Anti-VEGF antibody treatment of glioblastoma prolongs survival but results in increased vascular cooption

Abstract

Vascular endothelial growth factor (VEGF) is an important mediator of the intense angiogenesis which is characteristic of glioblastoma. While genetic manipulation of VEGF/VEGF receptor expression has previously been shown to inhibit glioblastoma growth, to date, no study has examined the efficacy of pharmacologic blockade of VEGF activity as a means to inhibit intracranial growth of human glioblastoma. Using intraperitoneal administration of a neutralizing anti-VEGF antibody, we demonstrate that inhibition of VEGF significantly prolongs survival in athymic rats inoculated in the basal ganglia with G55 human glioblastoma cells. Systemic anti-VEGF inhibition causes decreased tumor vascularity as well as a marked increase in tumor cell apoptosis in intracranial tumors. Although intracranial glioblastoma tumors grow more slowly as a consequence of anti-VEGF treatment, the histologic pattern of growth suggests that these tumors adapt to inhibition of angiogenesis by increased infiltration and cooption of the host vasculature.

Figure 1

(A) Individual glioma cells induce vascular process formation or sprouts 7 days post-tumor implantation. Vascular projections associated with a small capillary exhibit positive immunoreactivity for VEGF in close association with a group of tumor cells (counterstained with hematoxylin). In a parallel field (B), these projections also exhibit positive immunoreactivity for the angiogenic marker, integrin alpha V-beta 3, which was present on some individual tumor cells as well. Bar=10 µm. At 12 days post-implantation (C), the tumors measure approximately 1 mm in maximum diameter and are already densely vascularized as evidenced by CD31 immunoreactivity. Bar=60 µm. (D) A group of vessels in the tumor periphery appears to be growing in parallel, toward the tumor mass. These vessels also exhibited strong VEGF immunoreactivity. Bar=30 µm. (E) There were no VEGF-immunoreactive vessels or cells in normal basal ganglia. Also, VEGF staining was blocked when a control peptide was added (not shown). (F) Graphic representation of G55 tumor growth after injection into the basal ganglia of athymic rats. Measurements represent maximum tumor diameter. By day 21, greater than 90% of the animals was sacrificed or succumbed to increased intracranial pressure. Growth curve determinations were repeated in three independent experiments with similar results.

Figure 2

Kaplan-Meier survival analysis of the outcome of athymic rats with intracranial human glioblastoma treated with anti-VEGF antibody. In this experiment, two groups of six rats received intraperitoneal injections of anti-VEGF antibody (600 µg/injection) or PBS every other day starting 2 hours after tumor implantation. The median survival in the control group was 18.5 days. The anti-VEGF-treated animals survived nearly twice as long, median survival 34.5 days (P < .0001). In four of four experiments, systemic anti-VEGF treatment was associated with survival prolongation in animals with intracranial glioblastoma.

Figure 3

(A and B) Systemic anti-VEGF treatment causes a marked reduction in the vascularity of intracranial human glioblastoma. There was a greater than four-fold reduction in vascular density as evidenced by CD31 immunoreactivity in glioblastoma tumors grown in animals treated with anti-VEGF (mean±SEM, n = 6 tumors/group) (P < .0001). Bar=60 µm.

Figure 4

Systemic anti-VEGF treatment is associated with a marked increase in the incidence of TUNEL-positive cells in intracranial human glioblastoma. (A) There were no TUNEL-positive cells when the enzyme terminal deoxynucleotidyl transferase was omitted from the reaction mix. (B) The TUNEL assay demonstrated the presence of spontaneous apoptosis in control tumors. (C) There was a marked increase in apoptosis in tumors in animals treated with anti-VEGF antibody (mean±SEM, n = 9 tumors/group) (P < .011). Bar =60 µm.

Figure 5

Hematoxylin and eosin staining of control and anti-VEGF-treated tumors reveals that while control tumors (A) exhibit sharp tumor margins and grow as well-circumscribed non-invasive masses, the anti-VEGF antibody-treated tumors were markedly more heterogeneous with more invasive-appearing borders (B) and exhibited a striking increase in the formation of satellite tumors (C). Satellites, composed of groups of tumor cells, approximately 5 to 50 in number and between 50 and 500 µm in diameter, were detected on the periphery of the primary tumor. Bar = 80 µm (A and B); 120 µm (C).

Figure 6

(A) Normal vascularity of rat basal ganglia as detected by CD31 immunohistochemistry (B). Greater than 90% of the satellite tumors associated with blood vessels, as evidenced by CD31 immunohistochemistry. Bar=60 µm.