Article Figures & Data
Figures
- Fig. 1.
PlGF-induced migration and D16F7 inhibitory effects on C6 cells transfected with human VEGFR-1. (A) Analysis of human VEGFR-1 transcript levels in transfected C6 clones. VEGFR-1 mRNA levels in C6-derived clones transfected with control (C6-CTR1, C6-CTR2) or VEGFR-1–expressing (C6-MF1, C6-MF2, C6-MF3) vectors were analyzed by RT-PCR. Amplified products were separated on 1% agarose gels, and results are representative of one out of two independent experiments. The human melanoma GR-Mel and M14 cell lines were used as positive and negative controls, respectively. MW, molecular weight markers. (B) Analysis of human VEGFR-1 protein in transfected C6 clones. VEGFR-1 protein levels in C6-derived clones transfected with control or VEGFR-1-expressing vectors were analyzed by Western blotting. (C) Cell migration of transfected C6 cells in response to PlGF. Migration of C6-CTR2 and C6-MF1 cells in response to serum-free medium or PlGF (50 ng/ml) was analyzed after 18 hours of incubation, in the absence or presence of 5 μg/ml D16F7. Results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: comparisons between PlGF C6-MF1 and all the other groups, ***P < 0.001; comparisons between NS C6-MF1 and C6-CTR2 groups, +++P < 0.001. (D) Influence of an isotype-matched control Ab (IgG1) on the chemotactic response of C6-MF1 cells to PlGF. Cell migration in response to PlGF (50 ng/ml) was analyzed after 6 hours of incubation, in the absence or presence of 5 μg/ml control IgG1. NS, nonstimulated cells. Differences between PlGF and PlGF+IgG1 were not significant.
- Fig. 2.
D16F7 inhibits VEGF-A or PlGF-induced chemotaxis and ECM invasion of C6 cells overexpressing VEGFR-1. (A) Migration of C6-MF1 cells in response to PlGF or VEGF-A was analyzed after 6 hours of incubation, in the absence (NT, not treated) or presence of 5 μg/ml D16F7. NS, nonstimulated cells. Histogram represents the mean ± S.D. (n = 3) of migrated cells/microscopic field. The results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: PlGF vs. NS, PlGF vs. D16F7, or PlGF vs. PlGF+D16F7, ***P < 0.001; VEGF-A vs. NS, VEGF-A vs. D16F7, or VEGF-A vs. VEGF-A+D16F7, +++P < 0.001; differences between NS, D16F7, PlGF+D16F7, and VEGF-A+D16F7 were not significant. (B) Photographs from a representative migration experiment with C6-MF1 cells in response to PlGF or VEGF-A. Original magnification, 100×. (C) For spheroid invasion assay, C6-MF1 cells were embedded in collagen I in the absence or presence of D16F7 (10 µg/ml) and PlGF or VEGF-A (50 ng/ml). Relative invasion was quantified as the spheroid area difference (in square millimeters) at each of the indicated time points minus day 0. Data are expressed as the mean ± S.D. of relative invasion (n = 5–8). NS, nonstimulated cells. The results of statistical analysis were as follows: PlGF vs. NS and PlGF vs. PlGF+D16F7 at 24, 48, and 72 hours, ***P < 0.001; VEGF-A vs. NS and VEGF-A vs. VEGF-A+D16F7 at 24 and 48 hours, ++P < 0.01; at 72 hours, +++P < 0.001. Differences between NS and PlGF+D16F7 or NS and VEGF-A+D16F7 were not significant. (D) Representative pictures of spheroids taken at 24, 48, and 72 hours. Original magnification, 40×.
- Fig. 3.
Inhibition by D16F7 of VEGF-A- and PlGF-induced phosphorylation of VEGFR-1 and Erk1/2 in C6 cells overexpressing VEGFR-1. Western blotting of total or phosphorylated VEGFR-1 (pVEGFR-1) at tyrosine 1213 and total or phosphorylated Erk1/2 (pErk) in untreated or D16F7 (10 µg/ml) pretreated C6-MF1 cells in response to 100 ng/ml PlGF (A) or VEGF-A (B). Histograms represent the densitometric quantification of band intensities in the corresponding immunoblots, expressed as the pVEGFR-1/VEGFR-1 ratio relative to an untreated control, after normalization for β-actin expression. The normalized pVEGFR-1/VEGFR-1 ratio in untreated cells was considered equal to 1. (C) Histogram represents the mean ± S.D. (n = 3) percentage inhibition values of PlGF- or VEGF-A-induced VEGFR-1 or Erk1/2 phosphorylation in C6-MF1 cells after treatment with 10 µg/ml D16F7, calculated from immunoblot densitometric analysis.
- Fig. 4.
D16F7 treatment inhibits in vivo growth of C6-MF1 cells injected intramuscularly. (A) Safety analysis of D16F7 treatment. Toxicity of D16F7 mAb treatment was evaluated on the basis of net BW reduction in nude mice treated for 2 weeks with vehicle (CTR) or 20 mg/kg D16F7 on alternate days (eight animals/group). Data represent the BW variation with respect to the first day of treatment, to which the arbitrary value of 100 was assigned. (B) Antitumor activity of D16F7 against intramuscular xenograft. C6-MF1 cells were injected intramuscularly in the left hind limb of nude mice and, 6 days after tumor challenge, animals were treated with vehicle (CTR) or with D16F7 mAb (20 mg/kg). Results are the mean ± S.D. (n = 8) of tumor volumes. Data are representative of two independent experiments with similar results. Student’s t test analysis, ***P < 0.001.
- Fig. 5.
D16F7 treatment prolongs the survival of animals orthotopically injected with C6-MF1 glioma cells. (A) C6-MF1 cells were injected intracranially into nude mice (n = 11). Survival curves were generated by a Kaplan-Meier product-limit estimate, and statistical differences between the various groups were evaluated by log-rank analysis: CTR vs. 10 or 20 mg/kg D16F7, P < 0.001. (B) H&E staining of brain histologic sections from CTR mice and 20 mg/kg D16F7 mAb–treated mice. Histograms represent the mean ± S.D. area of the tumor occupied by cancer cells (original magnification, 4×) or the number of mitotic and apoptotic (original magnification, 40×) tumor cells/high-power fields (n = 20). Red arrows in the CTR panel (original magnification, 60×) indicate mitoses; black arrows in the D16F7 panel (original magnification, 60×) point to apoptotic cells. (C) Immunostaining with anti-CD31 antibody of brain histologic sections. Arrows point to highly vascularized regions within CTR tumor mass, and the square indicates a faint vessel in the section from 20 mg/kg D16F7-treated mice. Original magnification, 10×. Histogram represents the mean ± S.D. number of vessels/high-power field (n = 20). Student’s t test analysis: **P < 0.01; ***P < 0.001.
- Fig. 6.
Mechanism of action of the anti–VEGFR-1 D16F7 mAb. The anti–VEGFR-1 D16F7 mAb interacts with a receptor site corresponding to amino acids 149–161 of human VEGFR-1 (Graziani et al., 2016), which is different from that involved in VEGF-A or PlGF binding (Davis-Smyth et al., 1998; Christinger et al., 2004). Thus, the D16F7 mAb does not prevent ligand interaction with the transmembrane or soluble receptor. (A) The mAb hampers receptor homodimerization and signal transduction with consequent inhibition of tumor and microenvironment cell functions. (B) D16F7 mAb preserves the soluble VEGFR-1 (sVEGFR-1) the ability to sequester receptor ligands (decoy function) and inhibits endothelial cell migration in response to sVEGFR-1. Akt, protein kinase B; Erk, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase.
Additional Files
Data Supplement
- Supplemental Figures -
Figure S1: D16F7 mAb inhibits VEGFR-1 phosphorylation in response to PlGF and VEGF-A in C6-MF3 cells.
Figure S2: D16F7 mAb specifically recognizes both human and murine VEGFR-1 protein in a concentration-dependent manner.
Figure S3: Histological analysis of brain sections from long-term surviving mice treated with D16F7.
- Supplemental Figures -
In this issue
In Vivo Antiglioma Activity of a Novel Anti–VEGFR-1 mAb
