Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma

Abstract

Bevacizumab, an antibody against vascular endothelial growth factor (VEGF), is a promising, yet controversial, drug in human glioblastoma treatment (GBM). Its effects on tumor burden, recurrence, and vascular physiology are unclear. We therefore determined the tumor response to bevacizumab at the phenotypic, physiological, and molecular level in a clinically relevant intracranial GBM xenograft model derived from patient tumor spheroids. Using anatomical and physiological magnetic resonance imaging (MRI), we show that bevacizumab causes a strong decrease in contrast enhancement while having only a marginal effect on tumor growth. Interestingly, dynamic contrast-enhanced MRI revealed a significant reduction of the vascular supply, as evidenced by a decrease in intratumoral blood flow and volume and, at the morphological level, by a strong reduction of large- and medium-sized blood vessels. Electron microscopy revealed fewer mitochondria in the treated tumor cells. Importantly, this was accompanied by a 68% increase in infiltrating tumor cells in the brain parenchyma. At the molecular level we observed an increase in lactate and alanine metabolites, together with an induction of hypoxia-inducible factor 1α and an activation of the phosphatidyl-inositol-3-kinase pathway. These data strongly suggest that vascular remodeling induced by anti-VEGF treatment leads to a more hypoxic tumor microenvironment. This favors a metabolic change in the tumor cells toward glycolysis, which leads to enhanced tumor cell invasion into the normal brain. The present work underlines the need to combine anti-angiogenic treatment in GBMs with drugs targeting specific signaling or metabolic pathways linked to the glycolytic phenotype.

Fig. 1.

Quantification of tumor progression and contrast enhancement. Representative images of control (A, D, G, and J) and bev-treated animals (B, E, H, and K). Tumor volume was assessed from T2-weighted images (A and B) to determine tumor doubling time (TDT) (C), Ki67 immunostaining (D and E), and quantification thereof (F). Control values were set at 100%. (G and H) Postcontrast T1-weighted images. (I) Tumor volumes assessed from postcontrast T1-weighted images were similar to T2-weighted images. (J–L) Reduction of contrast agent uptake (CE) in the treated group as evidenced by the mean tumor area under the curve (AUC). Ctrl: controls; Tr: treated. (Scale bars: ±SE.) *P < 0.05.

Fig. 2.

DCE-MRI analysis of bev-treated glioblastomas. Tumor perfusion maps of representative control (A, D, G, and J) and bev-treated animals (B, E, H, K). Bevacizumab led to a significant reduction of blood flow, F_b (A–C); of blood volume per unit of tissue, v_b (D–F); and of the blood-to-tissue extraction constant, K_trans (G–I). (J–L) The extravascular extracellular space (interstitial space volume) per unit of tissue, v_e, was not significantly modified. Ctrl: controls; Tr: treated. (Scale bars: ± SE.) *P < 0.05, **P < 0.01, ***P < 0.001. Colors range from blue (low values) to red (high values).

Fig. 3.

Changes in blood vessel morphology and tumor cell invasion after bev treatment. Immunostaining for von Willebrand factor (vWF) (A and B) and quantification thereof (C), indicating a significant reduction in the density of medium and large blood vessels and in total vessel number after bev treatment. (Scale bar: 200 μm.) Nestin-stained composite images (D and E) reveal a more homogeneous appearance of the treated compared with untreated tumors, also reflected in corresponding T2-weighted MRI images (F). Large vessels (“V”) appear as dark tortuous lines in nestin and T2-weighted images and necrotic areas (“N”) as brighter spots. Quantification of the nestin-positive cells outside the tumor core (G and H) shows a 68% increase in cell invasion after treatment (I). mi.v: microvessels; in.v: intermediate-sized vessels; ma.v: macrovessels; Ctrl: controls; Tr: treated. (Scale bars: ± SE.) ***P < 0.001.

Fig. 4.

Histological and ultrastructural changes after bev treatment. Hematoxylin- and eosin-stained sections of GBM xenografts (A–D). In control tumors (A and C), typical hallmarks of GBM growth are visible: necrosis (“N”) and microvascular proliferations (arrowheads) in the tumor center (A) and periphery (C), but not in treated tumors (B and D). The endothelium appears more normal in treated tumors (arrows). [Scale bar (A–D): 50 μm.] (E–H and J) Transmission electron microscopy (TEM) images of GBM xenografts. Microvascular proliferation and endothelial cell sprouting (red arrows) in control tumors (E). Treated tumors show more normalized blood vessels, yet no mature blood–brain barrier (F). A denser cellular composition in the tumor core in control tumors (G) compared with the treatment group (H) where several lytic areas were observed (red arrow). Cells from the invasive front (J) had a more elongated morphology, suggesting a subpopulation within the tumor. Dividing cells in the invasive front (white arrow in J). [Scale bar (E–H and J): 5 μm.] Quantification of mitochondria per cell from TEM micrographs (I). Ctrl: controls; Tr: treated. (Scale bars: ±SE.) **P < 0.01.

Fig. 5.

Molecular changes induced in GBM xenografts after bev treatment. Western blot for HIF1α in control and bev-treated glioblastoma xenografts (A) and quantification thereof (B). Signal normalization with a human-specific nestin antibody (n = 7). (C) Schematic of key regulatory molecules associated with receptor tyrosine kinase activation induced after bev treatment (genes marked in dark pink were up-regulated >1.5-fold; genes in light pink were up-regulated >1-fold ; genes in gray were unchanged; and genes in white were not on the array). (Scale bars: ±SE.) *P < 0.05. (See also Tables S2 and S3.) Ctrl: controls; Tr: treated. AKT1/2: protein kinase B/Bβ; APC: adenomatous polyposis coli; AXIN: axis inhibition protein; βcat: β1 Catenin (CTNNB1); EGFR: epidermal growth factor receptor; Erk: mitogen-activated protein kinase 1 (MAPK1); FOX01: forkhead box O1; FZD: Frizzled homolog; GLUT: glucose transporter; Grb2: growth factor receptor-bound protein 2; GSK3B: glycogen synthase kinase 3 β; IGFR: insuline-like growth factor receptor; LEF1: lymphoid enhancer-binding factor 1; mTOR: FK506 binding protein 12-rapamycin associated protein 1; PDGFR: platelet-derived growth factor receptor; PI3K: phosphoinositide-3-kinase; PTEN: phosphatase and tensin homolog; RAS: RAS protein superfamily; p70s6k: ribosomal protein S6 kinase; SHC: (Src homology 2 domain containing) transforming protein 1; Sos: Son of sevenless; TCF3: transcription factor 3; TSC1/2: tuberous sclerosis 1/2.