expressed different levels of CD133 (data not shown).
P3, EGFRwt
+, and EGFRvIII
+ GSC lines were previously described [
31]. Cells were cultured in neurobasal medium (NBM) supplemented with 2 mM GlutaMAX and 1× B-27 (Life Technologies, Carlsbad, CA), 1× penicillin/streptomycin (Lonza, Basel, Switzerland), 1 U/ml heparin (Sigma-Aldrich) and 20 ng/ml FGF-2 (hereafter referred to as complete NBM).
The human umbilical vascular endothelial cells (HUVEC) were isolated from freshly delivered umbilical cords as previously described [
32] and cultured in EGM-2.
The human GR-Mel and M14 melanoma cell lines, used as positive and negative controls for VEGFR-1 or VEGFR-2 transcripts, were obtained and cultured as previously described [
33].
Human GBM cell lines were authenticated by STR profiling (BMR genomics, Padova, Italy) and GSCs lines were periodically tested for the expression of phenotypic markers [in P3-derived cells, EGFR amplification or mutation; in GSCs, the above described markers].
Generation of GBM cell lines overexpressing VEGFR-1
Cell clones were obtained by limiting dilution from U87 cells and one clone was transfected with the pBLAS49.2 or pBLAS49.2/VEGFR-1 plasmids. The pBLAS49.2/VEGFR-1 construct was obtained by cloning of VEGFR-1 cDNA from pcDNA3/VEGFR-1 plasmid (a generous gift of Dr. K. Ballmer-Hofer, PSI, Zurich) into pBLAS49.2 vector (InvivoGen, San Diego, CA). Transfection was performed using lipofectamine 2000 (Invitrogen, Camarillo, CA), as described by the manufacturer, and transfected cells were selected in blasticidine (Invitrogen) containing culture medium. Antibiotic resistant clones were isolated by ring cloning and U87 clones maintained in the presence of 2.5 μg/ml blasticidine. VEGFR-1 expressing subclones were identified by RT-PCR and Western blotting.
Analysis of VEGFRs transcripts
Quantification of membrane VEGFR-1 and VEGFR-2 transcripts was performed by quantitative real-time reverse transcriptase –polymerase chain reaction (qRT-PCR) according to the dual-labeled fluorigenic probe method and using an ABI Prism 7000 sequence detector (PerkinElmer, Groningen, the Netherlands), as previously described [
34]. Expression levels were calculated by the relative standard curve method. Primers used were as follows: VEGFR-1, forward 5′-ACCGAATGCCACCTCCATG-3′ and reverse 5′-AGGCCTTGGGTTTGCTGTC-3′; VEGFR-2, forward 5′-GTCTATGCCATTCCTCCCCC-3′ and reverse 5′-GAGACAGCTTGGCTGGGCT-3′. For each sample, the level of VEGFR-1 or VEGFR-2 transcripts was normalized to that of 18S RNA (TaqMan® Gene Expression Assay, Applied Biosystems, Foster City, CA) and referred to the values of the VEGFR-1 and VEGFR-2 negative M14 cell line, to which the arbitrary value of 1 was assigned.
In VEGFR-1-transfected cells detection of VEGFR-1 transcript was confirmed by RT-PCR analysis. The cDNA preparation followed by PCR amplification to evaluate VEGFR-1 expression was performed as previously described [
35], utilizing an annealing temperature of 58 °C and the following primers: human VEGFR-1, forward primer 5′-CTCCTGAGTACTCTACTCCT-3′, reverse primer 5′-GAGTACAGGACCACCGAGTT-3′ (640 bp fragment); human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward primer 5′-TCCCATCACCATCTTCCA-3′, reverse primer 5′-CATCACGCCACAGTTTCC-3′ (380 bp fragment).
Quantification of VEGF-A and PlGF in GBM cell culture conditioned media by ELISA
Conditioned media from GBM cells were obtained by incubating semi-confluent cultures for 24 h in 0.1% BSA/DMEM medium without FBS. These conditions did not significantly affect cell viability. Supernatants were concentrated at least 10-fold in Centriplus concentrators (Amicon, Beverly, MA). Cells were detached from the flasks with PBS/EDTA. Cytokine secretion values were normalized by the total number of cells.
Quantification of the amount of VEGF-A and PlGF in the conditioned medium was performed using goat anti-VEGF-A or anti-PlGF IgGs (R&D Systems, Abingdon, UK), at a concentration of 10 μg/ml in PBS, to coat Maxisorp Nunc immunoplates (Nunc, Roskilde, Denmark). Detection of the cytokines was performed with biotinylated goat anti-VEGF or anti-PlGF IgGs (0.4 μg/ml; R&D Systems) followed by incubation with streptavidin alkaline phosphatase conjugate (1:10,000) (Roche, Monza, Italy) and alkaline phosphatase reaction. Optical density at 405 nm was measured in a 3550-UV Microplate reader (Bio-Rad, Hercules, CA).
Western blotting
Proteins were run in 10% SDS-polyacrylamide gels and transferred to supported nitrocellulose membranes by standard techniques. Immunodetection was performed using the following antibodies: mouse monoclonal anti-VEGFR-1 (clone D2, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-EGF receptor (EGFR) (528, 1:1000; Santa Cruz Biotechnology); mouse monoclonal antibody anti-EGFRvIII (L8A4; 1:1000; Absolute Antibody, Oxford, UK); rabbit polyclonal anti-phosphorylated VEGFR-1 at tyrosine 1213 (1:500; R&D Systems); rabbit polyclonal anti-Erk1&2 (1:1000; Genetex, Irvine, CA); rabbit polyclonal anti-phospho-Erk1&2 (Thr/Tyr185/187, 1:1000; Invitrogen); or rabbit polyclonal anti-β-actin (1:10,000; Sigma Aldrich) primary antibodies. Anti-mouse or anti-rabbit Ig/Horseradish peroxidase secondary antibodies and ECL Western blotting detection reagents from GE Healthcare (Milan, Italy) were used to identify the proteins of interest.
Chemotaxis assay and spheroid invasion assay
In vitro migration assay was performed using Boyden chambers equipped with 8 μm pore diameter polycarbonate filters (Nuclepore, Whatman Incorporated, Clifton, NJ) coated with 5 μg/ml gelatin (Sigma-Aldrich), as previously described [
36,
37]. Treatment with D16F7 was carried out by incubating the cells in the presence of the indicated mAb concentrations in a rotating wheel for 30 min at room temperature. Cells (2 × 10
5/chamber) were then loaded in the upper compartment of Boyden chambers and migration assay, toward stimuli (50 ng/ml VEGF-A or PlGF) present in the lower compartment, was done in the absence or in the presence of D16F7 mAb or, in selected experiments, of an equivalent amount of a species- and isotype-matched control antibody (mouse IgG1, R&D Systems) for 18 h. Migrated cells, attached to the lower side of the filters, were stained with crystal violet counted in triplicate samples for a total of 12 high power (200× magnification) microscopic fields.
For spheroid invasion assay, tumor cells (25,000–30,000 cells/ml) were suspended in DMEM-1640 containing 10% FBS (for GBM cell lines) or in complete NBM (for P3-derived GSC lines), supplemented with methyl cellulose (0.24% final concentration; Sigma-Aldrich), seeded in 96-well round bottom cell culture plates (100 μl/well; Corning® Costar® Ultra-Low attachment multi-well, Sigma-Aldrich) and centrifuged at 3000 rpm for 90 min [
31]. Plates were then incubated for 24 h under standard culture conditions (5% CO
2, at 37 °C) to allow spheroid formation. Spheroids were collected, embedded individually in 100 μl of matrigel (reduced growth factor basement membrane matrix, Pathclear, Cultrex, Gaithersburg, MD) in 0.1% BSA/DMEM or NBM medium, with or without VEGF-A or PlGF (50 ng/ml) and/or D16F7 mAb, and plated in each well of a 96-well flat bottom plate, previously coated with 50 μl of matrigel. Five to ten replicates were set up for each experimental group. After matrigel solidification at 37 °C, 100 μl of invasion medium, with or without VEGF-A, PlGF or EGF (50 ng/ml), were added and plates incubated at 37 °C for up to 72 h. Spheroids were visualized and photographed using a Nikon Eclipse TS100 microscope in conjunction with a Nikon DS-Fi1 high resolution camera (Melville, NY). Measurements were performed using Adobe Photoshop CS6 software. Relative invasion area was defined as area of spheroids (in mm
2) at each time point minus area on day 0.
Preliminary experiments on U87 and U87-MF24 cell migration in response to PlGF and in the presence of graded concentrations of D16F7 indicated that the IC50 values were 1.54 ± 0.22 μg/ml and 2.49 ± 0.56 μg/ml, respectively. Therefore, based on these results we selected the mAb concentrations to be tested in the functional assays.
Cell proliferation assay
Cell proliferation was evaluated in 96-well plates using the tetrazolium compound MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulphophenyl)-2H–tetrazolium, inner salt] from Promega (Madison, WI), as previously described [
38]. Briefly, increasing numbers of GBM cells, suspended in complete medium containing graded concentrations of D16F7 up to 20 μg/ml of D16F7 or control antibody or without antibodies, were dispensed into flat-bottom 96-well plates and grown at 37 °C in a 5% CO
2 humidified atmosphere. Six replica wells were used for every condition. After 3 days, 20 μl of MTS solution were added to each well and cells were incubated at 37 °C for 2 h. Absorbance was read at 490 nm (reference wavelength 655 nm) using a 3550-UV Microplate reader (Bio-Rad).
Statistical analyses
Statistical analysis of the differences between pairs of groups was performed by Student’s t test. For multiple comparisons ANOVA analysis, followed by Bonferroni’s post-test, was used. Statistical significance was determined at α = 0.05 level. Differences were considered statistically significant when p < 0.05.
Discussion
In the present study we demonstrate for the first time that the novel anti-VEGFR-1 mAb D16F7, which diminishes receptor activation by VEGF-A and PlGF, inhibits chemotaxis and ECM invasion of human GBM and patient-derived GSC lines.
Our data suggest that VEGFR-1 itself can transmit signals that promote GBM cell invasiveness. Importantly, since D16F7 does not reduce VEGFR-1 interaction with its ligands while inhibiting receptor homodimerization, the mAb is considered to display inhibitory effects on VEGFR-1 activation in a non-competitive fashion [
15]. Moreover, D16F7 does not hamper soluble VEGFR-1 ability to act as decoy receptor for VEGF-A and PlGF. This is particularly important considering the role of the soluble receptor in controlling tumor progression. In fact, in GBM low soluble VEGFR-1/VEGF-A ratio has been related to higher aggressiveness compared with astrocytomas [
47].
Characterization of GBM lines showed that VEGF-A and PlGF are secreted by most of the cell lines tested, suggesting that an autocrine loop may occur in VEGFR-1 expressing GBMs through activation of the receptor tyrosine kinase activity, in accordance with a previous study [
39]. Indeed, since we found that VEGFR-1 is frequently detected in GBM specimens, D16F7 is expected to interrupt the autocrine loop that favors tumor aggressiveness.
Although required for inflammatory reactions associated with tumor growth and metastasis and for monocyte migration [
48,
49], VEGFR-1 kinase activity is weakly induced upon ligand binding and receptor signaling has not been fully elucidated in tumor cells [
43]. Potential tyrosine phosphorylation sites have been identified in VEGFR-1 [
17,
44] and their role in receptor activation in GBM has been only recently investigated [
50]. Tyrosine 1213, which is regarded as the main auto-phosphorylation site responsible for activation of intracellular pathways [
9,
44,
45], became phosphorylated in a highly VEGFR-1-expressing GBM cell line upon exposure to exogenous VEGF-A or PlGF [
50]. In our study with U87-derived cells over-expressing VEGFR-1, exposure to VEGF-A or PlGF causes substantial receptor phosphorylation at tyrosine 1213 and pre-treatment with D16F7 prevents VEGFR-1 auto-phosphorylation in response to both ligands. Conversely, it has been reported that an anti-PlGF antibody only partially affected growth factor-induced VEGFR-1 auto-phosphorylation at this amino acid residue [
50]. Therefore, our data strongly suggest that blockage of VEGFR-1 activity is more efficiently achieved using D16F7 mAb, which avoids receptor activation by both VEGF-A and PlGF. Moreover, in our model VEGFR-1 auto-phosphorylation is followed by downstream phosphorylation of Erk1/2 that is counteracted by D16F7 treatment.
Analysis of VEGFR-1 in GSC lines indicates a quite variable expression of the receptor. In particular, ~17% (3 out of 18) of the GSCs tested demonstrate high levels of VEGFR-1 transcript that result in remarkable amounts of the corresponding protein detected on immunoblot. In this context, our results are in line with a recent study in a limited number of patient-derived GSC samples showing VEGFR-1 staining by immunocytochemistry analysis [
40].
The specificity of D16F7 against the chemotactic and invasive response to VEGF-A and PlGF was confirmed by the lack of antibody activity against cells responding to ligands that do not bind VEGFR-1 (i.e. EGF). Meanwhile, D16F7 markedly inhibits VEGFR-1 ligand-induced motility of GSCs expressing mutant EGFR (i.e., #74 and P3-derived EGFRvIII
+ GCSs) as well as GSCs over-expressing EGFRwt (i.e., P3-derived EGFRwt
+ GSCs). Actually, GBM cells harboring EGFRvIII mutation have recently been found to possess an angiogenic phenotype in vivo due to upregulated secretion of VEGF-A compared with cells over-expressing EGFRwt, which instead showed an enhanced invasive behavior [
31].
Tumor cell invasion, angiogenesis, and genetic intra-tumor heterogeneity are hallmarks of GBM that reflect major factors involved in treatment failure [
51‐
54]. Indeed, EGFR amplification and mutation are invariably expressed in a heterogeneous manner, and the presence of EGFRvIII in a minor population of GBM cells has been shown to confer a more aggressive tumor phenotype through paracrine mechanisms [
53]. It has recently been demonstrated that EGFR amplification is an early event in GBM development, while EGFRvIII subsequently emerges during disease progression to drive a more aggressive tumor that becomes dependent on angiogenesis for growth [
31]. Moreover, studies performed in a large cohort of GBM patients have indeed shown that VEGFR-1 is detected in tumor vessels and at significantly higher levels compared with lower grade gliomas [
55]. In this context, since D16F7 can interact with VEGFR-1 expressed by tumor cells as well as by endothelial cells, the advantage of D16F7 in the control of GBM growth is two-fold: the mAb may inhibit tumor cell invasion and angiogenesis.
VEGF-A and PlGF produced by GBM cells can also stimulate angiogenesis and induce accumulation of VEGFR-1-positive bone marrow-derived myeloid cells in glioma tissues [
24]. These cells are involved in neovessel formation and ECM invasion by secreting MMP and angiogenic factors or other cytokines that promote tumor cell survival [
24,
56]. Since D16F7 is able to suppress bone marrow mobilization of myeloid progenitors [
15], this property may additionally contribute to restraining GBM progression.