Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity (original) (raw)
In vitro determination of differential drug sensitivity. Before undertaking our in vivo experiments, we established a dose of vinblastine at which significant antiproliferative effects on endothelial cells, but not tumor cells, were observed. To do so, we optimized growth conditions to achieve comparable levels of proliferative activity in 2 human neuroblastoma cell lines (SK-NM-C and SK-N-AS) and HUVECs. All 3 cell lines were grown in DMEM with 10% FBS, but the HUVECs were grown on gelatinized plates and in the presence of additional growth factors (bFGF and EGF). The untreated controls show similar levels of [3H]thymidine incorporation for all 3 cell lines, thus eliminating the concern that the differences in proliferation may be inherent (Figure 1). At the higher concentrations of vinblastine used (e.g., 100–400 ng/mL), all 3 cell populations were strongly inhibited, especially HUVECs. In striking contrast, at the lowest concentrations (e.g., 0.78 ng/mL) vinblastine retained almost the same degree of inhibitory activity against HUVECs, whereas antiproliferative activity against 2 tumor cell lines was lost (Figure 1). The source of this striking differential sensitivity is not clear, but it should be noted that at least 1 of the tumor cell lines, SK-N-MC, is positive for multidrug resistance–associated protein (MRP), which can result in vinblastine resistance (33). These in vitro findings suggest that the lowering of the usual MTD used in the clinic may not compromise vinblastine activity against (dividing) endothelial cells present in tumor blood vessels.
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.
In vivo tumor growth assessment. Building on this in vitro difference in sensitivity to vinblastine, we went on to evaluate the effects of lower doses of vinblastine in an in vivo model, using an increased dose frequency to maximize the potential for endothelial injury. Xenografts of either SK-N-MC neuroepithelioma or SK-N-AS neuroblastoma cell lines were implanted subcutaneously in the flanks of 4- to 6-week-old CB-17 SCID mice and grown to approximately 0.75 cm3 before initiation of treatment. The SK-N-MC cell line, which expresses MRP (33), was chosen for long-term experiment to assess for the development of drug resistance. In contrast, the SK-N-AS, a highly tumorigenic and vascular neuroblastoma cell line, which has not been studied, to our knowledge, for its chemoresistance, was chosen for the short-term experiment and tumor perfusion studies.
The major findings in the long-term experiment, using SK-N-MC human neuroblastoma xenografts were as follows: (a) The first treatment group, treated with DC101, an anti–flk-1 receptor antibody shown previously to inhibit growth of different kinds of human xenografts in mice and in murine tumor models (5), showed the anticipated effectiveness in inhibiting tumor growth (Figure 2, top panel, full circles; Table 1, day 15), but this effect was not sustained. (b) The findings in the second treatment group (vinblastine alone) were even more surprising. This agent, traditionally thought to act by inhibiting tumor cell proliferation through inhibition of tubulin assembly, even though used at “subclinical” low dose, produced significant, albeit not sustained, regression of tumor growth (Figure 2, top panel, filled squares; Table 1, day 15). (c) This growth delay in the vinblastine group was further potentiated with the simultaneous treatment of the anti–flk-1 antibody, DC101. The combination treatment induced an initial response comparable to the other treatment groups but then caused further, long-term, tumor regressions (Figure 2, top panel, full upward triangles; Table 1, experiments 2 and 3). (d) To date, the mice in combination therapy group have not manifested any resistance to the treatment or recurrence of disease, despite almost 7 months of continuous treatment. The mice remain healthy, with almost no evidence of tumor, except for a small, barely palpable remnant in 1 of the mice. The tumor sizes between the control and treatment group were statistically significant (P = 0.05) at days 30–45 and between the treatment groups at days 30–80.
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.
There is a marked initial response to both single agents such that in SK-N-MC xenografts, the response to DC101 at 15 days is almost equal to that of the combination-treatment group. The case of SK-N-AS shows the limitations of measuring tumor volume by external dimensions (Day 15, Vbl/DC101-shaded box). The ulcerated, scabbed tumor retains its external dimensions despoite evidence of vascular compromise (see Fig. 5), ulceration and necrosis. Over time there is a delayed divergence effect such that neither of the single agents alone is able to result in sustained tumor regressions whereas combination treatment induces a lasting tumor regression. Sacrificed refers to animals which were euthanized required by our institutional guidelines. ND, time points not done
The response in the SK-N-AS was even more rapid producing significant differences in tumor growth within 2 weeks of treatment (Table 1) without an induction regimen. The macroscopic changes in tumor vascularity (see Figure 5b) are supported by the findings in 1 tumor perfusion study (see later here).
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.
Also of interest, the 3 remaining mice in the long-term experiment were taken off the long-term combined therapy at 210 days of treatment and did not show any evidence of tumor relapse/regrowth 3 months later (G. Klement et al., unpublished observations).
Toxicity evaluation. Antivascular therapy would be expected to show minimal toxicity in the postnatal stage of development. To evaluate this aspect of DC101/vinblastine combination therapy, the health status of the mice was monitored. Weight was plotted at regular intervals and considered a surrogate for evaluation of systemic well-being, anorexia, or failure to thrive. As shown in Figure 2 (bottom panel), no significant differences in weights were seen between the 4 groups. The weight curve of the DC101 group parallels very closely that of the control group. The vinblastine group showed some weight gain retardation, but the differences never became significantly different from controls, and the mice continued to grow. Similarly, the toxicity profile in the combination treatment group was very similar to those in the single-agent groups, with the exception of a transient episode of weight loss associated with diarrhea. The episode lasted approximately 2–3 weeks and was unlikely to be due to the therapy as the mice recovered without interruption of treatment.
Other usual signs of drug toxicity in mice, such as ruffled fur, anorexia, cachexia, skin tenting (due to dehydration), skin ulcerations, or toxic deaths (21), were not seen at the doses used in our experiments. Diarrhea, a common sign of vinblastine toxicity when doses of 10 mg/kg (21) are used, was generally not observed, except for the previously mentioned transient episode.
Histopathological analysis. To elucidate further the mechanisms involved in the tumor regression after treatment with vinblastine, DC101, or the combined therapy, tissue histopathology assessment was undertaken. The typical tissue architecture of untreated SK-N-MC tumors is shown in Figure 3 (control panels). Cancer cells with high nuclear to cytoplasmic ratio form cuffs around central vessels, and apoptotic cells characterized by pyknotic nuclei and cytoplasmic blebbing, are only evident as a thin rim at the periphery of the cuffs. The nuclei of these cells stain strongly for terminal deoxynucleotidyl transferase (TUNEL) reactivity, as expected for cells undergoing apoptosis. Vinblastine alone or DC101 treatment alone both show an increase in the width of the apoptotic rims (Figure 3), suggesting the cells most distal to the tumor vasculature are primarily affected, but a large percentage of viable tumor cells still survive in the center of the cuff. In both single-agent groups, specimens were collected at the time mice had to be sacrificed for ethical concerns regarding tumor burden. Despite the evident increase in apoptosis, there was a net tumor growth. In contrast, histology of the combined therapy group, as would be predicted by the regression in tumor size in this treatment group at the time of analysis (7.5 weeks of treatment), shows overwhelming loss of both cell viability and preexisting tumor architecture (Figure 3, bottom panels). There is a close similarity of the appearance of H&E and TUNEL stain. Interestingly, we observed signs of endothelial cell toxicity in all of the treatment groups (Figure 4). Rather than a typical single layer of flattened endothelial cells surrounding the vascular lumen in untreated group (Figure 4a), we observed edema (Figure 4, b and c) and detachment from surrounding basement membrane (Figure 4, c–e) leading to complete vascular wall disintegration and tumor cell death (Figure 4f). Even though the actual examples depicted in Figure 4 are those in the DC101 group, their appearance is shared between the groups, and it is only the degree of tumor vessel regression that results in the seen differences in tumor size.
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).
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.
Tumor perfusion by assessment of intravascular fluorescence. To explore further the possibility that tumor regression induced with treatment using DC101 and/or vinblastine was indeed due to the vascular injury, rather than a direct antitumor cell effect, we assessed tumor perfusion directly by using a FITC-dextran fluorescence method. Mice carrying established subcutaneous SK-N-AS human neuroblastoma xenografts (∼0.25 cm3) were randomized into 4 groups and treated systemically with either saline control, DC101, vinblastine, or combination therapy for 10 days. FITC-dextran was injected into the lateral tail vein and equilibrated throughout the vascular compartment. The majority of the blood-borne dextran, because of its 148-kDa size, remains intravascular, and despite some perivascular losses due to changes in vascular permeability and the possibility of interstitial hemorrhages, the fluorescence is reflective of the overall volume of blood passing through the tumor vasculature. Because our therapy is chronic in its nature, changes in intratumoral vascular/blood volume are likely to represent structural changes rather than transient fluxes in vascular permeability. By these criteria, DC101 alone caused a 47% decrease in tumor perfusion, vinblastine alone resulted in a 41% decrease, and the combination of the 2 drugs resulted in 65% perfusion inhibition (Figure 5a). Of interest is the appreciable difference in gross vascularity in the corresponding tumor specimens (Figure 5b).
Effects of chemotherapy treatments on in vivo angiogenesis. The direct assessment of tumor vasculature does not provide any clues as to whether the apparent vascular inhibition within the tumor is primary (cause) or secondary (consequence) to the tumor regression. Evidence for the former would provide support for the hypothesis that low-dose vinblastine treatment alone is potentially antiangiogenic and that the extent of this antiangiogenic effect may be further enhanced by concurrent treatment with DC101. Again, the ratio between intra- and extravascular volume within the tumor could be also somewhat affected by transient changes in vascular permeability (34). To address these questions, we repeated the same fluorescence measurement using an in vivo Matrigel plug angiogenesis assay (ref. 32; personal communication, J. Ahern, Merck & Co., West Point, Pennsylvania, USA). Four treatment groups were treated with an identical therapeutic regimen as in the tumor perfusion experiment, and the regression of vascularity in subcutaneously implanted Matrigel pellets was quantitatively assessed by measuring the fluorescence of circulating FITC-labeled dextran (Figure 6). DC101 inhibited the bFGF-induced vascularization to 50% and vinblastine alone to 62.5% of the positive control. There was again an enhanced effect of the combination therapy, which reduced the Matrigel pellet fluorescence down to 29.2% of control, a level only marginally different from the negative control (Matrigel not supplemented with growth factors).
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 (A_P_ = 0.05).