The angiogenesis inhibitor tnp-470 reduces the growth rate of human neuroblastoma in nude rats

ABSTRACT A new animal experimental model of human neuroblastoma is described. The model involves xenotransplantation of a poorly differentiated human neuroblastoma cell line (SH-SY5Y) to the

subcutaneous tissue in the hind leg of nude rats (WAG rnu/rnu). Injection of 20 million cells suspended in 0.2 mL of medium in each hind leg yielded an 89% tumor take (41/46) in 23 nude

rats. Tumor take was evident after 2 wk. The tumors grew exponentially and reached a volume of 5.2 ± 1.6 mL 4 wk after transplantation. The tumor cells retained their morphologic phenotype

at the ultrastructural level after transplantation and were immunohistochemically positive for neuron-specific enolase and for chromogranins A and B. Subcutaneous injections of the

angiogenesis inhibitor TNP-470 (10 mg/kg of body weight) every other day gave a treated/control quotient for mean tumor volume of 0.34 after 12 d of treatment. This implies that angiogenesis

inhibition may be of value as a complement to chemotherapy in the treatment of human neuroblastoma. The presented animal experimental model is designed for investigations of the effects of

chemotherapy, angiogenesis inhibitors, radiotherapy, and/or surgery on the growth rate of human neuroblastoma. SIMILAR CONTENT BEING VIEWED BY OTHERS EFFECTS OF A MONOCLONAL ANTIBODY AGAINST

(PRO)RENIN RECEPTOR ON GLIOMAGENESIS Article Open access 16 January 2023 ANGIOGENIN AND PLEXIN-B2 AXIS PROMOTES GLIOBLASTOMA PROGRESSION BY ENHANCING INVASION, VASCULAR ASSOCIATION,

PROLIFERATION AND SURVIVAL Article 13 April 2022 ANGIOGENIC SIGNALING PATHWAYS AND ANTI-ANGIOGENIC THERAPY FOR CANCER Article Open access 11 May 2023 MAIN Neuroblastoma is an embryonal

cancer derived from immature neurons of sympathetic ganglia and the adrenal medulla(1). It remains one of the most malignant cancers of infancy and early childhood. The annual incidence is

between six and eight cases per million children under the age of 15 y. The median age of onset is 2 y(2). Despite an aggressive multimodality treatment involving chemotherapy, radical

surgery, and radiotherapy, the prognosis of neuroblastoma is poor(3–5). Animal experimental models are therefore needed to develop more effective modes of treatment. Xenotransplantation of

human cancers to immunodeficient animals has been shown to constitute a reliable and reproducible experimental model(6, 7). The poorly differentiated neuroblastoma cell line SH-SY5Y is a

suitable cell line for such transplantation experiments, because it is stable and well characterized morphologically and functionally(8, 9). The nude rat is a suitable host for

transplantation, because it is more robust and less prone to infections than the nude mouse, well characterized immunologically(10), and of a size suitable for the experiments in question.

To create an animal model for studying new modes of treatment of neuroblastoma, we have transplanted neuroblastoma cells into immunodeficient rats of the strain WAG rnu/rnu. Neuroblastoma is

a rapidly growing, well vascularized tumor. The growth of solid tumors is considered to be angiogenesis-dependent(11, 12). The fumagillin analog TNP-470 (AGM-1470) is a specific inhibitor

of endothelial cell mitosis and a potent angiogenesis inhibitor(13–16). The aims of this study were to investigate _1_) the characteristics of the SH-SY5Y/WAG rnu/rnu xenotransplant model

and _2_) the effects of the angiogenesis inhibitor TNP-470 on the experimental tumors. METHODS _TNP-470_. TNP-470 was a kind gift of Takeda Chemical Industries Ltd., Osaka, Japan. Its

structure has already been reported(14). TNP-470 was stored at -20°C. For _in vitro_ study, it was dissolved first in DMSO, and then diluted with culture medium to a cascade of

concentrations from 10 μg/mL to 10 pg/mL. The final concentration of DMSO in test dishes was 0.1%. For _in vivo_ study, TNP-470 was suspended immediately before treatment in 1% ethanol and

5% gum arabic in saline to the appropriate concentration. _NEUROBLASTOMA CELLS_. The adrenergic neuroblastoma cell line SH-SY5Y(8, 17) was kindly provided by Dr. June Biedler, The Memorial

Sloan-Kettering Cancer Center, New York. The cells were grown at 37°C in Eagle's minimum essential medium (Labsystems, Stockholm, Sweden), supplemented with 10% FCS (Sigma Chemical Co.,

St. Louis, MO), 1 mM L-glutamine, penicillin (100 IU/mL), and streptomycin (50 μg/mL), in a humidified 95% air/5% CO2 atmosphere. The medium was changed twice a week, and confluent cultures

were subcultivated after 10 min of treatment with 0.25% trypsin and 0.02% EDTA. Cells were cultured on Nunclon Delta dishes(culture area 56.7 cm2, A/S Nunc, Roskilde, Denmark). To produce a

larger number of tumor cells, a confluent dish was seeded either on a Nunclon TripleFlask or a Nunc Bio-Assay dish, both with a culture area of 500 cm2. The cells were harvested when

confluent. They were found by culture and by DNA staining to be free of mycoplasms. For s.c. injections, two preparations were made. A single-cell suspension was prepared by treatment of the

culture with 0.25% trypsin and 0.02% EDTA for 10 min. To obtain a cell aggregate suspension, the cells were mechanically dispersed using a scraper (Cell Lifter, Costar Corporation,

Cambridge, MA). Both preparations were suspended and centrifuged at 300 × _g_ for 10 min. Before injection the pellet was suspended in culture medium. In the single-cell suspension, viable

(_i.e._ trypan blue-excluding) cells were counted in a hemocytometer to achieve a final concentration of 100× 106 cells/mL. For the cell aggregate suspension, the number of viable cells was

extrapolated by using confluent dishes and the same dilution as above. The suspension was placed on ice. The cell viability with time in the cell concentration used for injections was

investigated, as xenotransplantation may be hampered by low take rates. A low take rate can partly be explained by the rapid decline observed in the number of trypan blue-excluding cells

after 4 h. Trypsinization of the cultures yielded mainly free cells in suspension, whereas the mechanically dispersed cell preparations yielded both aggregates of cells and free cells.

_CYTOTOXICITY ASSAY_. Cytotoxicity of TNP-470 against the neuroblastoma cell line was assessed by a semiautomatic fluorometric microculture cytotoxicity assay(18). TNP-470 in dilution series

was added to cultures 1 d after plating, and the number of surviving cells relative to the number of cells in untreated control cultures (100%) was assessed 3 d later. _NUDE RATS_.

Twenty-three nude rats (WAG rnu/rnu; Animal Department, BMC, Uppsala University, Sweden), 11 males and 12 females, were used for xenografting at the age of 8-10 wk (BW, 108-178 g). The rats

were housed in an isolated room at a temperature of 25°C with a 12-h light, 12-h dark cycle. They were fed _ad libitum_ with water and radiation-sterilized food (R3, Lactamin, Stockholm,

Sweden). The rats were handled with gloves and a faceguard to avoid infections. The experiments were approved by the regional ethics committee for animal research. No animal was excluded

from the study, with the exclusion criteria applied, namely loss of weight at two consecutive measurements and the development of an open wound over the tumor. _XENOGRAFTING_. The animal was

anesthetized with 2% halothane(ISC Chemicals Ltd., Avonmouth, UK) supplemented with 50% N2O in oxygen. At injection a 23-gauge cannula was used, and care was taken to deposit the suspension

s.c. without piercing the muscle fascia, and not to lose cells by leakage from the injection site. A small, delineated wheal appeared at injection. Twenty million cells suspended in 0.2 mL

of medium were injected in each hind leg. _QUANTIFICATION OF TUMOR GROWTH_. When tumor take was evident on palpation and/or was visible, the animal was anesthetized with 2% halothane(ISC

Chemicals Ltd.) supplemented with 50% N2O in oxygen. The tumor length (along the tumor long axis), width (perpendicular to the long axis), and height (protrusion above the skin level) were

measured with a caliper by the same investigator throughout the experiments (E.W.). It was not possible to make accurate measurements without anesthesia. Measurements were made every other

day at 1000 h to noon. Tumor volume was calculated by length × width2 × 0.44. Several algorithms have been proposed for calculating volumes of s.c.-grafted tumors(19). The constant 0.44 was

deduced from our data (now comprising 112 tumors) and may be specific for our xenotransplant model only. Tumor height was also measured, but was not as reproducible and reliable as tumor

length and width (data not shown). The true tumor weight and volume were recorded at autopsy. _TREATMENT WITH TNP-470 AND CONTROL GROUP_. When a tumor in an animal had reached a volume of

0.3 mL (designated d 0), the animal randomized to one of two groups, which received injections of TNP-470 or no treatment(controls). If both tumors in an animal reached a volume of 0.3 mL on

the same day, both tumors were followed, and if not, only the largest one. Twelve animals (15 tumors), 6 males and 6 females, were treated for 12 d with TNP-470. This was injected s.c. once

every other day in the neck, in a dose of 10 mg/kg of BW. The site of injection was changed each time to avoid skin erosion. Eleven animals (13 tumors), 5 males and 6 females, served as

controls. _PERFUSION FIXATION AND AUTOPSY_. Animals were anesthetized by an intraperitoneal injection of sodium barbital (60 mg/kg of BW, Mebumal, ACO, Stockholm, Sweden) and subjected to

perfusion fixation or vascular casting on d 26-37 after xenotransplantation. Vascular casts will be considered in a separate communication. For perfusion fixation, the left femoral artery

was cannulated. The cannula was connected to a pressure-recorder, calibrated to record pressures between 0 and 200 mm Hg. One milliliter of saline with 300 IU of heparin was given through

the cannula to prevent intravascular coagulation and hypovolemia. The thoracic cage was opened, and a 16-gauge i.v. cannula(Viggo, Helsingborg, Sweden) was inserted in the thoracic aorta.

The right atrium was incised for free escape of blood. Perfusion fixation was performed with 2.5% glutaraldehyde in PBS, pH 7.3-7.4, 37°C, for transmission electron microscopy, or 4%

paraformaldehyde in Millonig's buffer, pH 7.3-7.4, 37°C, for light microscopy and immunohistochemistry. Perfusion was commenced within 2 min after opening of the thoracic cage. The

intraarterial pressure was kept at 100-140 mm Hg during perfusion. Usually, 350 mL of the fixative were sufficient for blood wash-out and fixation. After fixation, the tumors were dissected

out, and their weight and volume were recorded as described by Elias and Hyde(20). The thorax and abdomen were opened and checked for metastases. The liver and lungs were cut into 2-mm

slices and checked for macroscopic metastases, and the mesentery, spleen, and paraaortic lymph nodes were also inspected. Bone marrow biopsy specimens were taken from the iliac crest.

_IMMUNOHISTOCHEMISTRY_. Specimens perfusion-fixed in formaldehyde were dehydrated and embedded in paraffin. Sections were cut at 5 μm and put on diaminoalkyl-silane-treated glass slides(21),

deparaffinated, microwave-treated for 15 min (750 W) in citrate buffer, blocked in 0.3% hydrogen peroxide for 30 min, and blocked in 0.1% BSA for 30 min. The primary antibodies (NSE;

monoclonal mouse anti-human NSE, 1:50, DAKO-NSE H14, Dako A/S, Glostrup, Denmark. CgA; monoclonal mouse anti-human CgA, 1:300, catalog. no. 1199021, Boehringer Mannheim GmbH, Mannheim, FRG.

CgB; polyclonal rabbit anti-human CgB, 1:1000, Department of Clinical Chemistry, Uppsala, Sweden.) were applied in 0.1% BSA overnight. The secondary antibodies (NSE and CgA; polyclonal,

biotinylated rabbit anti-mouse immunoglobulins, 1:200, E 354, Dako A/S. CgB; polyclonal, biotinylated swine antirabbit immunoglobulins, 1:200, E 353, Dako A/S) were applied in 0.1% BSA for

30 min. Detection was with ABComplex/HRP (K 355, Dako A/S), 1:100 with 0.1% BSA for 30 min, followed by development with diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) and

counterstaining with hematoxylin. Sections from a human pancreas (NSE) or a human jejunum (CgA and CgB) served as positive controls, and omission of the primary antibody as a negative

control. When not stated otherwise, solutions were diluted in PBS. _STATISTICS_. Data were processed in Statistica 4.1 (StatSoft Inc., Tulsa, OK) for Macintosh on a Macintosh PowerBook 540c

personal computer. Differences between groups were analyzed with the Mann-Whitney _U_ test. RESULTS _NEUROBLASTOMA CELLS_. Karyotyping was done to ensure that no genotypic shift or

contamination had occurred during previous passages. Karyotyping was successful and identical in 10/18 metaphase plates (G-banding technique). The SH-SY5Y genotype was 47, XX, 1q+, 7q-, -22,

+2 marker chromosomes, which is in good agreement with the original report(17) and a more recent one(22). All SH-SY5Y cells in the present study are from passage 28. _IN VITRO CYTOTOXICITY

OF TNP-470_. The _in vitro_ cytotoxicity of TNP-470 was determined to ensure that the effect of TNP-470 is angiostatic and not cytotoxic. Tumor cell growth was inhibited first at 1 mg/mL.

Plasma concentrations of TNP-470 were not analyzed in our experiments, but plasma concentrations of TNP-470 in nude rats by reversed-phase HPLC after s.c. injections are less than 1 μg/mL

(Katsuichi Sudo, Takeda, data on file). _EXPERIMENTAL TUMORS_. The tumor take or growth did not differ when trypsinized or scraped cells were used (data not shown), and for simplicity

trypsinized cells are currently used. Subcutaneous injection of 20 million cells in 0.2 mL of medium produced tumors in 89% of the animals,_i.e._ in 41 of 46 injections. A 0.4-mL injection

(_i.e._ 40 million cells) did not result in a higher take rate (data not shown) and is therefore not justifiable, especially in view of the cost of raising so many cells. A larger volume

than 0.4 mL increased the risk of leakage from the injection site. There was no difference in tumor take or growth between animals injected in one hind leg and those injected in both (data

not shown), and consequently all animals were injected in both hind legs. The growth rate of the untreated experimental tumors is given in Fig. 1. Females tended to have a slightly later

(17.5± 3.5 d, _n_ = 13) tumor take (_i.e._ when tumors were≥0.3 mL in size) than males (14.9 ± 2.0 d, _n_ = 15)(_p_ = 0.04). After tumor take the growth rates were similar (data not shown).

_LIGHT MICROSCOPY_. Light microscopy showed that the tumors were separated from the skin by a thin (50-150 μm), highly vascularized connective tissue capsule (Fig. 2a). The tumors often grew

invasively into the superficial gluteal muscle and the biceps femoris muscle (Fig. 2b). The center of the tumors displayed apoptosis, necrosis, and hemorrhage. There was almost no

connective tissue. In tumors treated with TNP-470, the tumor cells formed cuffs (10-15 cell layers thick) around tumor vessels (Fig. 3a), a pattern not observed in controls (Fig. 3b).

Quantitative measurements showed that the fraction of apoptosis, necrosis, and hemorrhage in TNP-treated tumors was 48% after 12 d compared with 23% in controls (data not shown). Stereologic

quantification of microvascular counts showed a 36% decrease in vascular density in the viable part of the tumors (data not shown). _TRANSMISSION ELECTRON MICROSCOPY_. Transmission electron

microscopy was performed to ascertain the neuroendocrine nature of the tumor cells and showed that they had retained their ultrastructural characteristics after transplantation (Fig. 4).

_IMMUNOHISTOCHEMISTRY_. Immunohistochemistry demonstrated that SH-SY5Y cells both in culture and in the experimental tumors expressed NSE, CgA, and CgB. No regional or distant neuroblastoma

metastases were found at autopsy or by immunohistochemistry of lung tissue and bone marrow (iliac crest). _TREATMENT WITH TNP-470_. Treatment with TNP-470 resulted in a reduced tumor growth

rate (Fig. 1), and the T/C quotient was 0.34 after 12 d of treatment (Fig. 5). A dosage of 20 or 30 mg TNP-470/kg of BW resulted in a rapid loss of BW by more than 25% in a separate toxicity

study (data not shown). With the dosage of 10 mg of TNP-470/kg of BW used in the present study, the BW was retained, but did not increase. DISCUSSION There is a clinical need for more

effective treatment modalities of human neuroblastoma. Because tumor growth and metastasis are angiogenesis-dependent(12), it is important to investigate the effect of angiogenesis

inhibitors on the growth rate of neuroblastomas. An animal experimental model suitable for such experiments is described in detail in this report. It involves a stable, well defined human

neuroblastoma cell line, which is comparatively simple to culture and lacks N-_myc_ amplification. Clinically, N-_myc_ amplification has been associated with poor prognosis(23), and it can

be argued that the group of patients with N-_myc_-amplified tumors would benefit most from new treatment modalities. We did, however, intentionally chose the SH-SY5Y cell line for our animal

experimental model, because approximately 75% of all neuroblastomas lack N-_myc_ amplification(24). Also, the negative prognostic value of N-_myc_ amplification has recently been

questioned(25). The cells retain a high viability during xenotransplantation, as reflected in a high tumor take rate (89%). Other cell lines may be more resistant to storage and transport,

but in practice, harvesting of cells and injection of animals must be meticulously planned and made as rational as possible. The tumors grow exponentially and invasively in the host, and the

xenografted tumor cells retain their neuroblast phenotype. The nude rat is comparatively resistant to bacterial infections despite its immunodeficiency, and its size makes it suitable for

surgical procedures as well as for injections, blood sampling, irradiation, and physiologic investigations, for example. It is concluded that the presented model is relevant and yields

reproducible results. Metastases were not observed during the short study period. Whether this was due to primary tumor suppression of micrometastases(26), or whether SH-SY5Y does not

metastasize in this xenotransplant model, is at present not known. However, in a pilot experiment, excision of the primary tumor in three rats did not result in metastatic growth after 60 d.

Serum from 17 tumor-bearing rats has also been tested on bovine capillary endothelial cells _in vitro_ and had no effect on the proliferation of the endothelial cells (M. O'Reilly,

personal communication). This implies that SH-SY5Y does not metastasize. In this respect the model diverges from human neuroblastoma, which metastasizes early and extensively(1). It can be

argued that our model is less suitable due to its lack of metastases. This was again an intentional choice by us, because the lack of metastases is advantageous in that it permits simple

measurement of tumor volume with or without treatment, all other factors being equal. Hence, the model is relevant and designed for direct measurements of tumor volume. Inhibitors of

angiogenesis _in vitro_ and _in vivo_ have been developed(12, 26). As yet no “units of angiostatic activity” for comparisons of the potency of different angiogenesis inhibitors on the growth

rate of human tumors have been reported. We have chosen TNP-470 because it specifically inhibits endothelial cell growth(13–16), has a low toxicity(27, 28), and has been shown to have a

tumoristatic effect in animal experimental models involving different species, including rats(29). Other reasons for our choice are that no tachyphylaxis or resistance to the drug has been

observed (J. Folkman, personal communication) and that phase II clinical trials of TNP-470 in patients with solid tumors are currently being performed. Angiostatic treatment in pediatric

patients is likely to impair growth, but it must be kept in mind that current treatment regimens also impair growth and that TNP-470 is a representative of the first generation of

angiogenesis inhibitors. Treatment with TNP-470 resulted in a 66% reduction of neuroblastoma growth after 12 d (six injections) of treatment. This is the first report of an inhibitory effect

of TNP-470 on the growth rate of human neuroblastoma. A T/C quotient of 0.34 is similar to that reported for human and murine tumors with TNP-470(27). It is possible that a higher dose of

TNP-470 than 10 mg/kg of BW would reduce the T/C quotient further, but the“therapeutic window” for TNP-470 in immunodeficient rats was narrow. Also, the T/C quotient would have been reduced

further if the experiments had been prolonged, but this would reflect the expontential growth of the untreated tumors rather than an increasing efficiency of angiogenesis inhibition. The

tumor growth reduction observed is likely to be caused by inhibition of angiogenesis, because _1_) TNP-470 inhibits endothelial cell proliferation in picogram amounts(14), whereas the cell

line used was inhibited first at microgram amounts, and _2_) TNP-470-treated tumors exhibited a reduced micovascular density. Not only did TNP-470 reduce the micovascular density, but also

increased the fraction of necrosis. It is concluded that TNP-470 reduces the growth rate of human neuroblastoma in this xenotransplant model, and hence that angiogenesis inhibition may be a

valuable complement to chemotherapy in the treatment of human neuroblastoma. In summary, a new animal experimental model for human neuroblastoma is presented, and a reduction in tumor growth

rate after treatment with the angiogenesis inhibitor TNP-470 is documented. We will use this model to investigate the effects of cytotoxic drugs alone and in combination with TNP-470 and

other angiogenesis inhibitors, because recent studies(30) have shown synergistic effects of, and even tumor eradication with, angiogenesis inhibitors, cytotoxic drugs, and irradiation.

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references ACKNOWLEDGEMENTS Excellent technical assistance was provided by Barbro Einarsson, Ulrika Larsson, Lennart Lindström, Leif Ljung, and Marianne Ljungkvist. We are also grateful for

statistical advice of Stefan Matson. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Human Anatomy, University of Uppsala, Uppsala, Sweden Erik Wassberg & Rolf Christofferson

* Department of Medicine, University Hospital MAS, Malmö, Sweden Sven Påhlman * Department of Oncology, University Hospital, Uppsala, Sweden Erik Wassberg & Jan-Erik Westlin *

Department of Pediatric Surgery, Children's Academic Hospital, Uppsala, Sweden Rolf Christofferson Authors * Erik Wassberg View author publications You can also search for this author

inPubMed Google Scholar * Sven Påhlman View author publications You can also search for this author inPubMed Google Scholar * Jan-Erik Westlin View author publications You can also search

for this author inPubMed Google Scholar * Rolf Christofferson View author publications You can also search for this author inPubMed Google Scholar ADDITIONAL INFORMATION Supported by the

Swedish Medical Research Council (Project No. 00070), HRH Crown Princess Lovisa's Association for Child Medical Care, the Children Cancer Foundation of Sweden, the Lions Cancer Fund at

Akademiska Hospital, and funds from the Faculty of Medicine at Uppsala University. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Wassberg, E., Påhlman,

S., Westlin, JE. _et al._ The Angiogenesis Inhibitor TNP-470 Reduces the Growth Rate of Human Neuroblastoma in Nude Rats. _Pediatr Res_ 41, 327–333 (1997).

https://doi.org/10.1203/00006450-199703000-00004 Download citation * Received: 02 July 1996 * Accepted: 18 October 1996 * Issue Date: 01 March 1997 * DOI:

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