Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity

Abstract

Various conventional chemotherapeutic drugs can block angiogenesis or even kill activated, dividing endothelial cells. Such effects may contribute to the antitumor efficacy of chemotherapy in vivo and may delay or prevent the acquisition of drug-resistance by cancer cells. We have implemented a treatment regimen that augments the potential antivascular effects of chemotherapy, that is devoid of obvious toxic side effects, and that obstructs the development of drug resistance by tumor cells. Xenografts of 2 independent neuroblastoma cell lines were subjected to either continuous treatment with low doses of vinblastine, a monoclonal neutralizing antibody (DC101) targeting the flk-1/KDR (type 2) receptor for VEGF, or both agents together. The rationale for this combination was that any antivascular effects of the low-dose chemotherapy would be selectively enhanced in cells of newly formed vessels when survival signals mediated by VEGF are blocked. Both DC101 and low-dose vinblastine treatment individually resulted in significant but transient xenograft regression, diminished tumor vascularity, and direct inhibition of angiogenesis. Remarkably, the combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during the course of treatment, which lasted for >6 months. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Figure 1

Differential in vitro sensitivity to vinblastine of HUVECs and human neuroblastoma cells (SK-N-MC and SK-N-AS). Proliferation rates of the 3 cell lines were assessed by measuring [³H]thymidine incorporation across a wide range of vinblastine concentrations. The most significant differences in sensitivity to vinblastine are evident at lower concentrations, whereas both neuroblastoma cell lines continued to incorporate thymidine at 80–90% of control rates, whereas HUVECs rates fell to 6.2% of control. Averages of 8 wells per dose and the corresponding SDs are shown.

Figure 2

Induction of solid tumor regression by nontoxic, antiangiogenic combination therapy with low-dose vinblastine and anti–flk-1 antibody (DC101). Top panel: Established xenografts of human neuroblastoma (SK-N-MC) were treated by a putative antivascular regimen of low-dose vinblastine (induction: 0.75 mg/m² bolus intraperitoneally; 1 mg/m² per day continuous subcutaneous infusion for 3 weeks; maintenance: 1.5 mg/m² every 3 days) alone or in combination with an anti–VEGF-R2 antibody (DC101; 800 μg every 3 days). There is an appreciable tumor growth inhibition by each of the single agents, which is comparable, at least initially, with that of the combination treatment group. The benefit of the combination treatment is most evident after prolonged treatment, when lasting and complete tumor regression is observed. The data are a compilation of 2 independent experiments, with the initial experiment lasting 34 days and the second still ongoing (> 210 days). In both sets, 20 mice were randomized into 4 groups. Bottom panel: Lack of toxicity-dependent weight loss in mice bearing SK-N-MC tumor xenografts treated with “antivascular” vinblastine regimen alone or in combination with and anti–VEGF-R2 (DC101) antibody. There are no significant differences in weight between the groups, except for a transient (14–18 days long) episode of weight loss associated with diarrhea in the combination treatment group. The episode resolved without interruption of therapy. Average body weights (g) ± SD are plotted (n = 3–10 mice). ip, intraperitoneally.

Figure 3

Vinblastine, DC101, or combination therapy induces tumor cell apoptosis in perivascular cuffs of SK-N-MC tumor xenografts. H&E stain of formalin-fixed, paraffin-embedded sections. The typical tissue architecture (control, top two panels) shows perivascular cuffing by neoplastic cells and normal endothelial cell lining (arrows). Apoptotic cells (ap) are seen only at the periphery of the cuff, and their presence is confirmed by TUNEL staining (right-sided panels). In both single-treatment groups (vinblastine and DC101), widening of the apoptotic rims, and extension of the apoptotic figures into the cuff can be observed after 35 and 50 days of treatment, respectively. Viable malignant cells are still present within the tumor cuff in both single-agent groups. In contrast, histology of the combined therapy group reveals diffuse tumor cell death and total loss of preexisting architecture (bottom left-hand panel), a finding supported by the diffuse TUNEL stain in corresponding specimens (bottom right-hand panel).

Figure 4

Morphological features of vascular damage induced by antiangiogenic therapy. H&E stain of formalin-fixed, paraffin-embedded sections of the DC101 treatment group. These changes are common to all the treatment groups, but the prevalence and the severity of these changes were greatest in the combination treatment group. The typical slim single layer of endothelial cells, lining the vascular lumen in the untreated group (a, black arrows). Treatment with DC101 (35 days) and vinblastine (50 days) leads to various degrees of vascular wall disintegration. Edema and lymphocytic infiltration (arrows) are seen in both arterioles (b) and venules (c) of the tumor. Further injury resulted in detachment of endothelial cells from the underlying basement membrane (d) and coincided with tumor cell death in the perivascular cuff (e and f). A large majority of the perivascular cuffs in the combination treatment group correlated with changes seen in e and f. Horizontal bar = 100 μm.

Figure 5

Impact of antiangiogenic treatment regimen (Vbl+DC101) on integrity of tumor vasculature. (a) The decrease in intravascular FITC-dextran fluorescence reflects changes in tumor perfusion in established SK-N-AS neuroblastoma xenografts subjected to a 2-week course of treatment with anti–flk-1 (DC101) antibody, low-dose vinblastine, or combination of the 2. Both single-drug treatments caused a significant decrease in tumor perfusion, and this effect was enhanced by the combination therapy. Averages of 5 animals (bearing bilateral tumor xenografts) and their respective SDs are shown (*P = 0.05). (b) Tumor appearance in treated and untreated animals at the time of excision. Notable is the change in tumor vascularity in the single-treatment groups even before an appreciable change in tumor size. Groups were treated for 14 days before specimen collection.

Figure 6

Inhibition of angiogenesis in vivo by low-dose vinblastine in combination with anti–flk-1 antibody (DC101). Angiogenesis was induced in subcutaneously implanted Matrigel plugs (Mat) by admixing 500 ng/mL of bFGF (Mat+bFGF). The mice were treated with DC101 antibody (800 μg/mL) every 3 days, low-dose vinblastine (1 mg/m²) every 3 days, or combination therapy (Vbl/DC101). After 10 days of treatment, mice were injected intravenously with FITC-dextran; Matrigel plugs were removed; and the volume of new blood vessels was assessed by measurement of intravascular FITC content (normalized to FITC in the circulating plasma). Averages of 5 animals and their respective SDs are shown (^AP = 0.05).