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Experimental Therapeutics, Preclinical Pharmacology |
Division of Oncology at The Children’s Hospital of Philadelphia and Department of Pediatrics at the University of Pennsylvania, Philadelphia, Pennsylvania 19104 [A. E. E., K. D. K., N. I., G. M. B.]; Cephalon, Inc., West Chester, Pennsylvania 19380 [A. E. E., K. D. K., N. I., A. M. C., C. A. D., G. M. B.]; and College of Physicians and Surgeons of Columbia University, Division of Pediatric Oncology, New York, New York 10032 [D. J. Y.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most NBL tumor cells express one or more of the tyrosine kinase receptors TrkA, TrkB, and/or TrkC, which bind the neurotrophin nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3, respectively. The level of expression of the individual Trk receptors may regulate growth, differentiation, and cell death in NBL tumors. The presence of high TrkA expression (with or without TrkC) is associated with a favorable outcome in NBL tumors (1, 2, 3, 4, 5, 6) . In contrast, however, TrkB is expressed in unfavorable NBL tumors, especially those with MYCN amplification (7) . MBLs also express receptors of the TRK family, and TrkC expression is associated with a favorable outcome (8 , 9) .
The Trk tyrosine kinase inhibitor CEP-751 (KT-6587) is an indolocarbazole derivative that preferentially inhibits autophosphorylation and signaling of Trk family receptors at nanomolar concentration in vitro (10) . The purpose of this study was to determine the effect of CEP-751 on human NBL and MBL cell lines growing as xenografts in nude (nu/nu) mice.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
The NBL lines were available from the CHOP cell bank, and the MBL lines D283-Med and D341-Med were obtained from Drs. Henry Friedman (Duke) and Peter Phillips (CHOP). The DAOY MBL line was obtained from the American Type Culture Collection. The cell lines were grown in RPMI 1640 containing 10% fetal bovine serum maintained in 75-cm2 Costar flasks in a humidified atmosphere of 5% CO2 and air. For in vivo studies, cells were grown in 150-cm2 culture flasks to 
75% confluence, split to insure they were growing in log phase, and harvested in 24 h. Cells were harvested using 0.2% tetrasodium EDTA in PBS.
Animals.
Four-week-old female athymic NCR (nu/nu) mice were obtained from the National Cancer Institute (Frederick, Maryland). The Institutional Animal Care and Use Committee of the Joseph Stokes, Jr. Research Institute at CHOP approved all animal studies. Mice were maintained at four per cage under humidity- and temperature-controlled conditions and a light/dark cycle that was set at 12-h intervals. All animals were fed autoclaved Purina mouse chow and water ad libitum.
In Vivo Antitumor Experiments.
Tumors were measured in unanesthetized animals two times a week using a Vernier caliper. Tumors were measured in three dimensions d1 x d2 x d3, and calculations of tumor volume were made by multiplying the product of three dimensions by 
/6. Body weights were obtained, and the dose of compound was modified as the weight of the animal changed. For NBL cell lines, 2.5 x 107 cells/inoculate in RPM1 1640 were injected s.c. in the flank, together with an equal volume of Matrigel (Becton Dickinson; Refs. 12
and 13
). In the MBL lines D341 and D283, 1 x 107 cells were injected in a similar fashion. The success rate of tumor development after injection with tumor cells was generally from 75 to 100%. However, DAOY cells required passage as a xenograft through mice before their growth was sufficiently rapid to be used in the in vivo study. Tumor tissue was minced, homogenized in a tissue homogenizer, and then transferred to 10-ml syringes and forced through hypodermic needles of decreasing size down to 22 gauge, prior to injection. Subsequently, the time required for tumors to appear decreased from 80 to 24 days.
Two study approaches were used: (a) the treatment was started after the s.c. tumor was palpable at 0.2 cm3 (therapeutic study). Animals were assigned to treatment or control groups by matching tumor sizes in pairs, usually when at least six animals had measurable tumors; and (b) the treatment was started 4–6 days after the implantation, before tumors were palpable (prevention study). The day of initiating treatment varied with the growth rate of the cell line; D341 had the shortest time to tumor appearance (7 days), and treatment was started day 4 from inoculation, whereas CHP-134 tumors appeared in 15–17 days, therefore treatment was started day 6. Treatment with CEP-751 or vehicle control was administered b.i.d., 7 days/week.
Trk Studies.
The level of Trk expression in the cell lines used was measured by a semiquantitative reverse transcription-PCR method.4
All NBL and MBL cell lines (except DAOY) expressed low levels of TrkA and/or TrkB. To assess the importance of Trk expression for responsiveness to CEP-751, the SY5Y cell line was transfected with TrkB (14)
in a constitutive expression vector (15)
.
Apoptosis Detection by the TUNEL Method.
The apoptosis detection system measures the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12 dUTP at the 3'-OH DNA ends using the enzyme terminal deoxynucleotidyl transferase, which forms a polymeric tail (TUNEL assay; Ref. 16
). Using tumor touch preparations, the cells harvested from tumors on days 2, 4, and 6 after treatment were fixed with formaldehyde (4% in PBS), permeabilized with Triton X-100, and incubated with equilibration buffer, nucleotide mix, and terminal deoxynucleotidyl transferase enzyme. After incubation, DAPI was added to stain the cells. Slides were analyzed at 460 nM to see the blue DAPI-stained cells and at 350 nm to visualize the green apoptotic cells. The proportion of the green cells in the blue background was calculated, and the results were compared between treated and control tumor.
Statistical Analysis.
The Exact Wilcoxon-Mann-Whitney test of significance (17)
was used to compare the tumor size at a particular test day between CEP-751 treated and control groups.5
Also, each subject’s individual slope was calculated assuming that tumor size is a linear function of time, and the slopes in the two groups (CEP-751 versus vehicle) were compared using Student’s t test (18)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The analysis of slope (average daily tumor growth rate) for the treated animals was 0.17 cm3/day ± 0.051 compared with the control rate of 0.29 cm3/day ± 0.11 (P = 0.032). Similar differences were also apparent at day 12 (Table 1
; Fig. 1A
).
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; Fig. 1B
). The analysis of slope was applied to the animals that died early, because there was more than one tumor measurement to document early growth rate. For the CEP-751-treated animals, the slope was 0.151 ± 0.055 cm3/day versus 0.299 ± 0.068 in controls, but this difference did not reach significance (P = 0.12).
Twenty animals were injected with NBL-S, and by day 13, 15 of 20 had developed small tumors that grew slowly. Treatment was started on day 38 after injection, when most of the animals had measurable tumors. Two animals with internal tumors were not included. Seven were assigned to the treatment group (average tumor size, 0.23 cm3; range, 0.19–0.34 cm3), and six were assigned to the control group (average, 0.31 cm3; range, 0.23–0.37). One animal in each arm died of accidental death. The remainder were analyzed at day 17 of treatment. The treated tumors were significantly smaller than controls (0.94 ± 0.24 versus 5.17 ± 2.46, P = 0.016; Table 1
and Fig. 1C
). Similarly, there was a significant difference in the slope of the two curves (0.051 ± 0.034 treated versus 0.213 ± 0.143, P = 0.016).
NBL Prevention Studies.
Fourteen animals were injected with 2.5 x 107 IMR-5 cells/animal. One control animal died of accidental death early in the study. Treatment with 21 mg/kg b.i.d. was started on day 5 from injection of IMR-5 cells, before the tumors appeared. In six control animals, the tumors appeared by day 13, and in the seven treated animals, the tumors appeared by day 17. However, the overall delay in the median growth of tumors was not significant (P = 0.27). On day 27, the average tumor size in seven treated animals was smaller at 1.3 cm3versus 2.7 cm3 in the six-control group (Table 1
; Fig. 1D
), but this difference did not reach statistical significance (P = 0.062). Also, the analysis of slope showed a clear trend (0.118 ± 0.036 treated versus 0.188 ± 0.041 control), but this also did not reach significance (P = 0.073).
Animals were injected with 2.5 x 107 CHP-134 cells, and treatment (21 mg/kg b.i.d.) was started on day 6 prior to the appearance of tumor. A total of 12 tumors developed (6 in each group). In the CEP-751-treated animals, they appeared between days 15 and 46 (median, day 29) and in the control group between days 9 and 25 (median, day 15). However, because the treated tumors grew more slowly, it was not possible to compare tumor size in treated versus control on a specific day of treatment. Furthermore, tumors developed at two substantially different rates; six developed early (days 9–21), and six developed later (days 22–46). By analyzing the slopes of the total group, the daily growth of treated tumors was 0.06 ± 0.05 versus 0.122 ± 0.02 in the controls (P = 0.058). Little effect of treatment was seen on the tumor growth slopes in the early group (0.148 versus 0.169 cm3/day; P = 0.28), but in the later group, treated tumors grew at a significantly slower rate (0.022 versus 0.140 cm3/day; P = 0.049; Table 1
; Fig. 1, E and F
).
MBL Therapeutic Studies.
Sixteen animals were injected with D341 MBL cells, and 12 developed tumors by day 10. These animals started treatment on day 14; the six tumors in the control group ranged from 0.03 to 0.29 cm3 (median, 0.14 cm3), and the treated group ranged from 0.07 to 0.31 cm3 (median, 0.15 cm3). Two tumors developed internally (one in each group) and grew very rapidly at parallel rates, reaching 6.8 cm3 in 10 days of treatment. Excluding these two, on day 15 the average tumor size in the five treated animals was 1.98 cm3, whereas the average in the five control animals was 2.24 cm3, (Table 2
and Fig. 2A
), but this difference was not statistically significant (P = 0.60). The analysis of slope (0.199 versus 0.238 cm3/day) also did not show a significant difference (P = 0.75).
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), and analysis of slope (0.15 versus 0.17 cm3/day) was not significant (P = 0.67; Table 2
).
Sixteen animals were inoculated with D283, and all grew tumors; 8 control tumors size ranged from 0.3 to 0.5 cm3 (median, 0.35 cm3), and 8 treated tumors measured 0.3–0.45 cm3 (median, 0.34 cm3). Tumors appeared by day 11, and treatment was started 6 days later. The growth rate of the control tumors was slow, and by day 39, the largest tumor measured 2.4 cm3 (range, 1.0–2.4 cm3). The difference between the treated versus control tumors (1.04 and 1.54 cm3) was significant (P = 0.031; Table 2
and Fig. 2B
), and the analysis of slope (0.017 versus 0.031 cm3/day) was also significant (P = 0.021).
MBL Prevention Studies.
Eighteen animals were injected with 1 x 107 D341 cells/animal, and tumors appeared between day 3 and 13. Treatment was started on day 4 from injection. On day 16, there was a difference between the average tumor size in the treated versus control animals (0.9 cm3 and 2.17 cm3, respectively) with marginal significance (P = 0.10). The analysis of slope (0.092 versus 0.186 cm3/day) did not reach statistical significance (P = 0.126; Table 2
; Fig. 2C
).
Mechanism of Antitumor Activity.
SY5Y, an NBL line that expresses low levels of TrkB, was transfected with TrkB. Two of the resulting clones, G8 and G12 expressing low and high levels of TrkB, respectively, were inoculated into nude mice (15)
. Interestingly, the SY5Y-G12 clone expressing the highest level of TrkB had the fastest growth rate in untreated or vehicle-treated tumors, and the effect of CEP-751 was the greatest. The daily growth rates of SY5Y, SY5Y-G8, and SY5Y-G12 were 0.16, 0.20, and 0.21 cm3/day, and the significant difference between treated and control tumor size was 0.4, 0.1, and 0.01 cm3, respectively (Fig. 3)
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. A rare cell was seen with florescent staining on day 2 (0–3% of cells per high power field) and increasing numbers on days 4 (42%) and 6 (92%). No evidence of apoptosis was seen in the control group. The tumors prepared for TUNEL staining were also studied by light microscopy. In the treated tumors, there was no evidence of necrosis or maturation of the tumor.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CEP-751 is a relatively specific inhibitor of the Trk family of tyrosine kinases (TrkA, TrkB, and TrkC) at low concentrations. Indeed, this agent was shown to have antitumor activity both in vitro and in vivo in prostate cancer, ovarian cancer, and melanoma (10 , 11 , 22) . Given the important roles that members of the Trk family play in NBLs, this tumor appeared to be a very attractive candidate for treatment with CEP-751.
We tested the effect of this compound on four NBL cell lines growing as xenografts in nude mice, either by treating palpable tumors or by trying to prevent tumor development after inoculation. In the therapeutic studies, treatment with CEP-751 resulted in a statistically significant decrease in tumor growth in three lines (IMR-5, CHP-134, and NBL-S) in animals. In both models, the drug had no apparent effect for the first 7 days; then a substantial difference in the slope of tumor growth was observed. In the prevention studies, there was a noticeable delay in the development of tumors in the treated animals versus the controls. In the CHP-134 animals, the delay in appearance of tumors was so pronounced that many of the control animals had died from their tumor burden before the treated animals developed palpable tumors.
To determine to what extent the level of Trk receptors played a role in the degree of response, SY5Y cells expressing very low levels of TrkB were transfected with TrkB. Cells from two of the resulting clones were grown in nude mice, which were then treated with CEP-751. The SY5Y-G12 clone with the highest expression of TrkB was the most sensitive to treatment; therefore, the effect of CEP-751 may be mediated at least in part through its inhibition of TrkB.
It has been suggested that Trk expression supports not only the growth and differentiation of neuronal cells but is also important for cell survival. If Trk receptors and/or their ligands are involved in a survival pathway for NBL cells, treatment with CEP-751 would be expected to result in increased cell death. To examine this hypothesis, tumors were harvested at several times posttreatment and were examined for evidence of apoptotic cells using the TUNEL assay. There was no apparent difference after 1 day of treatment; however, there were more apoptotic cells in the treated tumors after 4 and 6 days compared with the corresponding controls. These results suggest that CEP-751 may induce apoptosis in these tumors, presumably as a consequence of blocking the autophosphorylation and signaling of the Trk receptor(s) being expressed.
MBLs are neuroectodermal tumors of the central nervous system. Recent studies have suggested that these tumors also express members of the Trk family, and high expression of TrkC was correlated with improved survival (8 , 9) . Therefore, we tested the effect of CEP-751 on three MBL cell lines growing as xenografts in nude mice in vivo. The tumors treated with CEP-751 were generally smaller than those in the control group. Studies of D283, which grew more slowly, did show a therapeutic effect of the compound. The difference in D341 was not statistically significant, and a difference in DAOY was not seen. Two of the three lines showed minimal TrkA expression, and DAOY expressed none. Thus, its effect in D283 could be attributable to the effect of CEP-751 on TrkA or on other tyrosine kinases. Additional studies are required to fully characterize these possibilities.
Although CEP-751 was developed because of its ability to inhibit autophosphorylation and signaling of Trk receptors, it also inhibits platelet-derived growth factor, epidermal growth factor receptor, and protein kinase C activity (10) and may inhibit other, as yet unidentified tyrosine, serine, or threonine kinases as well. Furthermore, because this is a small, lipophilic molecule, it should enter cells readily and could have wider effects than its ability to inhibit selected tyrosine kinases. Despite the potential for nonselective activity, the toxicity of this compound appears to be minimal at these concentrations. The compound has a short half-life in animals, requiring the twice-daily treatment, but the half-life in humans is much longer, which may enhance its therapeutic effect (23) . Also CEP-701, an analogue of CEP-751, is presently in Phase I clinical trials in the United States for solid tumors in adults (24 , 25) .6 CEP-701 is effective when given p.o., making it an attractive candidate for use in a pediatric population.
In summary, CEP-751 reduced in vivo growth of NBL cells growing as xenografts using two different treatment paradigms. It also resulted in a decrease in growth of MBL xenografts, with borderline significance. Evidence of in vivo antitumor efficacy using established NBL cell lines suggests that this compound may be even more effective in primary tumors from untreated patients, which express higher levels of Trk family receptors than cell lines. Furthermore, the apparent greater efficacy in slower growing tumors and in retarding tumor appearance suggests that its therapeutic efficacy may be greater in states of minimal residual disease. Thus, CEP-751, or potential derivatives of this compound, appear promising as therapeutic agents for tumors such as NBLs and possibly MBLs, either alone or in combination with other agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NIH Grant NS-34514 (to G. M. B.), a grant from the Audrey E. Evans Endowed Chair (to G. M. B.), and funds from the Children’s Cancer Research Foundation. 
2 To whom requests for reprints should be addressed, at Division of Oncology, Children’s Hospital of Philadelphia, Abramson Research Center, Room 902-D, 3516 Civic Center Boulevard, Philadelphia, PA 19104-4318; Phone: (215) 590-2817; Fax: (215) 590-3770; E-mail: brodeur{at}email.chop.edu
3 The abbreviations used are: NBL, neuroblastoma; MBL, medulloblastoma; CHOP, Children’s Hospital of Philadelphia; b.i.d., twice a day; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; DAPI, 4,6-diamidino-2-phenylindole.
4 N. Ikegaki et al., unpublished observations.
5 The software used to perform this test was authored by C. R. Mehta and N. R. Patel, entitled "StatXact3 for Windows: Statistical Software for Exact Nonparametric Inference. User Manual," pp. 216–226. Cambridge, MA: CYTEL Software Corporation, 1995.
6 S. J. Miknyoczki, A. Klein-Szanto, C. A. Dionne, and B. A. Ruggeri. The Trk tyrosine kinase inhibitor CEP-701 (KT-5555) exhibits significant antitumor efficacy in preclinical xenograft models of human pancreatic ductal adenocarcinoma, submitted for publication.
Received 11/25/98; revised 6/ 2/99; accepted 6/25/99.
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