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The Antiangiogenic Agent Linomide Inhibits the Growth Rate of von Hippel-Lindau Paraganglioma Xenografts to Mice

Departments of Endocrinology and Metabolism [D. J. G.], Bone Marrow Transplantation and Cancer Immunobiology [I. R., L. W., S. S.], and Department of Molecular Biology [I. S., L. E. B.], Hebrew University-Hadassah Medical Center, Jerusalem, Israel 91120, and Department of Biological Regulation, Weitzmann Institute of Science, Rehovot, Israel 76100 [M. N., R. A.]

	ABSTRACT

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The aim of this study was to ascertain the potential usefulness of the antiangiogenic compound linomide for treatment of von Hippel-Lindau (VHL)-related tumors. Paraganglioma tissue fragments obtained at surgery from a VHL type 2a patient were transplanted s.c. to male BALB/c nu/nu (nude) mice: (a) 2–3-mm fragments for "prevention" experiments; and (b) 2–3-mm fragments allowed to grow to 1 cm for "intervention" studies. Both groups received either 0.5 mg/ml linomide in drinking water or acidified water and were followed until tumor diameter reached 3 cm or for 4 weeks. In both the prevention and intervention experiments, a significant diminution of tumor size and weight was observed in the drug-treated animals. In vivo nuclear magnetic resonance analysis of tumor blood flow in linomide-treated animals showed localization of blood vessels almost exclusively to the periphery of the poorly vascularized tumors with a significant reduction of both vascular functionality and vasodilation. Histological examination of tumors from linomide-treated animals revealed marked avascularity. Treated animals also displayed a 2.4-fold reduction of tumor vascular endothelial growth factor mRNA levels. Taken together, our data indicate that in VHL disease, therapy directed at inhibition of constitutively expressed VEGF induction of angiogenesis by VHL tumors may constitute an effective medical treatment.

	INTRODUCTION

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VHL³ disease is an autosomal dominant cancer syndrome characterized clinically by retinal angiomas, cerebellar and spinal cord HBs, RCCs, pheos, and less commonly, pancreatic islet cell tumors and epididymal cysts. This multiorgan involvement can appear in various combinations; however, in an affected kindred, the clinical manifestations of the disease are usually invariable and breed true. Thus, on the basis of phenotype, VHL can be subdivided to: type 1, with RCC; type 2A, with pheo without RCC; and type 2B, with pheo and RCC. The penetrance of expression of the components characteristic of an affected kindred is very high; for example, it has been shown that >90% of patients will develop retinal angiomas, HBs, or RCCs by the age of 70 years (1) . The frequency of VHL in the population has been estimated to be 1:32,000; thus, in the United States alone, close to 8000 patients have VHL. The gene associated with VHL has recently been cloned (2) , and germ-line mutations in the VHL gene can be detected in virtually all patients with VHL (3) . These can be micro- and macrodeletions or point mutations scattered throughout all three exons of the VHL gene (4) . The VHL gene encodes a 213-amino acid peptide pVHL. In the past 2 years, it has become apparent that pVHL, considered to be a tumor suppressor gene, has pleotropic effects in the cell; pVHL prevents the binding of the elongin (SIII) transcriptionally active A subunit to the two regulatory subunits (B and C) in vitro, thus inhibiting transcription elongation by RNA polymerase II (5) . pVHL has recently been shown to play an important role in fibronectin extracellular matrix assembly (6) . Finally, in tumor cell lines derived from VHL patients, mutated pVHL is associated with constitutively elevated VEGF mRNA (7) , unresponsive to normal normoxia/hypoxia regulation (8) . Reintroduction of wild-type pVHL results in restoration of VEGF regulation (8) and inhibition of tumor growth in vivo (9 , 10) . Thus, mutated pVHL-related overexpression of VEGF and its receptors in the well-vascularized VHL tumors (11 , 12) might implicate unregulated VEGF-induced angiogenesis as a major pathogenetic pathway leading toward tumor formation.

There is no effective medical therapy for prevention of VHL or treatment of the clinical components of the disease once they appear. To date, treatment relies on clinical tumor surveillance. Tumors (RCC and pheo) are dealt with surgically, HBs either by conventional or radiosurgery, and retinal angiomas by laser therapy. Because a considerable number of tumors are inoperable (for example, metastatic RCC or bulbar/intrathecal spinal HB) and because of the high recurrence rate of tumors in VHL, a chemopreventive modality would be highly desirable. Linomide (quinoline-3-carboxamide), a potent immunomodulator, has also been shown to have a marked antiangiogenic effect, reducing vascularity in both transplanted normal (13) and neoplastic tissues (14) . This antiangiogenic effect is accompanied by an antitumoral activity in a variety of murine and human tumors (15, 16, 17, 18) . Moreover, linomide has recently been shown to specifically inhibit VEGF elicited migration and growth of vascular endothelial cells in vitro (19) and angiogenesis in breast carcinoma xenografts expressing a VEGF transgene (20) . The aim of this study was to ascertain the potential usefulness of linomide for treatment of VHL-related tumors, because these characteristically express high levels of VEGF.

	MATERIALS AND METHODS

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Establishment of a Transplantable VHL Tumor Line.
Paraganglioma tissue fragments obtained at surgery from a patient with VHL type 2A (harboring a missense mutation resulting in replacement of Val by Phe at position 166 of the VHL protein; Ref. 21 ) were transplanted s.c. to male BALB/c nu/nu (nude) mice. In 4 of 20 mice, tumors appeared after 7 months. Tissue fragments obtained from these four mice were secondarily transplanted s.c. to nude mice. Tumors were identified as originating from the original chromaffin tumor by positive stains for chromogranin A and neuron-specific enolase (data not shown). All animal experiments were conducted according to the stipulations of the local Animal Care Committee.

Treatment Groups.
For "prevention" experiments, fragments 2–3 mm in diameter were implanted s.c. under the neck skinfold of nude mice. For "intervention," the fragments were allowed to grow to 1 cm in diameter prior to initiation of therapy. In both experimental groups, animals received either 0.5 mg/ml linomide in drinking water or acidified water as control and were subsequently followed until tumor diameter reached 3 cm (because of increased morbidity and mortality of animals with tumors superceding this size) or for 4 weeks.

Assessment of the Effect of Linomide Therapy on Tumor Growth at Termination of Experiments.
Prior to tumor excision, animals were anesthetized, and tumor volume was calculated as follows: the maximal tumor diameter (X axis) and two additional diameters bisecting the X axis in the Y and Z axes were measured. The mean value of these three parameters served as an average value for calculation of the tumor volume. Tumors were then removed and weighed, and samples were processed for histopathological examination and for total RNA extraction. In some experiments, in vivo MRI assessment of tumor vascularity was performed.

Histochemistry.
Sections of paraffin-embedded tumor tissue were cut and placed on precoated slides (Sigma). Slides were deparaffinized in two changes of xylene (10 min) and rehydrated gradually from 100% ethanol to PBS. H&E staining is used to examine hemorrhage and necrosis. Blood vessel endothelial cells were visualized by incubation with anti-von Willibrand factor. Anti-von Willibrand factor was used at 1:1000 after 20 min of trypsinization (0.1% at 37°C) and blocking with 10% goat serum/0.1% BSA. The primary antibody was followed by antirabbit horseradish peroxidase, and antibodies were visualized with 3-amino-9-etylcarbazole (Sigma) in 50 mM Tris (pH 5.0).

RNA Extraction and Northern Analysis.
Total RNA was extracted by the guanidine thiocyanate method/phenol chloroform extraction procedure (22) . RNA was denatured with glyoxal and electrophoresed through 1% agarose gels. RNAs were then transferred onto nylon membranes (GeneScreen Plus) by capillary blot and hybridized to cDNA labeled with ³²P by the random priming method. For standardization, rRNAs were stained with methylene blue prior to hybridization. Relative quantities of the respective RNAs were assessed by scanning of the Northern blots. The hybridization probe for VEGF was a 1.8-kb mouse cDNA fragment that detects human VEGF transcripts at high sensitivity (23) .

MRI Assessment of Tumor Vasculature.
MRI experiments were performed on a horizontal 4.7T Bruker Biospec spectrometer using an actively RF decoupled surface coil, 2 cm in diameter, imbedded in a Perspex board, and a bird cage transmission coil. Mice were anesthetized (75 mg/kg ketamine + 3 mg/kg xylazine, i.p.) and placed supine with the tumor located on the center of the surface coil. MRI data were analyzed on a Indigo-2 work station (Silicon Graphics, Mountain View, CA) using paravision software (Bruker, Rheinstetten, Germany).

Vascular function (VF) was derived from images acquired during inhalation of carbogen (95% oxygen, 5% CO₂) and air-CO₂ (95% air, 5% CO₂; Eq. A ):

(1)

Where TE is the echo time, Y is the fraction of oxyhemoglobin, b is the volume fraction of blood, and C_MRI = 599 s^-1 at 4.7 T (24) . This parameter measures the capacity of erythrocyte-mediated oxygen delivery from the lungs to each pixel in the image (24 , 25) .

Vasodilation (VD) was derived from air and air-CO₂ images (Eq. B) :

(2)

Positive VD corresponds to increased signal intensity by hypercapnia, attributable to elevated blood oxygenation (and/or increase blood flow), whereas vascular steal will result in negative VD values (24) .

Statistical Analysis.
Group comparisons were performed using Students’ two-tailed t test.

	RESULTS

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Inhibition of Tumor Progression by Linomide.
Animals administered 0.5 mg/ml linomide in the drinking water commencing at the time of transplantation ("prevention" experiment) showed a marked retardation of tumor growth as compared with control animals (Fig. 1) . This inhibition of tumor growth was also reflected by a significant difference in tumor weights at the end of the experiment (Fig. 2) . Fig. 3 shows an intervention experiment in which the tumors were allowed to attain 1 cm in diameter, at which time linomide treatment was started at the same dose as in the prevention experiments. A marked diminution of tumor growth was noted with mean tumor volumes (±SE) for the control group at 13–14 days of 16,815 ± 4364 mm³ and for the treated group at 14–16 days of 1858 ± 778 mm³ (n = 5 for each group, P < 0.001).

View larger version (15K): [in this window] [in a new window]   Fig. 1. The effect of linomide (0.5 mg/ml) on tumor volume in nude mice implanted with VHL-derived paraganglioma. Tumor tissue fragments of 2–3 mm in size were implanted s.c. under the neck skinfold, and animals (n = 8 for each group) were started on 0.5 mg/ml linomide in the drinking water at day 0. Control animals received acidified water. From day 21 on, a significant difference in tumor volumes was observed between the two groups (P < 0.04 or less for each time point). Data are expressed as means, representative of two qualitatively similar experiments; bars, SE.

View larger version (9K): [in this window] [in a new window]   Fig. 2. The effect of linomide (0.5 mg/ml) on tumor weight in nude mice implanted with VHL-derived paraganglioma. The figure shows the wet weights (wt.) of the tumors at the end of the experiment depicted in Fig. 1 .

View larger version (16K): [in this window] [in a new window]   Fig. 3. The effect of linomide (0.5 mg/ml in drinking water) on nude mice with established VHL tumors. Tissue fragments were implanted as described in the legend for Fig. 1 , and tumors were allowed to attain 1 cm in diameter prior to commencement of the drug. Each plot depicts the course of an individual animal. , controls; •, linomide treated. Two animals in the control and linomide groups were removed from the study at 15 and 25 days, respectively, for other purposes. This experiment is representative of two qualitatively similar experiments. Animals were followed until tumor diameter reached 3 cm or for 4 weeks.

View larger version (74K): [in this window] [in a new window]   Fig. 4. MRI analysis of the effects of linomide on tumor vasculature. A and B, control animal tumors; C and D, tumors from linomide-treated animals (0.5 mg/ml in drinking water from day 0; analysis at day 35). A and C, vascular functionality (VF) derived from gradient echo MRI upon hyperoxygenation; B and D, vasodilation (VD) induced by hypercapnia, a marker of vascular maturation. The absolute values of VD and VF are noted on the color bar. Data were acquired at 4.7 T on a Bruker Biospec spectrometer (TE, 10 ms; TR, 230 ms; slice thickness, 1 mm; inplane resolution, 150 µm)

View larger version (144K): [in this window] [in a new window]   Fig. 5. Histology of tumors treated with linomide shows a reduction in vascular density and increased tumor cellularity. H &E staining of tumor treated with linomide (A) compared with controls (B). Anti-von Willibrand staining of tumors also shows a much less complex and less dense network of blood vessels after linomide (C) compared with controls (D).

View larger version (47K): [in this window] [in a new window]   Fig. 6. Northern blot analysis (upper panel) of VEGF mRNA levels in implanted VHL tumors in nude mice treated with linomide. Total RNA was extracted from tumors at the end of a prevention experiment as described in the legend for Fig. 1 and analyzed for VEGF mRNA levels as detailed in "Materials and Methods." The three left lanes and three right lanes depict levels from three randomly selected individual animals in control and linomide-treated groups, respectively. Lower panel, densitometric quantitation of the mean relative abundance of the mRNA, indicating that treatment with linomide resulted in a 2.4-fold reduction of tumor mRNA in the treated group; bars, SE. The size of the transcript is 3.7 kb, the major band seen in VEGF mRNA Northern analyses.

	DISCUSSION

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In the present study, linomide treatment did not cause tumor regression and was more effective as a chemopreventive rather than as a chemotherapeutic modality of treatment, similar to its impact on the progression of experimental prostatic cancer (30 , 31) . It is possible that a higher dosage of the drug might have resulted in a superior therapeutic effect; however, this could not be achieved because the mice will not ingest water with higher concentrations of linomide⁴ and parenteral administration is of reduced efficacy (18) . Two recent studies report the lack of effect of linomide in Phase II clinical trials in patients with advanced (metastatic) RCC (32 , 33) . In view of the angiostatic rather than angiocidal effect of linomide, as demonstrated in our studies and studies by others (30 , 31) , these results are not entirely unexpected. Patients at risk for VHL disease (i.e., carriers of VHL gene germ-line mutations) are subject to life-long tumor surveillance because of the high penetrance of the various tumor phenotypes in such mutation carriers. Therefore, it is expected that such VHL-related tumors will be detected very early in the future, at a clinical stage at which medical rather than surgical therapy might be contemplated. For example, VHL patients who develop RCC are not operated immediately but are followed by imaging and undergo tumor resection with nephron-sparing surgery only when the tumor size exceeds 3 cm in diameter (34) . The rationale for this clinical approach is the relative benign course of small tumors on the one hand (<3-cm diameter) and the inevitable need for recurrent surgery because of multicentricity of RCC in VHL patients, on the other (35) . In this setting, the antiangiogenic effect of linomide could prove effective in inhibition of RCC growth and possibly arrest tumor progression at a relatively early and indolent stage. Because our results are based on observations in a VHL paraganglioma, our results might also be relevant for sporadic pheo, because the VHL gene has been shown to be involved in a high proportion of such tumors (36) . Our findings require validation in other VHL tumor types, RCC in particular, because management of this tumor is most likely to benefit from early antiangiogenic therapy. This task may prove to be quite daunting, in light of the relative rarity of VHL disease coupled with the difficulty often encountered in establishment of primary human tumor xenografts.

In conclusion, our studies show that antiangiogenic medical therapy shows promise for hitherto medically untreatable VHL disease and might also be useful for unresectable sporadic pheo. This experimental system should serve as paradigm for treatment of other cancers in which VEGF-driven angiogenesis plays a role in the pathogenesis of the disease.

	FOOTNOTES

 
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.

¹ To whom requests for reprints should be addressed, at Department of Endocrinology and Metabolism, Hadassah University Hospital, P. O. Box 12000, Jerusalem, Israel 91120. Phone: 972-2-6777648; Fax: 972-2-6437940; E-mail: gross{at}vms.huji.ac.il

² Present address: Department of Pathology, Beth Israel-Deaconess Medical Center, Boston, MA 02275.

³ The abbreviations used are: VHL, von Hippel-Lindau disease; HB, hemangioblastoma; RCC, renal cell carcinoma; pheo, pheochromocytoma; VEGF, vascular endothelial growth factor; MRI, magnetic resonance imaging.

⁴ Unpublished observations.

Received 5/12/99; revised 8/13/99; accepted 8/16/99.

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