Mol Cancer Ther. 2004;3:335-343
© 2004 American Association for Cancer Research
TNP-470 promotes initial vascular sprouting in xenograft tumors
Jianzhong Huang1,
Jason S. Frischer1,
Tamara New2,
Eugene S. Kim1,
Anna Serur1,
Alice Lee2,
Angela Kadenhe-Chiwishe1,
Daniel A. Pollyea2,
Akiko Yokoi1,
Jocelyn Holash3,
George D. Yancopoulos3,
Jessica J. Kandel1 and
Darrell J. Yamashiro1,2
Divisions of 1 Pediatric Surgery and 2 Pediatric Oncology, College of Physicians and Surgeons of Columbia University, New York, NY; and
3 Regeneron Pharmaceuticals, Inc., Tarrytown, NY
Requests for Reprints: Darrell Yamashiro, Pediatric Oncology, Irving Pavilion 7, 161 Fort Washington Avenue, New York, NY 10032. Phone: (212) 305-2176; Fax: (212) 305-5848. E-mail: dy39{at}columbia.edu
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Abstract
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TNP-470 (AGM-1470), an analogue of fumagillin, was one of the first molecules proposed to have antiangiogenic properties. This concept was based on its ability to inhibit both endothelial proliferation in vitro and tumor growth in vivo in a number of xenograft models. Yet, subsequent investigations indicated that the biochemical activities associated with TNP-470 are not selective for endothelial cells. Moreover, recent evidence suggests that this agent inhibits tumor growth in vivo, but without a corresponding decrease in angiogenesis. Therefore, we performed a detailed comparison of TNP-470 to a validated antiangiogenic agent, a VEGF inhibitor termed VEGF-Trap, using a xenograft model of Wilms tumor. Treatment with TNP-470 for 5 weeks significantly suppressed xenograft growth (83%). Surprisingly, this inhibition was not associated with a decrease in angiogenesis, but instead with an increase in tiny neovessels. To determine whether this was a direct effect of TNP-470 on tumor vessels, we examined its effect in a short-term assay using large tumors with established vasculature. In contrast to treatment with VEGF-Trap, which led to rapid vessel regression and tumor hypoxia, tumors exposed to TNP-470 for 1 day displayed increased capillary sprouting, with significantly increased microvessel density, vessel length, and branch points. TNP-470 did not induce tumor hypoxia as demonstrated by minimal pimonidazole staining and VEGF expression. TNP-470 did, however, cause a marked increase in apoptosis of tumor cells. Our results indicate that the antitumor effects of TNP-470 cannot be attributed to prevention of neoangiogenesis, but instead to its direct action on tumor cells.
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Introduction
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Targeting tumor vasculature has recently been validated as a strategy for the treatment of some human cancers (1). One of the first putative antiangiogenic molecules described was TNP-470 (AGM-1470), an analogue of fumagillin, a naturally secreted antibiotic of the fungus Aspergillus fumigatus fresenius (2). Fumagillin was initially identified by its ability to inhibit endothelial proliferation in vitro. Subsequently, its TNP-470 analogue was shown to inhibit tumor growth in a number of xenograft models (2–6). Taken together, these studies suggested that TNP-470 might prevent tumor growth by impeding angiogenesis.
On the basis of these experiments, TNP-470 aroused much interest as a potential therapeutic agent in human cancer. Subsequent investigation of the biochemical targets of TNP-470 demonstrated that these are not inherently selective for endothelial cells. TNP-470 blocks methyionyl aminopeptidase-2 (MetAP2), an intracellular enzyme necessary for the process of protein myristolation, thus preventing membrane proteins from being translocated to the cell surface (7, 8). Inhibition of MetAP2 blocks cell-cycle progression in both endothelial and cancer cells (9). Consistent with these findings, some recent reports indicate that this agent inhibits tumor growth in vivo, but without an unambiguous corresponding decrease in tumor vasculature (10–14). In addition, initial clinical trials TNP-470 produced inconclusive results, without clear evidence of either reduced tumor growth or vascularity (15, 16). In particular, alterations in serum levels of surrogate markers for antiendothelial activity were not detected (16).
To resolve these ambiguities, we investigated the effect of TNP-470 on vasculature in both microscopic and large tumors, comparing this to the validated antiangiogenic agent VEGF-Trap. In these studies, both TNP-470 and VEGF-Trap significantly inhibited growth of microscopic tumor implants in a xenograft model of Wilms tumor. However, blood vessel growth was not suppressed by TNP-470, whereas it was almost completely prevented by VEGF-Trap. Instead, we detected a unique pattern of remodeling of tumor vasculature after TNP-470 administration, characterized by rapid and striking proliferation of microvessels. In addition, TNP-470 rapidly induced tumor cell apoptosis, whereas VEGF blockade selectively induced apoptosis of vascular cells.
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Materials and Methods
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Cell Line
Cultured human Wilms tumor cells (SK-NEP-1, American Type Culture Collection, Manassas, VA) were maintained in 75-cm2 flasks with McCoy's 5A medium (Mediatech, Fisher Scientific, Springfield, NJ), supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin (Life Technologies, Inc., Grand Island, NY). Cells were grown at 37°C in 5% CO2 until confluent and harvested by trypsinization, counted with trypan blue staining, and washed and resuspended in sterile saline solution (PBS, Life Technologies) at a concentration of 107 cells/ml.
Tumor Implantation
All experiments were approved by the Institutional Animal Care and Use Committee of Columbia University. Four- to 6-week-old female NCR nude mice (Taconic Farms, Germantown, NY) were housed in a barrier facility and acclimated to 12-h light-dark cycles for at least a day before use. The left flank was prepared sterilely after anesthetizing the mice with i.p. ketamine (50 mg/kg) and xylazine (5 mg/kg). An incision was made exposing the left kidney, and an inoculum of 106 tumor cells (0.1 ml) was injected with a 25-gauge needle. The flank muscles were closed with a single 4-0 Polysorb suture (US Surgical, Norwalk, CT) and the skin closed with staples.
Administration of TNP-470 in Nonestablished Tumors
TNP-470 for the experiments with the Wilms tumor xenografts was provided by the Developmental Therapeutics Program of the National Cancer Institute. Mice with implanted tumor cells were maintained for 1 week, and then randomly divided into two groups. TNP-470 (30 mg/kg) or vehicle was injected i.p. three times a week. Animals were observed for adverse effects, and euthanized at 6 weeks. At the termination of the experiment, mice treated with TNP-470 had a mean body weight of 17.8 g versus 19.8 g in the control mice (P = 0.01).
Rapid Vasculature Regression Assay
SK-NEP-1 tumor cells were implanted intrarenally in nude mice, and tumors allowed to grow for 6 weeks. Mice were then randomly divided into four groups, and then treated on Day 0 with vehicle, TNP-470 at 30 mg/kg or 100 mg/kg every other day (Day 0, 2, 4), or VEGF-Trap 500 µg (Days 0, 4), a soluble decoy receptor construct that incorporates domains of both VEGFR-1 and VEGFR-2 and binds VEGF with high affinity (17). Mice were sacrificed at Days 1 and 5, and tumor vasculature evaluated by lectin perfusion as described below. All mice had large tumors, with the average tumor weight ± SD for Day 1 of 8.4 ± 3.1 g, and for Day 5 of 7.7 ± 2.1 g.
Fluorescein Angiography
Selected anesthetized mice underwent fluorescein angiography via left ventricular puncture using 5% FITC-dextran (Sigma Chemical Co., St. Louis, MO) before sacrifice. Fluorescein-perfused tumors were cut into 100-µm sections using a vibratome device, cooled, and evaluated by fluorescent microscopy.
Lectin Perfusion
Before euthanasia, selected mice underwent left ventricular injection of fluorescein-labeled Lycopersicon esculentum lectin (100 µg in 100 µl PBS, Vector Laboratories, Burlingame, CA). The vasculature was fixed by infusion of 1% paraformaldehyde and then washed by perfusion of PBS. Lectin-perfused tumors were cut into 40-µm sections using a vibratome device, and evaluated by fluorescent microscopy as described below.
Digital Image Analysis
Digital images from the fluorescein-labeled lectin studies were acquired from a Nikon E600 fluorescence microscope (10x objective) with a Spot RT Slider digital camera (Diagnostic Instruments, Sterling Heights, Michigan) and stored as TIFF files. Quantitative assessment of angiogenesis and tumor vessel architecture was performed by computer-assisted digital image analysis as described by Wild et al. (18). Changes in vessel architecture were evaluated by quantifying branch points/nodes, end points, and total vessel length, after application of a common threshold value, inverting the image, morphological erode, and skeletonization using a combination of Adobe Photoshop (Adobe Inc., Mountain View, CA) and Image Processing Tool Kit (Reindeer Graphics, Inc., Raleigh, NC) as described (18). Statistical comparison was performed using Kruskal-Wallis analysis.
Immunohistochemistry
Five-micrometer-thick sections cut from selected specimens of tumor-bearing kidney were baked, deparaffinized in xylene, and rehydrated. Endogenous peroxidases were quenched in 3% hydrogen peroxide (Sigma) for 20 min. A rat anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM-1) monoclonal antibody was used (Research Diagnostics, Inc., Flanders, NJ) at a 1:50 dilution. Monoclonal anti-
smooth muscle actin (SMA) antibody (Sigma) was used at a 1:200 dilution.
TUNEL Assay
Mounted paraformaldehyde-fixed specimens underwent TUNEL (terminal deoxyribonucleic d-UTP nick end labeling) assay for apoptosis using the ApopTag Red In Situ Apoptosis Detection kit (Intergen Company, Purchase, NY). Five-micrometer-thick sections were baked, deparaffinized in xylene, and rehydrated. Antigen retrieval was performed with trypsin for 40 min. Samples were incubated with equilibration buffer, and then with reaction buffer containing TdT enzyme. Signals were visualized using anti-digoxigenin conjugated to rhodamine, and slides mounted in medium containing DAPI (Vector). Digital images were obtained with the Nikon E600 fluorescence microscope (20x objective) with a Spot RT Slider digital camera. All processing was done on the red channel, with a common threshold value applied, and the image inverted using Adobe Photoshop. To eliminate background and allow for computer quantification, spots less then 20 pixels in size were removed, and the remaining marks counted using the Image Processing Tool Kit.
In Situ Hybridization
Selected tumor tissue was initially preserved in 4% paraformaldehyde overnight, transferred to 17% sucrose, and embedded in OCT compound and frozen. Sections were probed with 35S-labeled cRNA, using a probe spanning codons 57–192 of human VEGF as described (19).
Hypoxia Studies
One hour before sacrifice, mice were injected i.p. with pimonidazole (Hypoxyprobe-1, 60 mg/kg/dose; Chemicon International, Inc. Temecula, CA). Tumor tissue was excised and fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 µm. The sections were deparaffinized, rehydrated, and washed with PBS containing 0.1% BRIJ-35 (Fisher Scientific) then exposed for 10 min to 3% hydrogen peroxide to quench endogenous peroxidases. The slides were incubated with Pronase (Biomeda Corp., Foster City, CA) for 40 min at 40°C; with CAS blocking serum for 30 min; and then for 1 h at room temperature, with 1:50 dilution of antibody, Hypoxyprobe-1-MAb1 (Chemicon), a mouse monoclonal IgG1 (MAb) that recognizes pimonidazole adducts in hypoxic tissues. The sections were incubated with a biotinylated rabbit anti-mouse secondary antibody (Zymed), and then with a streptavidin peroxidase. A substrate chromagen, AEC (aminoethyl carbazole), was used to visualize the signal.
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Results
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Intrarenal implantation of the Wilms tumor cell line SK-NEP-1 results in large, richly vascularized tumors (20–23). We tested the effect of TNP-470 on the development of microscopic tumor implants by starting treatment 1 week after implantation, using TNP-470 (30 mg/kg) three times per week for 5 weeks. TNP-470 significantly suppressed Wilms tumor growth, causing an 83% reduction in xenograft weights (0.59 versus 3.43 g, P = 0.004; Fig. 1A
). For comparison, we evaluated the effects of VEGF-Trap, a composite decoy receptor based on VEGFR-1 and VEGFR-2 regions fused to an Fc segment of IgG1, which is a potent inhibitor of VEGF (17). This validated antiangiogenic agent caused 97% inhibition of Wilms tumor growth as compared to controls in a parallel experiment (P < 0.0009; Fig. 1B).
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Figure 1. TNP-470 suppresses tumor growth. Wilms tumor xenografts were implanted intrarenally in nude mice, and treatment started 1 week later with TNP-470, 30 mg/kg, 3 times/week (n = 10) or PBS (n = 10) (A); or VEGF-Trap, 500 µg/dose, 2 times/week (n = 10) (B); or vehicle (n = 10). There was significant suppression of tumor growth with both TNP-470 (*,P = 0.004) and VEGF-Trap (*,P < 0.0009).
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We compared vasculature in these tumors by fluorescein-dextran angiography and by immunostaining for endothelial and vascular mural cells, using anti-PECAM-1 and anti-SMA antibodies, respectively. Despite significantly inhibiting tumor growth, TNP-470 administration did not decrease tumor vasculature, as assessed by fluorescein-dextran angiography (Fig. 2, Fl-D)
. Conversely, on closer examination, we observed a striking increase in the number of small microvessels in tumors that had been exposed to TNP-470, as compared to control tumors. Similarly, immunostaining for endothelium (Fig. 2, PECAM) and recruited vascular mural cells (Fig. 2, SMA) confirmed the presence of myriad sprouts in TNP-470-treated tumors, which were often seen arising from larger vessels. In contrast, vasculature was nearly absent in VEGF-Trap-treated xenografts, as demonstrated by fluorescein angiography and PECAM and SMA immunostaining (Fig. 2, VEGF-Trap). These results indicate, unlike the antiangiogenic agent VEGF-Trap, that TNP-470 administration reduces xenograft size but concurrently causes increased microvessel sprouting.
If TNP-470 acts to suppress tumor growth principally by inhibition of angiogenesis, then it would be predicted that administration would reduce tumor perfusion, causing hypoxia. Because expression of VEGF is exquisitely regulated by oxygen tension, it may serve as an indicator of the physiologic response of tumor cells to hypoxia (24). We compared VEGF expression in Wilms tumor xenografts exposed to TNP-470 or VEGF-Trap over 5 weeks by in situ hybridization. A similar low-level, diffuse pattern of VEGF expression was observed in control and TNP-470-treated tumors (Fig. 3, A and B) , whereas expression was strikingly up-regulated in VEGF-Trap-treated tumors. This result suggests that TNP-470, in contrast to VEGF-Trap, did not induce vascular insufficiency and consequent hypoxia in these xenografts. Therefore, we postulated that TNP-470 might act on tumor cells directly, and examined apoptosis by TUNEL assay. TNP-470-treated Wilms tumor xenografts at 5 weeks displayed significantly more apoptotic bodies than controls (Control 10.5/field versus TNP-470 61.7/field, P = 0.03). Taken together, these results suggested that 5 weeks of TNP-470 administration concurrently increased vascular sprouting and tumor cell apoptosis in tumors developing from microscopic implants, yet without evidence of vascular insufficiency.
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Figure 3. TNP-470 does not increase VEGF expression. VEGF expression, as determined by in situ hybridization, was similar in the control tumors (A), compared with the TNP-470-treated tumors (B). There was a marked increase in the VEGF-Trap-treated tumors (C). Bar = 400 µm.
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To determine whether these results were due to a direct effect on tumor cells or vasculature, we examined the short-term effects of TNP-470 on large tumors with established vasculature, comparing them directly that of VEGF-Trap. We established Wilms tumor xenografts in nude mice and allowed them to grow without intervention for 6 weeks. This resulted in large tumors with an average weight of 8 g at the time of sacrifice. TNP-470 was administered at two dose levels (30 or 100 mg/kg), and effects on tumor vasculature, apoptosis, and hypoxia examined after 1 or 5 days. For comparison, we administered VEGF-Trap (500 µg/dose) to an independent cohort of mice. We have recently demonstrated that vessels in established Wilms tumors strikingly regress 1 day after injection of VEGF-Trap (25).
Mice were euthanized on Days 1 and 5 after receiving TNP-470 (Days 0, 2, 4), VEGF-Trap (Days 0, 4), or control injections. To define perfused vessel lumens, we injected fluorescein-labeled L. esculentum lectin intravascularly in tumor-bearing animals at the time of sacrifice. One day after the first injection of TNP-470, we observed striking proliferation of vascular sprouts at both dose levels (30 or 100 mg/kg), as compared to control tumors (Fig. 4)
. In contrast, VEGF-Trap administration resulted in a sharp decrease in lectin-outlined tumor vessels. Similar results were seen at Day 5 (data not shown).
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Figure 4. TNP-470 promotes immediate vascular sprouting in established Wilms tumors. Established Wilms tumors were treated with vehicle (Control), 30 mg/kg of TNP-470 (TNP30), 100 mg/kg of TNP-470 (TNP100), or VEGF-Trap. One day later, the mice were injected intravascularly with fluorescein-labeled L. esculentum lectin (Fl-L). In comparison to control tumors, TNP-470-treated tumors resulted in a marked increase in vessels. In marked contrast, VEGF-Trap administration resulted in significant decrease in lectin-outlined tumor vessels. Images were further processed and binarized (Bin) to black and white. To assess vessel architecture, binarized images were skeletonized (Skel) and scored by the computer for total vessel length, vessel end points, and nodes. Size bar = 200 µm.
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Microvessel density (MVD) and architecture were quantitatively assessed by use of computer-assisted digital image analysis as described by Wild et al. (18). Images were initially binarized to black and white, and a common threshold level applied that preserved vascular morphology (Fig. 4). As a measurement of MVD, we quantified the total amount of FL-positive pixels (Fig. 5) . After 1 day, TNP-470 significantly increased MVD at 30 mg/kg (182% of control, P = 0.032) and 100 mg/kg (244% of control, P = 0.004), while VEGF-Trap significantly decreased MVD (46% of control, P = 0.037). Similar results were obtained at Day 5, with TNP-470 significantly increasing MVD at 30 mg/kg (209% of control, P = 0.003) and 100 mg/kg (216% of control, P = 0.003), in contrast to sharp decrease in MVD caused by VEGF-Trap (60% of control, P = 0.01).
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Figure 5. TNP-470 increases microvessel (MVD), vessel length, and nodes. After binarization of the images, MVD was estimated by the total number of white pixels per field as determined by the method of Wild et al. (18). Results show the mean white pixel count per image ± SD for Day 1 and Day 5 (number of images is six to eight). After images were skeletonized, they were scored by the computer for total vessel length, vessel end points, and nodes (mean ± SD). TNP-470 caused a significant increase in MVD (pixel count), vessel length, and nodes (*,P < 0.05; **,P < 0.01; ***,P = 0.005). In contrast, VEGF-Trap significantly inhibited MVD, vessel length, ends, and nodes.
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Because measurement of MVD by pixel count will overestimate the contribution of large-caliber vessels, we normalized vessel width by skeletonizing the images, which enables measurement of total vessel length (Fig. 5). In addition, complexity of the vascular architecture was further analyzed by counting vessel ends (which correlates with total vessel number) and branch points (or nodes) per image. At both doses, TNP-470 significantly increased total vessel length, while VEGF-Trap decreased it. TNP-470 also caused a significant increase in the branch points, but not in vessel ends, providing evidence that this agent had induced microvascular sprouting from existing vessels; in contrast, VEGF-Trap caused a sharp decrease in branching (Fig. 5).
To determine which vessel components might participate in early TNP-470-stimulated sprouting, we examined the status of endothelial and recruited mural cells in TNP-470-treated tumors at 1 day by double-label immunostaining for PECAM and SMA (Fig. 6)
. Surprisingly, microvessel sprouts appeared to contain both PECAM and SMA-immunopositive cells, suggesting that apposition of mural cells occurred rapidly after or concurrently with endothelial sprouting.
To investigate whether TNP-470 caused early vascular insufficiency, we compared VEGF expression by in situ hybridization in xenografts exposed to this agent or VEGF-Trap at 1 and 5 days. A low-level, diffuse pattern of VEGF expression was observed (Fig. 7A)
at 1 and 5 days in control and TNP-470-treated tumors. In contrast, VEGF was rapidly up-regulated in VEGF-Trap-treated tumors. We also examined hypoxia directly, by injecting tumor-bearing animals with pimonidazole 60 min before sacrifice and immunostaining for pimonidazole adducts. Similarly, hypoxic tumor regions were widespread in the VEGF-Trap-treated tumors, but manifested as rare areas in TNP-470-treated and control tumors (Fig. 7B). These data provide evidence that even in the short term, TNP-470 did not induce tumor hypoxia, in marked contrast to VEGF-Trap.
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Figure 7. TNP-470 treatment does not increase either tumor hypoxia or tumor expression of VEGF. A, VEGF expression was examined by in situ hybridization at Day 1 and Day 5. There was a low-level diffuse expression of VEGF in control tumors, and in the TNP-470-treated tumors at both 30 mg/kg (TNP30) and 100 mg/kg (TNP100). This is in contrast to the marked up-regulation seen after VEGF-Trap treatment. Bar = 400 µm. B, hypoxia was assessed at Day 1 and Day 5 by injecting pimonidazole i.p. in tumor-bearing control, TNP-470-treated, or VEGF-Trap-treated mice just before sacrifice. Tumors were immunostained for adducts of pimonidazole. Hypoxic areas were markedly increased after VEGF-Trap-treated xenografts at both Day 1 and Day 5. In contrast, no change was observed in TNP-470-exposed tumors as compared to controls at either time point. Bar = 200 µm.
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To determine if TNP-470 acted directly on tumor cells, we examined apoptosis at 1 and 5 days (Fig. 8)
. After one injection of TNP-470, there was a marked increase in apoptotic bodies in comparison to control [Fig 8B; TNP-470 (30 mg/kg) 610%, P = 0.001; TNP-470 (100 mg/kg) 770%, P = 0.002]. Despite the marked inhibition of angiogenesis by VEGF-Trap at this early time point, there was not a significant increase in apoptosis seen in VEGF-Trap-exposed tumor cells (154%, P = 0.11) as compared with controls. Similar results were obtained at the Day 5 time point (Fig 3C). Double-labeling for PECAM-1 and TUNEL revealed that the apoptosis induced by TNP-470 occurs primarily in tumor cells (Fig. 8A), with only rare apoptosis seen in endothelial cells. This is in contrast to the results with VEGF-Trap, where apoptosis is often seen in endothelial cells. Our studies of VEGF-Trap treatment demonstrate that, while xenografts eventually involute if treatment is continued, the loss of tumor cells occurs subsequent to vessel regression (25). The present results indicate that TNP-470 inhibits tumor growth principally by a direct effect on tumor cells, rather than indirectly, through inhibition of the angiogenesis.
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Figure 8. TNP-470 treatment causes rapid tumor apoptosis. A, apoptosis within the tumor was determined by TUNEL assay. In comparison to control or VEGF-Trap, more apoptotic bodies were observed in tumors treated with TNP-470 30 mg/kg (TNP30) or TNP-470 100 mg/kg (TNP100). Bar = 100 µm. Double-labeling for TUNEL (red) and PECAM-1 (green) demonstrates that TNP-470 produces apoptosis predominantly in tumor cells. VEGF-Trap induces apoptosis within endothelial cells (arrow). Bar = 50 µm. B, apoptosis was quantified for Day 1 (closed bars, n = 6 each point) and Day 5 (open bars, n = 8 each point). TNP-470 at 30 mg/kg (TNP30) and 100 mg/kg (TNP100) caused a significant increase in apoptosis at both Day 1 and Day 5 (*P < 0.002), in contrast to VEGF-Trap.
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Discussion
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The development of new vascular networks depends on a series of interrelated events, including proliferation of endothelial cells, sprouting of neovessels from established vasculature, remodeling of new and preexisting vessels, and the recruitment of vascular mural cells. Some molecules (such as VEGF) are known to promote some or all of these processes, rendering VEGF a rational target for antiangiogenic drug development. However, some agents (like TNP-470) were initially screened by testing for inhibition of endothelial proliferation in vitro. Subsequent antitumor efficacy in vivo was then postulated to be due to effective antiangiogenesis. For example, TNP-470 suppresses endothelial proliferation in vitro and tumor growth in vivo. However, there has been increasing evidence that other cell types, including tumor and smooth muscle cells, are also sensitive to TNP-470 (13, 26–28), a lack of specificity which is consistent with the proposed biochemical targets of this molecule (29–31). Thus, the precise effect of such an agent on the different processes contributing to angiogenesis may require clarification.
In our studies, TNP-470 significantly altered tumor vasculature, causing a rapid, quantitatively significant increase in the number of perfused microvessels in as short a time as 1 day. This alteration is in sharp contrast to the similarly rapid decrease in vessel density observed after potent VEGF blockade. The morphological pattern of dense, fine vascular sprouts induced by TNP-470 persisted after 5 weeks of drug administration, never apparently undergoing the remodeling which is a key feature of effective angiogenesis. The reasons for this effect are unclear. TNP-470 may interfere with vessel remodeling, potentially disrupting feedback loops which normally limit the density of capillary sprouting. It is also possible that these effects occur indirectly, via disruption of normal tumor cell signaling, as there was no evidence of vascular insufficiency in TNP-470-treated tumors in our studies. Tumors exposed to TNP-470 displayed neither increased expression of VEGF nor increased immunostaining for pimonidazole adducts, indicating that tumors were not hypoxic. Yet TNP-470 significantly decreased xenograft weights, and increased tumor cell apoptosis. Taken together, these findings indicate that this agent may act as a direct negative regulator of tumor cell growth in vivo.
In summary, these observations indicate that TNP-470 acts via mechanisms distinct from agents that target proangiogenic cytokines, such as those that block VEGF. Instead, TNP-470 administration results in early proliferation of microvessel sprouts, a pattern that persists if administration continues. Thus, current evidence suggests that TNP-470 does not have antitumor effects due to antiangiogenic activity, but rather due to a direct action on tumor cells.
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Acknowledgments
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We are grateful to S. Zabski for technical assistance.
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Footnotes
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Grant support: Pediatric Cancer Foundation (J.J. Kandel and D.J.Yamashiro), Sorkin Fund (J.J.Kandel), and National Cancer Institute CA088951 (D.J. Yamashiro). D.A.Pollyea was sponsored by the Student Research Program of the American Pediatric Society and the Society for Pediatric Research.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6/20/03;
revised 12/ 3/03;
accepted 12/23/03.
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Abstract
Introduction
