Journal: Neuro-Oncology

Article Title: VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance

doi: 10.1093/neuonc/noy103

Figure Lengend Snippet: VEGFR2 tyrosine kinase expressed by glioblastoma cells remains active under bevacizumab therapy. (A) Western blot (WB) for VEGFR2 and tubulin in a panel of patient-derived glioblastoma cultures. (B) Quantitative RT-PCR analysis of VEGF-A and VEGFR2 expression in glioblastoma cells standardized to expression in human microvascular endothelial cells (HMVECs). Data are presented as mean ± SD, n = 3. The y -axis is log 10 transformed. (C) Viability of CPH017, IN1123, and 1966 cells treated with SU1498 (10 µM) or left unstimulated. Data are presented as mean ± SD, n = 3. Significance determined by 2-way ANOVA with Bonferroni post-test. (D) Representative WB of total and phosphorylated VEGFR2 and tubulin in CPH017 cells plated overnight in Neurobasal media without epidermal growth factor and basic fibroblast growth factor, then treated with either SU1498 (30 µM for 3 h), VEGF-A (40 ng/mL, 15 min), or the combination (30 µM SU1498 for 3 h followed by 40 ng/mL VEGF-A for 15 min). (E) Quantification of relative phosphorylated-VEGFR2/tubulin levels from WB analysis. Data are presented as mean ± SD, n = 3. Significance determined by one-way ANOVA with Dunnett’s post-test correction and ratio paired 2-sample t -test. (F) Viability of glioblastoma cells treated 7 days with bevacizumab (Bev) (0–2 mg/mL). Data are presented as mean ± SD, n = 3. Significance determined by 2-way ANOVA with Bonferroni post-test correction. (G) ELISA quantification of VEGF-A secretion by glioblastoma cells treated with Bev (0.5 mg/mL, 48 h) or IgG control. Data are presented as mean ± SD, n = 3. N.D. denotes that signal could not be detected. (H) Representative WB of total and phosphorylated VEGFR2 or tubulin in CPH017 and 1966 cells treated for 72 h with Bev (0.5 mg/mL) or IgG. * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001; NS: nonsignificant.

Article Snippet: Conditioned media were collected 48–72 hours later and subjected to Human VEGF-Quantikine enzyme-linked immunosorbent assay (ELISA) or Human VEGF-C-Quantikine ELISA (R&D Systems), following manufacturer’s instructions using a Synergy2 microplate reader (BioTek).

Techniques: Western Blot, Derivative Assay, Quantitative RT-PCR, Expressing, Transformation Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Neuro-Oncology

Article Title: VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance

doi: 10.1093/neuonc/noy103

Figure Lengend Snippet: VEGF-C is expressed under bevacizumab therapy and interacts with VEGFR2 in glioblastoma cells. (A) Quantitative RT-PCR analysis of VEGF-C and VEGFR3 expression in glioblastoma cells standardized to HDLECs. Data represented as mean ± SD; n = 2. The y -axis is log 10 transformed. N.D. = not detected. (B) ELISA quantification of VEGF-C secretion by glioblastoma cells treated 72 h with bevacizumab (Bev; 0.5 mg/mL) or IgG. Data represented as mean ± SEM; n = 3. Significance determined by paired 2-sample t -test. * P ≤ 0.05. (C) Correlation of VEGF-C ELISA quantification in matched samples of blood-plasma and conditioned media (72 h) from tumor-derived cell cultures of 7 glioblastoma patients. Significance was determined by Spearman’s correlation analysis. (D) Representative confocal images showing PLA signal (red), F-actin (gray), and 4′,6′-diamidino-2-phenylindole (blue) of glioblastoma cells. Staining with only VEGF-C antibody (AB) was used as a negative control. (E) Quantification of PLA dots in glioblastoma cells. Data are represented as mean ± SEM; n > 40 cells.

Article Snippet: Conditioned media were collected 48–72 hours later and subjected to Human VEGF-Quantikine enzyme-linked immunosorbent assay (ELISA) or Human VEGF-C-Quantikine ELISA (R&D Systems), following manufacturer’s instructions using a Synergy2 microplate reader (BioTek).

Techniques: Quantitative RT-PCR, Expressing, Transformation Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Derivative Assay, Staining, Negative Control

Journal: Neuro-Oncology

Article Title: VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance

doi: 10.1093/neuonc/noy103

Figure Lengend Snippet: VEGF-C regulates canonical VEGFR2 survival signaling. (A) WB analysis of downstream VEGFR2 signaling in CPH017 glioblastoma cells starved for 24 h followed by stimulation with VEGF-A (40 ng/mL) or VEGF-C (0.2 µg/mL). (B) Quantitative RT-PCR analysis of VEGF-C expression in glioblastoma cells 48 h following transfection with either siCtrl (nontargeting control) or siVEGF-C (VEGF-C targeting siRNA). Data are represented as mean ± SEM; n = 3 or 4. Significance determined with 1-sample t -tests setting the hypothetical value to 1. (C) WB for VEGF-C and tubulin in CPH017 cells 72 h following siCtrl or siVEGF-C transfection. (D) Quantitative RT-PCR analysis of VEGF-A expression in CPH017 and IN1123 cells 48 h following siCtrl or siVEGF-C transfection. Data are represented as mean ± SEM; n = 3 or 4. Significance determined with 1-sample t -tests setting the hypothetical value to 1. (E) ELISA quantification of VEGF-A in conditioned media from CPH017 glioblastoma cells transfected with siCtrl or siVEGF-C. Data are represented as mean ± SEM; n = 2. Significance determined by 1-way ANOVA with Dunnett’s post-test correction. (F) Viability of CPH017 and IN1123 glioblastoma cells transfected with siCtrl or siVEGF-C. Data are represented as mean ± SEM; n = 3–5. Significance determined by 2-way ANOVA with Bonferroni post-test correction. (G) WB of pro- and cleaved (Cl.) caspase-3 and tubulin in CPH017 and IN1123 glioblastoma cells 72 h following siCtrl or siVEGF-C transfection. (H) Viability of CPH017 and IN1123 glioblastoma cells 5 days after transfection with siCtrl or siVEGF-C alone and combined treatment with 0.5 mg/mL bevacizumab (Bev). Data are represented as mean ± SEM; n = 3. Significance determined by 1-way ANOVA with Dunnett’s post-test correction. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Conditioned media were collected 48–72 hours later and subjected to Human VEGF-Quantikine enzyme-linked immunosorbent assay (ELISA) or Human VEGF-C-Quantikine ELISA (R&D Systems), following manufacturer’s instructions using a Synergy2 microplate reader (BioTek).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay