[CANCER RESEARCH 61, 1527–1532, February 15, 2001]
`
`Antitumor Activity of the Rapamycin Analog CCI-779 in Human Primitive
`Neuroectodermal Tumor/Medulloblastoma Models as Single Agent and in
`Combination Chemotherapy1
`Birgit Geoerger, Karol Kerr, Cheng-Bi Tang, Kar-Ming Fung, Bruce Powell, Leslie N. Sutton, Peter C. Phillips, and
`Anna J. Janss2
`Division of Neuro-Oncology [B. G., K. K., C-B. T., P. C. P., A. J. J.], Department of Neurosurgery [L. N. S.], and Pathology [B. P.], Children’s Hospital of Philadelphia, and
`Department of Pathology [K-M. F.], Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104
`
`ABSTRACT
`
`We examined the cytotoxicity of the immunosuppressant agent rapa-
`mycin and its analogue CCI-779 in human brain tumor cell lines in vitro
`and in vivo as single agents and in combination with standard chemother-
`apeutic drugs. In the rapamycin-sensitive PNET/MB cell line DAOY,
`rapamycin exhibited additive cytotoxicity with cisplatin and with camp-
`tothecin. In vivo, CCI-779 delayed DAOY xenograft growth by 160% after
`1 week and 240% after 2 weeks of systemic treatment, compared with
`controls. Single high-dose treatment induced 37% regression of tumor
`volume. Growth inhibition of DAOY xenografts was 1.3 times greater
`after simultaneous treatment with CCI-779 and cisplatin than after cis-
`platin alone. Interestingly, CCI-779 also produced growth inhibition of
`xenografts derived from U251 malignant glioma cells, a human cell line
`resistant to rapamycin in vitro. These studies suggest that the rapamycin
`analogue CCI-779 is an important new agent to investigate in the treat-
`ment of human brain tumors, particularly PNET/MB.
`
`INTRODUCTION
`
`Rapamycin is an immunosuppressant agent that arrests cells in the
`G1 phase of the cell cycle and induces apoptosis. This carbocyclic,
`lactone-lactam macrolide antibiotic is pharmacologically active
`through binding to ubiquitous, predominantly cytosolic immunophilin
`receptors (e.g., FK506-binding protein-12). Binding of rapamycin to
`the mammalian target of rapamycin, also known as RAFT 1, RAPT 1,
`and FRAP (1–3), inhibits its kinase activity and subsequently de-
`creases phosphorylation and activation of p70 S6 kinase, translation of
`mRNA-encoding ribosomal proteins and elongation factors [eukary-
`otic initiation factor 4E binding protein (pH acid stable protein I) and
`eEF-2], and enzymatic activity of the cyclin-dependent kinase cdk2-
`cyclin E complex, resulting in a mid-to-late G1 cell cycle arrest (4–8).
`Antitumor activity of rapamycin as a single agent has been de-
`scribed in vitro and in vivo (9–13). Coadministration enhanced cis-
`platin-induced apoptosis in human cell lines (14) and produced addi-
`tive cytotoxicity with 5-fluouracil and cyclophosphamide in a Colon
`38 tumor model (13).
`PNET/MB3 are the most common malignant brain tumors in child-
`hood. With current treatment, including radio- and chemotherapy
`subsequent
`to surgical
`resection,
`the 5-year survival
`rate for
`PNET/MB exceeds 80% (15, 16). However, 30–50% of all patients
`experience tumor progression or late relapse with very low salvage
`rates (17). Therefore, improving the long-term survival of patients
`
`Received 6/29/00; accepted 12/15/00.
`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 B. G. is supported by the NIH (Grant POI-NS 34514), by the Dr. Mildred Scheel
`Stiftung Deutsche Krebshilfe e.V., and by the Jeffrey Miller Neuro-Oncology Research
`Fund.
`2 To whom requests for reprints should be addressed, at Children’s Hospital of
`Philadelphia, Division of Neuro-Oncology, 3400 Civic Center Boulevard, Philadelphia,
`PA 19104. Phone: (215) 590-5170; Fax: (215) 590-3709; E-mail: janss@email.chop.edu.
`3 The abbreviations used are: PNET/MB, primitive neuroectodermal tumor/medullo-
`blastoma; CPT, camptothecin; TUNEL, terminal deoxynucleotidyl transferase-mediated
`dioxygenin-11-dUTP nick end labeling.
`
`with high risk PNET/MB will require therapeutic strategies that
`augment the effects of current treatment.
`Rapamycin and its analogue, CCI-779, are attractive candidates for
`brain tumor therapy. Their unusual mechanism of action make it
`unlikely that
`these agents will
`interfere with the cytotoxicity of
`standard chemotherapeutic agents or exhibit cross-resistance. They
`are highly lipophilic (18) and thus able to penetrate the blood-brain
`barrier. Finally, rapamycin has minimal systemic toxicity in animals
`or humans (19–22). In vitro studies in our laboratory find that brain
`tumor cell lines can be exquisitely sensitive to rapamycin. For exam-
`ple, the PNET/MB cell line DAOY has an ID90 ⫽102 ng/ml. In
`contrast, the PNET/MB D283 and malignant glioma U251 cell lines
`are highly resistant, with ID90 values of 1.8 ⫻ 106 and 3.6 ⫻ 1019
`ng/ml, respectively.4
`Here, we describe preclinical investigations of rapamycin and CCI-
`779 in treatment of human brain tumors. The cytotoxicity of rapamy-
`cin was measured using brain tumor cell lines in culture; the cytotox-
`icity of CCI-770 was measured using s.c. brain tumor xenografts and
`in combination with cisplatin and CPT. Our results indicated that
`rapamycin in vitro and CCI-779 in vivo exhibited additive cytotoxicity
`in PNET/MB cells when combined with cisplatin or CPT. In vivo
`studies showed that the same dose of CCI-779 given in daily injec-
`tions over 2 weeks produced greater tumor suppression than when
`given as a single injection. Four weeks of daily CCI-779 injections
`were not superior to 2 weeks in the induction of tumor regression and
`growth retardation of human PNET/MB xenografts. Finally, CCI-779
`suppressed the growth of human U251 malignant glioma cells in vivo
`despite the resistance of the cell line to rapamycin cytotoxicity in
`vitro.
`
`MATERIALS AND METHODS
`
`Cell Cultures. DAOY and D283 human medulloblastoma cells were pur-
`chased from the American Tissue Culture Collection (Rockville, MD), and the
`glioma cell line U251 was kindly supplied by Dr. Henry Friedman (Duke
`University, Durham, NC). The cell lines were grown in Richter’s Zinc Option
`Media (Life Technologies, Inc.) supplemented with 10% FCS (Sigma, St.
`Louis, MO) and incubated at 37°C in humidified 5% CO2 (23).
`Drugs. Rapamycin and CCI-779 were obtained from Wyeth-Ayerst Re-
`search Laboratories (Princeton, NJ), CPT from Smith-Kline Beecham Phar-
`maceuticals, and cisplatin from Sigma Pharmaceuticals. Rapamycin and CCI-
`779 were stored as dry powder at ⫺20°C and suspended in 100% ethanol the
`day of use. CPT was stored as a 5-mM solution in DMSO and cisplatin as a
`5-mM solution in sterile water at ⫺20°C.
`In Vitro Assay for Cytotoxicity. Cytotoxicity of rapamycin, CPT, and
`cisplatin were assessed for unsynchronized DAOY and D283 cells by MTS
`tetrazolium Cell Titer 96 AQ Non-Radioactive Cell Proliferation assay (Pro-
`mega, Madison, WI). DAOY and D283 cells were plated in 96-well plates, 500
`cells/well. After 24 h, rapamycin, CPT, and cisplatin, diluted in serum-free
`Zinc Option Medium immediately before use, were added to the media as
`single agents or in combination (rapamycin plus CPT or rapamycin plus
`
`4 Unpublished data.
`
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`cisplatin). Each drug dose or combined drug dose was repeated five times.
`After 5 days of drug exposure, MTS solution was added to each well, and
`absorbance was measured at 490 nm. Experimental absorbance was computed
`by subtracting the absorbance of blank wells (media only). Fractional survival
`was computed as: experimental absorbance (mean of 5 trials/dose or dose
`combination)/absorbance for cells treated with drug free media (mean of 15
`trials).
`Analysis of in Vitro Drug Interaction. An additivity model was used as
`described previously (24). The additive cytotoxicity of a drug/dose combina-
`tion is defined as the sum of cell kill for each agent tested as a single drug, with
`a 90% confidence interval for this sum. Antagonistic cytotoxicity is defined as
`a drug interaction that caused less cytotoxicity than the sum of each agent used
`alone (i.e., less than additivity), and synergy is defined as the combined effect
`of drugs that is greater than the sum of the cytotoxicity for each agent used
`alone (i.e., greater than additivity). The treatment effect for each agent at a
`given dose was identified by means of single-agent dose-effect curves (25, 26).
`Cytotoxicity of single agents was characterized further by fitting dose-response
`curves to a linear model by means of regression analysis with transformations
`of the response (i.e., fractional survival) and the log of the dose. These
`transformations permit the calculation of 95% confidence intervals for single
`agent dose-response curves and the subsequent calculation of a 90% confi-
`dence interval for the expected additive cytotoxicity. Drug interaction was
`characterized by comparing the observed cytotoxicity of a drug combination
`with the expected additive cytotoxicity.
`In Vivo Human DAOY-MB and U251 Malignant Glioma Xenograft
`Models. Female athymic nude mice 4–6 weeks of age were purchased from
`the National Cancer Institute (Frederick, MD) and maintained in filter-top
`cages in an aseptic environment with laminar flow filtered ventilation at the
`Laboratory Animal Facility of the Joseph Stokes, Jr., Research Institute of the
`Children’s Hospital of Philadelphia. DAOY-MB xenograft experiments were
`performed with DAOY tumors that had undergone at least eight serial passages
`as xenograft tumors in the flanks of nude mice. The flank xenograft tumors of
`stock animals were dissected free of connective tissue and external vasculature
`in a sterile laminar flow hood. The tumors were coarsely minced and then
`passed through a 20-mesh screen in a tissue press. The xenograft homogenate
`was passed sequentially through a 20-, 21-, 22-, and 23-gauge needle. The
`tumor homogenate (0.15 ml) was injected s.c. through a 23-gauge needle into
`the right flank of each nude mouse. For U251 xenografts, U251 cells grown in
`in vitro cell cultures were harvested in log-phase and resuspended in Matrigel
`(Life Technologies, Inc.). Cells (1 ⫻ 107) in 0.15 ml per animal were injected
`into the right flank of athymic nude mice. The length and width (mm) of s.c.
`tumors were measured every 2–3 days with Vernier calipers (Fischer Scienti-
`fics). Tumor volume was calculated by the following formula: width (2) ⫻
`length/2 (27).
`Treatment with CCI-779 and/or Cisplatin. Nude mice bearing s.c.
`DAOY flank xenograft tumors were treated starting on day 10; animals bearing
`U251 xenografts were treated starting 3 weeks after tumor implantation. Stock
`dilution of CCI-779 50 mg/kg was diluted to a final concentration of 2 mg/ml
`using a diluent of 5% Tween 80 and 5% polyethylene glycol 400. The efficacy
`of different treatment schedules was tested in DAOY/MB xenografts. Each
`treatment group consisted of five to six animals. CCI-779 (20 mg/kg) was
`administered daily ⫻ 5 for 1, 2 or 4 weeks; or 100 mg/kg of CCI-779
`was administered on days 1 and 12. Drug interaction of CCI-779 in vivo was
`studied by injection of 5 mg/kg cisplatin as a single dose, with or without daily
`treatment of 20 mg/kg CCI-779 ⫻ 5 for 2 weeks. U251 xenograft tumors were
`treated with daily with 20 mg/kg CCI-779 ⫻ 5 for 2 weeks. Control animals
`bearing s.c. flank tumors were treated with diluent at equivalent doses and
`schedule.
`Tumor volumes were measured serially and normalized by the index of
`observed tumor volume in comparison to initial, pretreatment tumor volume
`(Vo/Vi). Growth delay end points were defined as the posttreatment interval at
`which tumor volume increased by 5-fold (27). The Wilcoxon-Gehan Test was
`used to evaluate differences in time to reach 5-fold initial tumor volume (28).
`Histology. Nude mice with DAOY-MB xenografts were treated on day 10
`after tumor inoculation with either 100 mg/kg CCI-779, 5 mg/kg cisplatin, or
`both, or diluent, as described above in treatment studies. Animals were killed
`electively 4 days after this treatment. Flank tumors were harvested, fixed in
`10% formalin, paraffin-embedded and cut into microsections. Sections were
`stained with H&E for morphology.
`
`Proliferation. Immunohistochemistry for Ki-67 antigen was performed to
`determine the proliferation indices of the xenograft tumors. A monoclonal
`MIB-1 antibody (1:50; Immunotech, Marseilles, France) was used, detected by
`the Animal Research Kit (ARK; DAKO Corporation, Carpinteria, CA) accord-
`ing to the manufacturer’s instructions, with biotinylated Fab antimouse anti-
`body, Streptavidin-horseradish peroxidase, and diaminobenzidine tetrahydro-
`chloride (DAKO Corporation). For quantification, tumor cells were counted at
`⫻1000. The proliferation index was expressed as the number of MIB-1
`positive cells/100 neoplastic cells.
`Apoptosis. TUNEL was performed to quantify apoptotic cell death. A
`modified protocol according to Gavrieli et al. (29) was used. In brief, tissue
`sections were deparaffinized, rehydrated, and treated with 10 ␮g/ml Proteinase
`K (Boehringer Mannheim, Indianapolis, IN) for 15 min at room temperature.
`DNA strand breaks were labeled by polymerization of 30 ␮M digoxigenin-11-
`dUTP (Boehringer Mannheim, Indianapolis, IN) to the 3⬘-OH sites, catalyzed
`by 0.3 units/␮l terminal deoxynucleotidyl transferase (Boehringer Mannheim,
`Indianapolis, IN) for 45 min at 37°C. Labeled DNA breaks were revealed by
`anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (Boeh-
`ringer Mannheim) at a dilution of 1:200. The labeled DNA strands were
`visualized using Fast Red (Sigma, St. Louis, MO). Terminal deoxynucleotidyl
`transferase or the biotinylated dUTP was omitted for negative controls; sec-
`tions of a postnatal day-8 rat brain were used for positive controls. To quantify
`TUNEL-positive cells, at least 2000 tumor cells were counted at 400X mag-
`nification. Labeled cells displaying compaction or segregation of the nuclear
`chromatin, or breaking up of the nucleus into discrete fragments, were counted
`as apoptotic cells. Labeled cells adjacent to or within necrotic areas were not
`counted. The apoptotic index (AI) was expressed as the number of TUNEL-
`positive cells/100 tumor cells.
`
`RESULTS
`
`Rapamycin Had Additive Effects with Cisplatin or CPT in
`Human PNET/MB Cell Lines in Vitro. DAOY cells incubated with
`rapamycin for 96 h demonstrated a dose-dependent reduction of cell
`viability that reached maximal effects at 1 ng/ml (Fig. 1A). We
`selected concentrations of rapamycin from 0.001–1 ng/ml to evaluate
`drug interaction with cisplatin and CPT. Rapamycin augmented cis-
`platin- and CPT-induced cytotoxicity in DAOY/MB cells in a dose-
`dependent fashion. To evaluate drug interaction objectively, the ad-
`ditivity model described in “Materials and Methods” was used; r2
`values for single-agent regression curves were 0.867, 0.941, and 0.888
`for survival curves of rapamycin, cisplatin, and CPT, respectively.
`Using this model, simultaneous treatment of DAOY/MB cells with
`rapamycin and cisplatin resulted in additive cytotoxicity; the efficacy
`of cisplatin and 1 ng/ml rapamycin was increased 100-fold relative to
`cisplatin alone (Fig. 1B). Similar results were observed when rapa-
`mycin was combined with CPT (Fig. 1C).
`Rapamycin did not augment the cytotoxicity of cisplatin or CPT in
`in vitro studies using D283 PNET/MB cells, a cell line resistant to
`rapamycin (Fig. 2 A and B).
`CCI-779 Induced Growth Delay in DAOY Xenografts. To eval-
`uate the antitumor activity of CCI-779 in DAOY flank xenografts,
`athymic nude mice were treated with four different treatment sched-
`ules. Results of these studies are summarized in Table 1. CCI-779
`administered at 20 mg/kg 5 days/week for 1 and 2 weeks yielded 1.6-
`and 2.4-fold delayed tumor growth (Fig. 3). Time to reach 5-fold
`tumor volume was significantly greater in animals treated for 1 week
`or 2 weeks compared with control animals (see Table 1). Retreatment
`of large tumors with CCI-779 for 2 weeks (20 mg/kg i.p. 5 days/week
`on days 29 to 42) restored growth inhibition but did not yield tumor
`regression (Fig. 3). Treatment with CCI-779 (20 mg/kg i.p.) 5 days/
`week for 4 weeks delayed time to reach 5-fold pretreatment volume
`by 174% compared with controls (see Table 1). Thus, prolonged
`treatment is not more efficacious than 2-week treatment. A growth
`delay ⬎50 days was observed in 20% of animals treated 2 or 4 weeks
`but not in those treated 1 week.
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`No significant toxicities were seen in this higher-dosed treatment
`group, however, weight loss ⬎10% initial body weight and dermatitis
`were observed in mice treated daily; the percentage of animals ex-
`hibiting toxicities increased with the duration of treatment. None of
`the animals died of chemotherapeutic-induced toxicity.
`CCI-779 Induced Growth Delay in U251 Malignant Glioma
`Xenografts. To determine whether the resistance to rapamycin ob-
`served in vitro occurs in vivo, we used xenografts derived from U251
`malignant glioma cells, a cell line resistant to rapamycin in vitro but
`reportedly sensitive in vivo (12). Animals bearing U251 tumor xe-
`nografts were treated with CCI-779 (20 mg/kg i.p. 5 days/week) for 2
`weeks. As shown in Table 1 and Fig. 5, time to grow to 5-fold
`pretreatment tumor volume was 40 days with treatment compared
`with 27 days in control animals, a delay of 148%.
`CCI-779 and Cisplatin Induced Antitumor Activity in DAOY
`Xenografts. To evaluate the interaction of CCI-779 with cisplatin in
`vivo, we treated mice bearing DAOY xenografts with cisplatin (5
`mg/kg i.p. once) alone or in combination with CCI-779 (20 mg/kg i.p.
`5 days/week) for 2 weeks. Time to reach 5-fold pretreatment volume
`was 10, 21, and 27 days in control mice, mice treated with cisplatin
`alone, and mice treated with cisplatin plus CCI-779, respectively (see
`Table 1 and Fig. 6).
`
`Fig. 1. DAOY sensitivity to cytotoxic effects of rapamycin in vitro. A, dose-response
`curve of rapamycin as a single agent. Fractional survival of DAOY cells determined by
`3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay is plotted against the
`dose of rapamycin (log scale). Data represent the mean of five values. Bars, SE. B,
`dose-response curves of rapamycin and cisplatin. Fractional survival of DAOY cells
`incubated with cisplatin alone (f), with cisplatin and rapamycin 0.001 ng/ml (‚),
`rapamycin 0.01 ng/ml (Œ), rapamycin 0.1 ng/ml (E), or rapamycin 1 ng/ml (F). Simul-
`taneous treatment of DAOY cells with cisplatin and rapamycin increases cytotoxic
`activity in a dose-dependent and additive fashion. Data are presented as the mean of five
`values. Bars, SE. C, dose-response curves of rapamycin and camptothecin. Fractional
`survival of DAOY cells incubated with CPT alone (f), with CPT and rapamycin 0.001
`ng/ml (‚), rapamycin 0.01 ng/ml (Œ), rapamycin 0.1 ng/ml (E), or rapamycin 1 ng/ml
`(F). Simultaneous treatment of DAOY cells with camptothecin and rapamycin increases
`cytotoxic activity in a dose-dependent and additive fashion. Data are presented as the
`mean of five values. Bars, SE.
`
`Treatment with a single high dose CCI-779 (100 mg/kg i.p) induced
`DAOY xenograft regression of 37% compared with initial tumor
`volume within 1 week (Fig. 4). However, unlike tumors treated with
`daily CCI-779, tumors treated with the single high dose resumed
`growth after 1 week. Animals bearing these tumors were given a
`second treatment with 100 mg/kg i.p. on day 12, but did not achieve
`a second growth regression. Time to reach 5 ⫻ pretreatment volume
`was 14 days in the treatment group compared with 10.5 days in
`controls (see Table 1).
`
`Fig. 2. D283 nonsensitivity to cytotoxic effects of rapamycin in vitro. A, dose-response
`curve of rapamycin and cisplatin. Fractional survival of D283 cells (derived from human
`PNET/MB) incubated with cisplatin alone (f), with cisplatin and rapamycin 0.01 ng/ml
`(Œ), rapamycin 0.1 ng/ml (E), or rapamycin 1 ng/ml (F) or rapamycin 10 ng/ml (⫻).
`Simultaneous treatment of D283 cells with cisplatin and rapamycin did not increase the
`cytotoxic activity of cisplatin alone. Data represent the mean of five values. Bars, SE. B,
`dose-response curves of rapamycin and camptothecin. Fractional survival of D283 cells
`incubated with cisplatin alone (f) with cisplatin and rapamycin 0.01 ng/ml (Œ), rapamy-
`cin 0.1 ng/ml (E), or rapamycin 1 ng/ml (F) or rapamycin 10 ng/ml (⫻). Simultaneous
`treatment of D283-MB cells with camptothecin and rapamycin did not increase the toxic
`activity of camptothecin alone. Data are presented as the mean of five values. Bars, SE.
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`Table 1 Summary of human brain tumor xenograft studies
`
`Xenograft
`cell line
`DAOY
`
`DAOY
`
`Dose
`
`Treatment agenta
`0.25 cc i.p.
`Control
`20 mg/kg i.p.
`CCI-779
`20 mg/kg i.p.
`CCI-779
`0.25 cc i.p.
`Control
`20 mg/kg i.p.
`CCI-779
`1.5 cc i.p.
`Control
`100 mg/kg i.p.
`CCI-779
`0.25 cc i.p.
`Control
`5 mg/kg i.p.
`Cisplatin
`5 mg/kg i.p. ⫹ 20 mg/kg i.p.
`Cisplatin ⫹ CCI-779
`0.25 cc i.p.
`Control
`5 mg/kg i.p.
`CCI-779
`a Refer to “Materials and Methods” for details of treatment.
`b Experimental group compared with relevant control.
`
`DAOY
`
`DAOY
`
`U251
`
`Schedule
`5 days/wk–2 wk
`5 days/wk–1 wk
`5 days/wk–2 wk
`5 days/wk–4 wk
`5 days/wk–4 wk
`Day 1
`Day 1
`5 days/wk–2 wk
`Day 1
`Day 1 ⫹ 5 days/wk–2 wk
`5 days/wk–2 wk
`5 days/wk–2 wk
`
`n
`5
`6
`6
`4
`6
`6
`6
`5
`5
`5
`4
`6
`
`Days to 5-fold
`pretreatment tumor
`volume
`15
`24
`36
`11.5
`20
`10.5
`14
`10
`21
`27
`27
`40
`
`Range (days)
`16–24
`20–30
`25–⬎50
`10.5–16
`12–⬎50
`9–13
`10–21
`9–12
`14–30
`19–32
`17.5–40
`32–⬎50
`
`Pb
`
`⬍0.05
`⬍0.0005
`
`⬍0.05
`
`⬍0.05
`
`⬍0.05
`⬍0.005
`
`0.07
`
`CCI-779 Induced Tumor Cell Death in Human PNET/MB
`Xenografts. Histological inspection showed that DAOY xenografts
`from control mice were composed of densely packed cells with
`hyperchromatic round-to-oval nuclei, nuclei surrounded by scant cy-
`toplasm, and frequent mitotic figures, as many as 20 mitotic figures/
`field using a 40 ⫻ objective. Tumor cells were arranged in solid sheets
`separated by delicate fibrovascular septae. Small regions of necrosis
`were often seen at the tumor center (Fig. 7A). The MIB-1 index was
`44–48/100 tumor cells, and TUNEL-positive cells occurred in 3.75/
`100 tumor cells.
`Tumors in mice treated with cisplatin alone were smaller, more
`necrotic (Fig. 7B), and exhibited more proliferation and apoptosis
`(20–41% of cells stained form MIB-1 and 5% were TUNEL-positive)
`when compared with tumors from control mice harvested the same
`day after implantation. DAOY xenografts in mice treated with CCI-
`779 alone (Fig. 7C) showed lower proliferation activity than those in
`controls (18 and 17% MIB-1-positive cells), but not increased apo-
`ptosis (2.5% TUNEL-positive cells). DAOY tumors from mice treated
`with cisplatin and CCI-779 were smaller than controls and exhibited
`large areas of necrosis, hyalinization, and hemosiderin deposition
`
`Fig. 3. Suppression of DAOY xenograft growth by CCI-779 used as a single agent.
`Athymic mice bearing human DAOY flank xenografts were separated into three treatment
`groups: control (f) and CCI-779 20 mg/kg 5 days/week i.p. for 1 week (‚) or for 2 weeks
`(E). Symbols along the abscissa indicate the time of CCI-779 injection for the latter
`treatment groups. Tumor size was normalized by dividing the observed tumor volume by
`the initial pretreatment tumor volume (Vo/Vi), and this was plotted over time with day 1
`representing the first day of treatment. CCI-779 treatment delayed growth to 5-fold initial
`tumor volume by 160% (1 week) and by 240% (2 weeks), compared with controls.
`Tumors of animals treated with CCI-779 for 1 week received a second course of CCI-779
`(20 mg/kg, 5 days/week) beginning day 29 as indicated by the ‚ along the abscissa.
`Retreatment resulted in a flattening of the growth curve.
`
`Fig. 4. Suppression of DAOY xenograft growth by a single dose of CCI-779. Athymic
`mice bearing human DAOY flank xenografts were separated into two treatment groups:
`control (f) or CCI-779 100 mg/kg i.p. on day 1 (‚). Time of treatment is indicated by a
`symbol beneath the abscissa. Tumor size was normalized by dividing the observed tumor
`volume by the initial pretreatment tumor volume (Vo/Vi), and this was plotted on a log
`scale. CCI-779 induced 37% tumor regression within 1 week, but only increased the time
`to grow 5-fold the initial tumor volume by 131%, as compared with controls.
`
`(Fig. 7D). Tumor cells surrounding the central necrosis exhibited
`18–38% MIB-1-labeling and 5.5% TUNEL-labeling.
`
`DISCUSSION
`
`Rapamycin and its analogue CCI-779 are new cytotoxic agents with
`the potential to improve the current treatment of pediatric brain
`tumors. The antitumor activity of rapamycin has been demonstrated in
`human rhabdomyosarcoma and neuroblastoma tumor cell lines in
`vitro (9, 11) and in B16 melanocarcinoma, Colon 38 tumors, CD8F1
`mammary tumors, EM ependymoblastoma, and U251 glioblastoma
`brain tumors in vivo (10, 12, 13).
`This report documents the additive cytotoxicity of rapamycin and
`CCI-779 when combined with a platinating agent or a topoisomerase
`I inhibitor, classes of drugs currently used clinically for PNET/MB
`(15, 30). We found that cytotoxic efficacy in vitro and tumor regres-
`sion in vivo were modulated by dosing schedule. Furthermore, this
`report is the first to document the cytotoxicity of rapamycin analogue
`CCI-779 in human PNET/MB and malignant glioma xenograft tumors
`both as a single agent and in combination with cisplatin. This supports
`previous reports that in vitro resistance does not accurately predict the
`efficacy of this agent in vivo and demonstrates that tumor toxicity can
`be increased by using combination chemotherapy without the risk of
`increased systemic cytotoxicity.
`The antitumor activity of CCI-779 in vivo was tested using four
`
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`distinct treatment schedules. Nude mice engrafted with human DAOY
`flank xenografts treated systemically with CCI-779 20 mg/kg daily 5
`times per week exhibited significant delay of tumor growth. Treat-
`ment for 2 weeks was superior to shorter and longer treatment inter-
`vals. CCI-779 retreatment during xenograft expansion after treatment-
`induced growth delay restored growth inhibition, suggesting efficacy
`even for bulky tumors. Single high-dose treatment with CCI-779
`achieved transient tumor regression, however, overall tumor growth
`delay was inferior to the equivalent dose given daily over a week.
`Previous studies examining antitumor activity of rapamycin in vivo
`used higher doses and different treatment schedules. For example,
`Houchens et al. (12) used 200, 400, and 800 mg/kg rapamycin i.p. on
`days 2, 6, and 10 to treat U251 malignant glioma xenografts, whereas
`Eng et al. (13) reported on the antitumor activity of 100 mg/kg i.p. for
`9 days or 400 mg/kg i.p. on days 2 and 9 in melanoma, ependymo-
`blastoma, and Colon 38 tumor models. In vitro results demonstrated a
`dose threshold of 1 ng/ml in DAOY, above which additional cytotox-
`icity is not achieved. In vivo results support the absence of a linear
`dose-response relationship in these tumors. Accordingly, our results
`indicate that high doses are not necessary for antitumor activity.
`In vitro studies of rapamycin identify an interesting pattern of
`tumor cell response: either exquisite sensitivity or profound resistance
`(9, 11). Evaluation of brain tumor cell lines in our laboratory show a
`similar pattern. Four PNET/MB cell lines were highly sensitive to
`growth inhibition by rapamycin in vitro (IC50 ⱕ 10 ng/ml), whereas
`three PNET/MB cell lines and the U251 malignant glioma cell line
`
`Fig. 5. Suppression of U251 malignant glioma xenograft growth by CCI-779. Athymic
`mice bearing flank xenografts derived from the in vitro nonsensitive human U251 glioma
`cell line were separated into two groups: control (f) and CCI-779 20 mg/kg 5 days/week
`i.p. for 2 weeks (‚). Symbols along the abscissa indicate time of CCI-779 injection in the
`treatment group. CCI-779 induced a tumor growth delay of 131% compared with controls.
`
`Fig. 6. Suppression of DAOY xenograft growth by cisplatin with or without CCI-779.
`Athymic mice bearing human DAOY flank xenografts were separated into three treatment
`groups: control (f), cisplatin (E), and cisplatin plus CCI-779 (‚). Cisplatin alone (5
`mg/kg i.p., day 1) induced a tumor growth delay of 210%, compared with controls.
`Simultaneous treatment with cisplatin (5 mg/kg i.p., day 1) and CCI-779 (20 mg/kg i.p.,
`5 days/week for 2 weeks) enhanced this delay by another 19%.
`
`Fig. 7. Impact of systemic cisplatin and/or CCI-779 on DAOY tumor histology. A,
`control. Micrograph of DAOY flank tumor excised from athymic mouse that had not
`received antitumor therapy. Tumor volume on day of excision (day 4 after treatment)
`was 131% larger than the initial tumor volume. The section illustrates homogeneous,
`cellular tumor cells with large nuclei and little cytoplasm. H&E stain, X25. B,
`cisplatin. Micrograph of DAOY flank tumor excised from athymic mouse treated with
`single-dose cisplatin (5 mg/kg i.p.). Tumor volume on the day of excision (day 4 after
`treatment) was 50% of the initial tumor volume. The tumor exhibits small areas of
`necrosis. H&E stain, ⫻25. C, CCI-779. Micrograph of DAOY flank tumor excised
`from athymic mouse treated with a single dose CCI-779 (100 mg/kg i.p.). Tumor
`volume on the day of excision (day 4 after treatment) was 89% of the initial tumor
`volume. The tumor exhibits small areas of necrosis comparable with those seen with
`cisplatin treatment. H&E stain, ⫻25. D, cisplatin plus CCI-779. Micrograph of DAOY
`flank tumor excised from athymic mouse treated with single-dose cisplatin (5 mg/kg,
`i.p., day 1) and single-dose of CCI-779 (100 mg/kg, i.p., day 1). Tumor volume on the
`day of excision (day 4 after treatment) was 32% of the initial tumor volume. The
`tumor exhibits a large area of central necrosis. H&E stain, ⫻25.
`
`were nearly resistant (IC50 ⬎ 1000 ng/ml).5 No cell lines tested
`exhibited intermediate sensitivity. The mechanism(s) determining re-
`sistance or sensitivity to rapamycin is still under investigation. Sen-
`sitivity of cancer cell lines may be attributable to the inhibition of
`insulin-like growth factor-I receptor-mediated signaling by rapamy-
`cin, which is essential for proliferation in some tumor cells (i.e.,
`alveolar RMS cell lines; Ref. 11). Cellular resistance to rapamycin has
`been correlated to paracrine growth factor signaling pathways (9), the
`failure to inhibit c-Myc induction, mammalian target of rapamycin
`mutants (11), and GLI zinc finger protein (31). Nevertheless, CCI-779
`treatment induced tumor growth delay in nude mice bearing U251
`xenografts, a cell line highly resistant to rapamycin in vitro. Houchens
`et al. (12) report similar results using rapamycin to treat U251
`xenografts, suggesting that resistance to rapamycin and its analogues
`documented in vitro does not accurately predict resistance in vivo.
`Rapamycin exhibits the same activity in the Colon 38 tumor model
`when administered i.p., i.v., i.m., and s.c.; upon p.o. administration, its
`activity was reduced but not abolished (13).
`Because of its high lipophilic index (18), rapamycin easily crosses
`the blood-brain-barrier, which is important for systemic administra-
`tion in the treatment of brain tumors. Pharmacological studies of
`rapamycin show that rapamycin is cleared rapidly from the blood after
`s.c. or p.o. administration and sequestered intracellularly, particularly
`in erythrocytes. It dissociates slowly from intracellular FK506-bind-
`ing protein-12 and is metabolized by cytochrome p450 3A enzyme
`(8). This may explain its long half-life in vivo (57–62 h in renal
`transplant patients; Ref. 32).
`Rapamycin induces reduction of tumor growth with little systemic
`toxicity as compared with other antitumor drugs. In our hands, ani-
`
`5 Manuscript in preparation.
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`RAPAMYCIN ANALOG CCI-779 IN PNET/MB BRAIN TUMORS
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`mals treated with a single dose of 100 mg/kg did not experience any
`obvious drug toxicity. With daily treatment for ⬎2 weeks (20 mg/kg
`5 days/week) animals experienced weight loss and dermatitis. Weight
`loss, increase in blood glucose concentrations, gastric ulceration, and
`thrombocytopenia have been described by others (19, 21, 33). Eng et
`al. (13) reported the LD50 of rapamycin in mice as 587 mg/kg, yet in
`an earlier study no significant side effects were mentioned when doses
`up to 800 mg/kg were used (12). Clinical Phase