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Combination Chemotherapy Including Combretastatin A4 Phosphate and Paclitaxel Is Effective against Anaplastic Thyroid Cancer in a Nude Mouse Xenograft Model

Departments of Endocrine Neoplasia and Hormonal Disorders (S.-C.J.Y.) and General Internal Medicine, Ambulatory Treatment, and Emergency Care (L.S., S.-C.J.Y.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Department of Hematology (M.S.), Guangdong Provincial People’s Hospital, Guangzhou, 510089 People’s Republic of China; Department of Pathophysiology (H.Y., J.P.), Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510089 People’s Republic of China; and Oxigene, Inc. (D.C.), Waltham, Massachusetts 02451

Address all correspondence and requests for reprints to: Sai-Ching Jim Yeung, M.D., Ph.D., The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 437, Houston, Texas 77030. E-mail: syeung{at}mdanderson.org.

	Abstract

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Context: Anaplastic thyroid cancer (ATC) is extremely aggressive, and no effective treatment is available. Combretastatin A4 phosphate (CA4P), a vascular disrupting agent, has limited activity against ATC in a clinical trial, and so does paclitaxel.

Objective: We hypothesized that a triple-drug combination including CA4P and paclitaxel would improve efficacy against ATC. Therefore, we evaluated two such combinations in vivo.

Setting: We used a nude mouse xenograft model with ARO and KAT-4 cells.

Interventions: The first combination consisted of CA4P, paclitaxel, and manumycin A (a farnesyltransferase inhibitor), and the second, CA4P, paclitaxel, and carboplatin.

Main Outcome Measures: Main outcome measures included tumor growth curves and tumor weights.

Results: Tumor growth curve analysis (linear mixed models, P < 0.05) and xenograft weight analysis (Kruskal-Wallis one-way ANOVA on ranks, post hoc pairwise comparison, Dunn’s test, P < 0.05) demonstrated that both triple-drug combinations were significantly better than placebo for both cell lines. Anti-bromodeoxyuridine immunostaining of xenograft sections from animals injected with bromodeoxyuridine before being killed showed that CA4P alone did not inhibit DNA synthesis, but manumycin A and paclitaxel did. CA4P decreased the depth of the viable outer rim of tumor cells on xenograft sections. Using electron microscopy, we found blebbing/budding of endothelial cells into capillary lumens and autophagy of tumor cells in CA4P-treated xenografts.

Conclusions: Both triple-drug combinations demonstrated excellent antineoplastic activity against ATC. The correlative findings in xenografts were consistent with vascular disruption but not direct inhibition of cell proliferation as the primary antineoplastic mechanism contributed by CA4P. These regimens warrant further investigation in clinical trials for ATC.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE U.S. INCIDENCE of thyroid cancer is rising; approximately 1500 people were expected to die from thyroid cancer in the United States in 2006 (1). Anaplastic thyroid cancer (ATC), which constitutes 1.6% all of thyroid cancers, is one of the most aggressive of all solid tumors. ATC patients have a median survival of 3–7 months and account for a significant portion of thyroid cancer deaths. Current therapies have done little to improve the outcome of ATC (2, 3).

Paclitaxel, an inhibitor of tubulin depolymerization, has shown activity against ATC in vitro and in animal studies (4). Subsequently, a prospective phase II clinical trial in ATC patients with persistent or metastatic disease despite surgery or local radiation therapy demonstrated significant but limited clinical activity against ATC (5).

Vascular disrupting agents take advantage of differences in the pathophysiology of tumor vs. normal tissue blood vessels (e.g. increased proliferation and fragility, differences in cytoskeleton, and up-regulated proteins) to cause a rapid and selective shutdown of the blood flow in tumors (6). In contrast to antiangiogenic drugs that block formation of new vessels in tumors, the vascular disrupting agents stop blood flow through preexisting vessels to deprive tumor cells of oxygen and nutrients (6, 7, 8). Vascular disrupting treatment, in combination with angiogenesis inhibition and direct cytotoxicity, can lead to improved activity against solid tumors (9). Combretastatin A4 phosphate (CA4P) is a tubulin-binding agent that inhibits tumor blood flow at doses less than 10% of the maximum tolerated dose (10); its rapid, selective, and extensive inhibition of tumor blood flow makes it a promising novel anticancer drug (11). CA4P has antineoplastic activity against ATC cell lines and xenografts (12). In a phase I trial, CA4P, given iv every 3 wk, induced remission of ATC for 30 months in one patient (13). A phase II trial showed that approximately 25% of ATC patients treated with single-agent CA4P experienced more than 3 months of progression-free survival (14). Although combining CA4P with other chemotherapeutic agents is expected to improve antitumor efficacy against ATC, the efficacy against ATC of a clinically safe combination (i.e. CA4P, paclitaxel, and carboplatin) (15) has not yet been evaluated in vivo.

We previously demonstrated that manumycin A, a farnesyltransferase inhibitor, had an antineoplastic effect in ATC, and the combination of paclitaxel and manumycin A further increased apoptosis (16). Our previous results also indicated that angiogenesis inhibition at least partially mediated the in vivo antineoplastic effects of manumycin A and paclitaxel (17). Therefore, the combination of CA4P, paclitaxel, and manumycin A is another triple-drug combination we chose to further investigate in vivo.

We hypothesized that a triple-drug combination including CA4P and paclitaxel was effective against ATC in vivo. On the basis of our previous work (16, 17) and the recent results of CA4P clinical trials (14, 15), we evaluated two triple-drug combinations (CA4P+paclitaxel+manumycin A and CA4P+paclitaxel+carboplatin) against ATC in a nude mouse xenograft model. Correlative studies of xenografts using electron microscopy and immunohistochemistry also shed new light on the in vivo action of CA4P against ATC.

	Materials and Methods

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Cell culture

Two human ATC cell lines, ARO (originally developed by G. J. F. Juillard at the University of California, Los Angeles, CA) and KAT-4 (originally developed by K. B. Ain at the University of Kentucky, Lexington, KY) (4), were kindly provided by the original investigators and were cultured as previously described (16). Both cell lines were free of Mycoplasma species and tested negative for a panel of pathogenic murine viruses (Research Animal Diagnostic Laboratory, University of Missouri-Columbia).

Nude mouse xenograft model

One million ATC cells suspended in RPMI 1640 medium were injected sc at the flank of each 5-wk-old nude (nu/nu BALB-c) mice bred at our institution. The mice were housed in Association for Assessment and Accreditation of Laboratory Animal Care-approved barrier facilities on a 12-h light, 12-h dark cycle, with food and water ad libitum. The mice were treated under approved protocols in compliance with the animal care and use guidelines of our institution, the U.S. Department of Agriculture, and the National Institutes of Health.

Drugs

Manumycin A and paclitaxel were purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in tissue culture-grade dimethylsulfoxide (Sigma) at appropriate concentrations before being diluted in tissue-culture medium to a final concentration of 0.5% (vol/vol) dimethylsulfoxide or less. Carboplatin was dissolved in sterile PBS. CA4P was kindly provided by Oxigene, Inc. (Waltham, MA) and was dissolved in normal saline at 10 mg/ml.

Treatment regimens

The drug treatments were administered in 7-d cycles, and control injections contained identical vehicles but no drugs. We first evaluated the combination CA4P+paclitaxel+manumycin A. Four days after xenografting, the mice were randomly assigned into four groups of eight mice: control, CA4P only, paclitaxel+manumycin A, or CA4P+paclitaxel+manumycin A. Manumycin A was injected ip at 3 mg/kg·d on d 1–5 [same cumulative dose per week as reported previously (17)], and paclitaxel was injected ip at 50 mg/kg·d on d 1 of each 7-d cycle [about 60% of the highest cumulative dose per week reported by Ain et al. (4)]. CA4P was administered ip on d 1–5, 2 h after the manumycin A or paclitaxel injection. We used 50 mg/kg·d of CA4P in the ARO xenografts. This dosage schedule resulted in observable toxicity; therefore, we subsequently used 30 mg/kg·d [an effective antivascular dose in rodents (18)] in the KAT-4 xenografts.

We next evaluated the combination CA4P+paclitaxel+carboplatin. In a phase I trial, a pharmacokinetic interaction between CA4P and carboplatin was noted when CA4P and carboplatin were administered on the same day (19), and modifying the dosing schedule to avoid the interaction rather than trying to modulate the interaction was recommended. Thus, a different dosage schedule for CA4P was used in this second set of experiments. Four days after xenografting, the mice were randomly assigned into eight groups of eight mice: control, CA4P, carboplatin, paclitaxel, CA4P+carboplatin, CA4P+paclitaxel, paclitaxel+carboplatin, or CA4P+paclitaxel+carboplatin. During each 7-d cycle, CA4P was injected ip at 100 mg/kg on d 1, carboplatin was injected ip at 50 mg/kg on d 2, and paclitaxel was injected ip at 15 mg/kg 1 h after carboplatin.

Animal monitoring

Tumor volumes were estimated by a² x bx 0.4, where a was the smallest diameter and b was the diameter perpendicular to a (20). Body weight, feeding behavior, and motor activity were monitored thrice weekly as indicators of general health. Animals with the following conditions were euthanized: more than 10% weight loss, motor retardation, inability to obtain food or water, ruffled hair, or largest diameter of the tumor more than 15 mm.

Immunohistochemical staining

When the experiments ended, the animals were euthanized, and the xenografts were dissected and weighed. Formalin-fixed xenografts were embedded in paraffin and sectioned according to standard techniques. Immunostaining was performed using the avidin-biotin peroxidase method (Vector Laboratories, Inc., Burlingame, CA). The primary antibodies were a rabbit polyclonal IgG against von Willebrand factor (Calbiochem, La Jolla, CA) and a rabbit polyclonal antibody that recognizes amino acids 41–160 near the N terminus of human carbonic anhydrase IX (CA IX) (Santa Cruz Biotechnologies, Santa Cruz, CA). The secondary antibody was biotinylated antirabbit IgG (Vector). The color was developed with 3,3'-diaminobenzidine tetrahydrochloride, and the slides were counterstained with hematoxylin. A negative control for each xenograft was performed by substituting normal goat serum for the primary antibody. To detect bromodeoxyuridine (BrdU) incorporation, we used the BrdU immunohistochemistry kit from Chemicon International, Inc. (Temecula, CA).

Transmission electron microscopy

Harvested xenografts were cut into 1.0- to 1.5-mm-thick slices and fixed with 2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h. The samples were washed with 1% cacodylate-buffered tannic acid, postfixed with 1% buffered osmium tetroxide for 1 h, and stained en bloc with 1% uranyl acetate before dehydration in ethanol, embedment in Spurr’s low-viscosity medium, and polymerization at 60 C for 2 d. Ultrathin sections, stained with uranyl acetate and lead citrate, were examined using a JEM 1010 transmission electron microscope (JEOL USA, Inc., Pleasanton, CA) at an accelerating voltage of 80 kV. Whole cells were imaged at a magnification of x6000. Changes in cytoplasmic organelle morphology were observed at x27,000.

Statistical analysis

The statistical analysis was performed with SPSS for Windows, version 12.0 (SPSS, Inc., Chicago, IL). The significance of differences among multiple groups was assessed using an ANOVA. When the data sets failed the normality test, the Kruskal-Willis one-way ANOVA on ranks was used. Post hoc pairwise comparison between groups was tested for significance using Dunn’s method. The null hypothesis was accepted if P < 0.05. Linear mixed models were used to analyze growth curves. Because each treatment group consisted of fewer than nine animals, only large differences could be detected. However, the results of recently reported statistics studies suggest that when only a limited number of samples is available, maximum P values of 0.10–0.20 may be appropriate for early-stage research (21, 22). Therefore, for this study, all appropriate P values were reported, and P values up to 0.20 were considered of interest for future investigations.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Combination of CA4P, manumycin A, and paclitaxel was effective against AT C

Toxicity. In mice with ARO xenografts, 50 mg/kg·d CA4P was administered for 5 d of each 7-d cycle. After 2 wk of therapy, one animal from the CA4P-only group and one animal from the triple-drug combination group had experienced dehydration noticeable by poor skin turgor; they were treated with normal saline (50 ml/kg·d) by tail-vein injection. We thus reduced the dosage of CA4P to 30 mg/kg·d for 5 d of each 7-d cycle for the KAT-4 mice. No clinical dehydration was observed at this dosage. An analysis of body weights among the treatment groups at the end of the experiments (for both cell lines) with one-way ANOVA revealed no significant differences.

Growth curve analysis of estimated tumor volumes. Caliper measurements of tumor dimensions were used to estimate the tumor volume, as described in Materials and Methods, and the growth data are plotted in Fig. 1, A and B. There were eight animals per group. Two animals with KAT-4 xenografts treated with the triple-drug combination experienced complete remission. These repeated measures were analyzed using linear mixed models, with the animals grouped by treatment (control, CA4P only, manumycin A and paclitaxel, or CA4P, manumycin A, and paclitaxel). An analysis of the fixed effects of treatment groups showed that the growth curves of the triple-drug combination group were significantly different from those of the placebo, CA4P, and manumycin A and paclitaxel groups (P < 0.001, P = 0.004, and P = 0.013 for the ARO xenograft groups, respectively, and P < 0.001 for all three KAT-4 xenograft groups).

View larger version (23K): [in this window] [in a new window]   FIG. 1. A and B, Tumor growth curves for CA4P, with or without manumycin A and paclitaxel. The mean estimated tumor size, represented by symbols (see symbol key in the left upper corner for group assignment), is plotted against the number of days after cancer cell xenografting. A, ARO xenografts; B, KAT-4 xenografts. M, Manumycin A; P, paclitaxel. C and D, Effect of the combination of CA4P, manumycin, and paclitaxel on ATC xenograft (C, ARO; D, KAT-4) growth rates. The box plots (tumor weight/number of days since xenografting) are shown. The treatment groups are shown on the horizontal axis. The error bars represent the fifth and 95th percentiles. Within the boxes, the red dashed lines represent the means, and the black lines represent the medians.

Combination of CA4P, carboplatin, and paclitaxel was effective against ATC

Toxicity. No treatment-related deaths or instances of dehydration were observed. An analysis of body weights among the treatment groups at the end of the experiments (for both cell lines) with the one-way ANOVA revealed no significant differences.

Growth curve analysis of estimated tumor volumes. The growth data for these experiments were based on the estimated tumor volume, calculated from the tumor dimensions; the data are plotted in Fig. 2, A and B. There were eight animals per group. These repeated measures were analyzed using linear mixed models with the animals as the subjects and the tumor size as the dependent variable and were grouped by treatment type. We analyzed the fixed effects of treatment groups and found that in both cell lines, the tumor size growth curves for the triple-drug group were statistically lower than those for the control group (P < 0.05). For the KAT-4 xenografts, the curves for the triple-drug group was also statistically lower than those for the three single-drug groups and the CA4P plus carboplatin group. The triple-drug group was near-significantly lower than the carboplatin plus paclitaxel group (P = 0.070) and the CA4P plus paclitaxel group (P = 0.053). For the ARO xenografts, the growth curves for the triple-drug group were significantly lower than those for all three single-drug groups.

View larger version (28K): [in this window] [in a new window]   FIG. 2. A and B, Tumor growth curves for CA4P, carboplatin, and paclitaxel, alone and in combinations. The mean estimated tumor size, represented by symbols (see symbol key in the left upper corner for group assignment), is plotted against the number of days after cancer cell xenografting. A, ARO xenografts; B, KAT-4 xenografts. Cpt, Carboplatin; P, paclitaxel. C and D, Effect of CA4P, carboplatin, and paclitaxel, alone and in combinations, on ATC xenograft growth rates. The box plots of the xenograft growth rate (tumor weight/number of days since xenografting) for ARO (C) and KAT-4 (D) are shown. The treatment groups are shown on the horizontal axis. The error bars represent the fifth and 95th percentiles. Within the boxes, the red dashed lines represent the means, and the black lines represent the medians.

CA4P did not inhibit incorporation of BrdU

To determine whether CA4P had a direct effect on tumor cell proliferation in vivo, in addition to its vascular disrupting effects, we injected 1500 mg/kg BrdU ip into mice 2 h after CA4P (50 mg/kg ip) on d 5 of the fifth cycle; 4 h later, the animals were killed, and the xenografts were resected, weighed, and fixed in 4% buffered formalin. Immunohistochemical staining of the paraffin sections with anti-BrdU antibody revealed that the tumors from the triple-drug group did not incorporate BrdU, whereas those from the CA4P-treated group did (Fig. 3). Therefore, 50 mg/kg CA4P did not severely suppress DNA synthesis or cell proliferation in the xenografts within the 6-h period after CA4P injection.

View larger version (86K): [in this window] [in a new window]   FIG. 3. Evaluation of BrdU incorporation (DNA synthesis). Immunohistochemical staining with BrdU antibody showed BrdU incorporation in the nuclei of cells in the crypts of a piece of ileum (positive control). The drug treatment groups of the xenograft sections are as labeled. M, Manumycin A; P, paclitaxel.

CA IX is expressed in tumor cells in the hypoxic regions of solid tumors (23, 24). We used sequential sections of xenografts for hematoxylin and eosin (H&E) staining, anti-von Willebrand factor immunostaining, and anti-CA IX immunostaining to determine the location of hypoxic and nonviable areas of the solid tumor relative to the distribution and location of blood vessels. As shown in Fig. 4A, viable tumor cells in the hypoxic areas of the xenograft tumors were not in the vicinity of any blood vessels or capillaries; these hypoxic cells bordered the necrotic central parts of the xenografts. The distance from the surface of a xenograft to the hypoxic region that marked the edge of the viable rim of cancer cells (represented by a red bar in Fig. 4B) was shorter in xenografts treated with CA4P (alone or in combination with carboplatin, paclitaxel, or both) than in xenografts not treated with CA4P. To confirm that CA4P treatment had thinned the outer rims of viable cancer cells in the xenografts, H&E-stained sections of all resected xenografts in the CA4P+paclitaxel+carboplatin experiment were examined using a microscope with eye pieces with ruler markings. For each xenograft, the thickness of the rims of viable cancer cells were measured at six random locations along the circumference of the tumor and averaged. The data for ARO cell xenografts are plotted in Fig. 4C and the data for KAT-4 cell xenografts in Fig. 4D. The three-way ANOVA showed that CA4P significantly (P < 0.05) thinned the rim thickness of viable cancer cells in both ARO and KAT-4 xenografts. Paclitaxel had a smaller but statistically significant thinning effect in KAT-4 xenografts but not ARO, and carboplatin did not significantly affect the thickness of viable rim in xenografts of both cell lines.

View larger version (53K): [in this window] [in a new window]   FIG. 4. CA4P caused thinning of the viable rims of cancer cells in xenografts. A, Left upper panel, H&E-stained slide; right upper panel, anti-von Willebrand factor (VWF)-immunostained slide; left lower panel, CA IX-immunostained slide; right lower panel, diagrammatic interpretation of the slides. These four panels are serial sections that show the same areas at the same magnification. B, CA IX immunostaining is shown. Each image is shown with the treatment group labeled above. The outer edge of the xenograft is oriented downward, and the inner necrotic area is oriented upward. The red bars represent the distance from the outer edge of the tumor to the inner hypoxic region. C and D, Bar charts of the thickness of the viable rims of cancer cells in ARO (C) and KAT-4 (D) xenografts are shown. The drug treatment groups are indicated by the table below the horizontal axis, with plus indicating the presence and minus indicating the absence of the corresponding drug. The error bars represent the 95% confidence intervals.

After CA4P treatment, endothelial cells change shape; this may be involved in vascular shutdown in vivo (25). As a consequence of vascular shutdown, hypoxia and nutrient deprivation are expected to induce autophagy in cancer cells. Autophagy may be a survival mechanism for short-term disruptions in the oxygen or nutrient supply; macroautophagy is a cell death mechanism that can occur either in the absence of apoptosis or concomitantly with apoptosis (26). To determine the in vivo effects of CA4P on ATC, ARO xenografts were resected 6 h after 100 mg/kg CA4P had been injected. The CA4P-treated tumors and untreated control tumors were cut into 1.0- to 1.5-mm-thick slices and processed for electron microscopy. Histomorphological changes in endothelial cells have been documented in xenograft tumors after treatment with a combretastatin analog (27). Using electron microscopy, we observed blebbing of endothelial cells in the capillaries of CA4P-treated xenografts but not in control xenografts (Fig. 5A; sites of endothelial cell shape changes indicated by red arrows). Macroautophagy was observed in the nearby cancer cells in CA4P-treated tumors but not in control tumors (Fig. 5B). Mitotic cancer cells were frequently observed in the control tumors. In summary, we found electron micrographic evidence that CA4P induced endothelial cell dysmorphism and macroautophagy in cancer cells, likely as a sequela of vascular disruption.

View larger version (60K): [in this window] [in a new window]   FIG. 5. Electron microscopic examination of xenografts. A, CA4P damaged endothelial cells in nude mouse xenografts. Cutouts showing only the endothelial cells are to the right of the original micrographs. The red arrows point to parts of the endothelial cells bulging into the capillary lumen. B, CA4P induced autophagy in cancer cells in xenografts. A schematic outline is shown to the right of each micrograph to facilitate interpretation. The red lines outline the nuclei. The blue lines outline the cell boundaries at low magnification and the mitochondria at high magnification. The green lines outline the autophagosomes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Farnesyltransferase inhibitors also have antiangiogenic activity among their multiple antineoplastic mechanisms (32). Our previous work demonstrated that paclitaxel+manumycin A is active against ATC in nude mice (16, 17) and antiangiogenesis plays an important role in the drugs’ in vivo action (16). CA4P+ paclitaxel+manumycin A had a superior in vivo antineoplastic effect to that of manumycin and paclitaxel or CA4P alone as shown in this report. This is not surprising, because Shaked et al. (33) reported that in tumor-bearing mice, vascular disrupting agents acutely mobilize circulating endothelial progenitor cells, which home to the viable tumor rim. Disrupting this process with antiangiogenic drugs should result in enhanced antitumor activity, and this may be the mechanistic rationale for the improved efficacy of CA4P when combined with antiangiogenic drugs (33).

During this translational investigation, we made interesting observations relating to the antineoplastic action of CA4P in vivo. We found that CA4P (50 mg/kg) did not inhibit DNA synthesis in xenografts to a large extent. Therefore, CA4P did not contribute to the combination of CA4P+paclitaxel+manumycin A through direct inhibition of ATC cell proliferation. We found that CA4P (100 mg/kg) induced dysmorphism of endothelial cells in electron micrographs; this is in agreement with the histomorphological data from xenografts treated with another combretastatin analog (27). Macroautophagy in cancer cells may result from nutrient deprivation after disruption of tumor blood flow. We also observed that the rims of viable cancer cells were thinner in CA4P-treated xenografts than in those without exposure to CA4P. Therefore, one may speculate that there are fewer viable cancer cells in a xenograft treated with CA4P+paclitaxel+carboplatin than in a xenograft of the same size/weight that has been treated with paclitaxel+carboplatin.

The significance of this report is the demonstration of antineoplastic activity of CA4P- and paclitaxel-based chemotherapy combinations against ATC in vivo. Correlative studies revealed endothelial changes, tumor cell macroautophagy, and thinning of the rim of viable cells in xenografts and corroborated tumor blood flow disruption as the primary antitumor mechanism of CA4P. There is a trend that CA4P+paclitaxel+carboplatin may be more efficacious than the two-drug combinations against KAT-4 xenografts, but this requires further investigation. Our results generated the specific hypothesis that CA4P+paclitaxel+carboplatin is more active than paclitaxel+carboplatin in ATC patients, which will require a large phase III multicenter clinical trial to have adequate statistical power to test.

	Acknowledgments

 
We thank Dr. C. D. Bucana and Mr. K. Dunner, Jr. (High Resolution Electron Microscopy Core Facility, The University of Texas M. D. Anderson Cancer Center), for their expert technical assistance.

	Footnotes

 
This research was partially supported by a grant to S.-C.J.Y. from Abbott Laboratories (Thyroid Research Advisory Council) and a contract with Oxigene, Inc. The animal facility and veterinary histopathology core facility at The University of Texas M. D. Anderson Cancer Center are partially supported by a Cancer Center Support Grant (CA16672).

Disclosure Statement: L.S., M.S., H.Y., and J.P. have nothing to declare. S.-C.J.Y. received a grant from Oxigene, Inc. D.C. is the Chief Scientific Officer of Oxigene, Inc.

First Published Online June 5, 2007

¹ S.-C.J.Y. and M.S. contributed equally and are considered first authors.

Abbreviations: ATC, Anaplastic thyroid cancer; BrdU, bromodeoxyuridine; CA IX, carbonic anhydrase IX; CA4P, combretastatin A4 phosphate; H&E, hematoxylin and eosin.

Received January 5, 2007.

Accepted May 24, 2007.

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