Eu.T J CanaT Ciin Oneal, Vol. 19, No. 6, pp. 799-805, 1983 .
`Primrd in Gn-at Britain.
`
`0277-5379183$3.00-ffi.OO
`© 1983 Pergamon Prtss Lid.
`
`Human Brain Tumor Xenografts in Nude Mice as
`a Chemotherapy Model*
`
`DAVID P. HOUCHENS,tt ARTEMIO A. OVEJERA,t SYLVA M. RIBLETt and DONALD E. SLAGEL§
`tBattelle Memorial Institute, 505 King Avenue, Columbus, OH 43201, U.S.A. and §Department of Surgery,
`University of Kentucky, Lexington, KY 40506, U.S .A.
`
`Abstract-Two human brain tumors which were previously established in nude
`mice were used to determine antitumor efficacy of various therapeutic agents. These
`tumors were a medulloblastoma (TE-671) and a glioma (U-251) with mass
`doubling times of 3.5 and 5.5 days respectively as subcutaneous implants in nude
`mice. Intracranial (i.e.) tumor challenge was accomplished by inoculating tissue
`culture-grown cells of either tumor into the right cerebral hemisphere to a depth of
`3 mm. Median survival time (MST) in untreated mice with 10 5 i.e. injected TE-671
`cells was approximately 30 days and 53 days in the U-251 tumor. With 2 X 105 U-251
`tumor cells the MST was 27-31 days. Groups of mice which had been inoculated
`with tumor were treated with various doses and schedules of antineoplastic
`compounds by the i.p. route. The TE-671 tumor responded to AZQ treatment with
`an increase in life span ([LS) of 37% compared to untreated controls and an !LS of
`30% with CCNU treatment. BCNU and PCNU were ineffective. With the U-251
`tumor BCNU produced an !LS of >60%, with 75% cures, >I 12% !LS with PCNU
`and 49% !LS with CCNU. Neither tumor responded to procarbazine, PALA,
`dianhydrogalactitol, D-0-norleucine or dibromodulcitol. The U-251 tumor was
`treated on various schedules and doses with BCNU and found to respond well on
`late as well as early treatment. A new drug (rapamycin) being investigated by the
`NCI was found to be very effective against the U-251 tumor. This model system
`should prove valuable in assessing the effects of various chemotherapeutic
`modalities against brain tumors.
`
`INTRODUCTION
`Tm: PROGNOSIS of brain neoplasia is generally
`poor. Although . tumors of the brain and cranial
`meninges account for only 1.5% of the total
`number of cancers in the U.S.,
`the median
`survival time for all stages and types of these
`tumors
`is
`less
`than
`I yr, and
`therapeutic
`approaches developed to date have not signifi(cid:173)
`cantly improved this survival pattern since 1950
`[I].
`Treatment of these tumors may include one
`modality or a combination of surgery, chemo(cid:173)
`therapy, radiation and/ or immunotherapy. Data
`indicate some significant effects with certain
`
`Accepted IO December 1982.
`*Supported in pan by Contract NOI-CM-67099 from the Drug
`Evaluation Branch, Developmental Therapeutics Program,
`National Cancer Institute. Pan of this work was presented in
`the Annual Meeting of the American Association for Cancer
`Research, Washington, DC, April, 1981.
`tTo whom requests for reprints should be addressed.
`
`chemotherapeutic agents [2-4] against experi(cid:173)
`mental animal brain tumors. However, several
`questions have been raised by various investigators
`as to the most appropriate tumor type and
`implant site to use in these systems. It is generally
`accepted that intracerebrally implanted gliomas
`more closely approximate human brain tumors,
`although the transplanted tumors may have a
`different blood supply than primary tumors.
`These concerns are valid as reflected by the fact
`that the murine ependymoblastoma model has a
`high rate of 'cures' with nitrosureas, which is not
`seen with human brain tumor patients [5 ].
`Numerous clinical trials have been performed
`using a variety of chemotherapeutic agents to
`treat brain neoplasia in humans [6, 7]. Both
`single-agent therapy studies [8] and combination
`treatment [9] have given some promise of control,
`but 'cures' are rarely achieved.
`With the advent of the growth and treatment of
`human tumor xenografts in athymic nude mice
`[IO] various high-priority drugs have been
`
`799
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`800
`
`D. P. Houchens et al.
`
`evaluated by the National Cancer Institute (NCI)
`against human breast, colon and lungtumorsasa
`part of the NCI tumor panel [11]. Shapiro et al.
`[12] reported the transplantation of seven human
`brain tumors from patients, both subcutaneously
`and intracerebrally, in nude mice. They showed a
`difference
`to chemotherapy response of
`the
`different tumors. Such an observation has been
`reported by others with subcutaneously im(cid:173)
`planted colon tumor xenografts of the same
`pathological
`type [13] and with melanoma
`xenografts [I 4].
`In the present study we report on the use of
`long-term tissue culture lines of human glioma
`and medulloblastoma implanted into nude mice
`for evaluating reponse to a variety of chemo(cid:173)
`therapeutic agents, including those which have
`been used in the treatment of human brain
`tumors. This was to determine if such a model
`could be used in the selection of drugs for clinical
`trials.
`
`MATERIALS AND METHODS
`Six- to eight-week-old female nude mice on a
`random-bred NIH Swiss background supplied by
`the Mammalian Genetics and Animal Production
`Branch of NCI were used in this study. They were
`maintained in autoclaved cages under polyester
`filters, given autoclaved feed and water ad libitum
`and kept in a laminar flow rack in quarters
`separate from other animals.
`The human tumors used in this study were a
`cerebellar medulloblastoma, TE-67 I, which was
`originally established in tissue culture from a 6-
`yr-old
`female without prior
`treatment by
`McAllister et al. in California [15] and furnished
`as a tumor line in nude mice by Dr. Beatrice
`Lampkin, Children's Hospital of Cincinnati, and
`a glioblastoma multiforme, U-251, which was
`originally established from a 75-yr-old male by
`Ponten [16] in Sweden and furnished by Dr.
`Darell Bigner of Duke University. Tissue cuhures
`of the TE-671 and U-251 lines were maintained as
`monolayers and grown at 37°C, 8% CO 2 in Eagle's
`minimum essential medium supplemented with
`5% calf serum and L-glutamine. Tumors were
`also maintained as xenografts by subcutaneous
`implantation of a 3-mm 3 fragment in the right
`subaxillary region of nude mice.
`The drugs used in this study were supplied by
`the Developmental Therapeutics Program, NCI.
`They were as follows: o-O-norleucine (DON)
`(NSC-7365 ); procarbazine (NSC-77213 ); 1-(2-
`chloroethy 1)-3-(2,6-dioxo-3-piperidy 1)-1-nitro(cid:173)
`sourea (PCNU) (NSC-95466); l ,4-cyclohexadiene-
`1,4-dicarbamic acid,2,5-bis( l-aziridinyl)-3 ,6-dioxo(cid:173)
`diethy l ester (AZQ) (NSC-182986); N-(phos(cid:173)
`phonacetyl)-L-aspartate, tetrasodium salt(PALA)
`
`(NSC-22413 l ); rapamycin (NSC-226080); and l ,3-
`bis(2-chloroethyl)-l-nitrosourea (BCNU) (NSC-
`409962). BCNU was dissolved in 95% ethanol
`(10% of final volume) and physiological saline.
`Methyl CCNU and CCNU were dissolved in 95%
`ethanol (10% of final volume), Emulphor(IO%of
`final volume) and physiological saline. All other
`drugs were dissolved or suspended in physio(cid:173)
`logical saline. All drugs were administered at the
`dosages and schedules indicated and were selected
`based on murine tumor model data from this
`laboratory and from the NCI and not from
`clinical treatment schedules.
`For intracranial tumor implantation the tissue(cid:173)
`cultured tumor cells were mechanically harvested
`by scraping, washed
`two
`times
`in Hank's
`balanced salt solution (HBSS) at 1000 revs/ min in
`a clinical centrifuge and resuspended in serum(cid:173)
`free HBSS to give a concentration of 1-2 X 105
`viable (trypan blue exclusion) cells per 0.025 ml.
`The mice were placed under anesthesia with
`sodium pentobarbital and the tumor implanted
`in the right cerebral hemisphere with a 26-gauge
`needle fitted with a sleeve that allowed only a 3-
`mm penetration.
`All mice were observed daily and median
`survival time was determined for each group. The
`percentage
`increase
`in
`life span (ILS) was
`
`determined by the formula TCC X 100, where T
`
`and Care the median survival time for the treated
`group and control group respectively. An ILS of
`>25% was considered as indicative of activity as
`described by NCI protocols for evaluation of
`chemotherapeutic agents in a murine brain tumor
`[2].
`
`RESULTS
`Table I shows the median survival time of the
`intracranial tumor-implanted control animals
`from four experiments each for TE-67 l and U-25 l
`tumors. All mice that died with both TE-671 and
`U-251
`inoculation
`exhibited
`neurological
`symptoms such as limb paralysis for several days
`before death. Additionally, most animals showed
`some enlargement of the head, with definable
`tumor masses extruded through the needle tract in
`the skull and under the scalp.
`Tables 2 and 3 show the results of experiments
`with TE-671 and U-251 respectively; treated with
`various antineoplastic compounds.
`With the TE-671 (Table 2) CCNU was toxic at
`the 30 mg/ kg dose but was somewhat effective in
`prolonging life at the 20 mg/ kg dose, while
`15 mg/ kg was ineffective. If the treatment was
`delayed 10 days after implantation the 20 mg/ kg
`dose was not effective. AZQ was toxic at 40, 20 and
`15 mg/ kg, but produced a 37% ILS when
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`Chemotherapy of Brain Tumor Xenografts
`
`801
`
`Table I. Deaths in untreated control nude mice inoculated intra(cid:173)
`cranially with human medulloblastoma or glioma cells
`
`Tumor•
`
`Experiment
`No.
`
`No.
`0£ mice
`
`MSTt
`(days)
`
`TE-671
`medulloblastoma
`
`U-251
`glioma
`
`I
`2
`3
`4
`
`I
`2
`3
`4
`
`5
`II
`9
`5
`30
`
`5
`II
`10
`12
`38
`
`34 .0
`31.0
`27.0
`24 .0
`29.4 (average)
`
`52.0
`57 .0
`56.5
`47 .0
`53 .0 (average)
`
`*Each mouse was injected with I X 105 tumor cells.
`tMST = median survival time.
`
`Table 2. Effects of various antineoplastic compounds on an intracranially implanted human
`medulloblastoma (TE-671) in nude mice•
`
`Drugt
`
`NSC No.
`
`o-O-Norleucine
`
`7365
`
`Procarbazine
`
`77213
`
`CCNU
`
`79037
`
`PCNU
`
`95466
`
`Dibromodulcitol
`
`104800
`
`Dianhydroga lacti tol
`
`132313
`
`AZQ
`
`182986
`
`PALA
`
`BCNU
`
`224131
`
`409962
`
`Dose
`(mg/ kg/inj.)
`
`40
`20
`
`500
`250
`500
`
`30
`20
`15
`20
`
`10
`
`200
`100
`200
`
`4
`
`40
`20
`15
`7.5
`7.5
`
`500
`400
`
`30
`15
`
`Schedule
`
`Q4D X3
`
`Q4DX 3
`
`single
`injection
`
`single
`injection
`
`Q4DX3
`
`Q4DX3
`
`Q4DX3
`
`Q4DX 3
`
`single
`injection
`
`First day
`or
`treatment
`
`Increase in
`life spant
`(%)
`
`3
`3
`
`3
`3
`10
`
`3
`3
`3
`10
`
`3
`3
`10
`
`3
`
`3
`3
`3
`3
`10
`
`3
`3
`
`0
`15
`
`23
`0
`0
`
`0 (toxic)§
`30
`0
`0
`
`0
`
`19
`15
`0
`
`3
`
`0 (toxic)§
`0 (toxic)§
`0(toxic)§
`37
`0
`
`0
`15
`
`0
`0
`
`• Five mice per group implanted with I± JO' tumor cells in 0.025 ml volume.
`tDrugs injected intraperitoneally.
`tincrease in life span-percentage median survival time 0£ treated group divided by median survival time of
`untreated tumor control group minus 100. Active if > 25%.
`§Toxic-mice died with a decrease in median survival time compared to control mice.
`
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`802
`
`D. P. Houchens et al.
`
`Table 3. Effects of various antineoplastic compounds on an intracranially implanted human glioma ( U-251)
`in nude mice•
`
`Drugt
`
`NSC No.
`
`o-O-Norleucine
`
`7365
`
`Procarbazine
`
`CCNU
`
`PCNU
`
`77213
`
`79037
`
`95466
`
`Dibromodulcitol
`
`104800
`
`Dianhydroga lactitol
`
`132313
`
`AZQ
`
`PALA
`
`BCNU
`
`186986
`
`224131
`
`409962
`
`Dose
`(mg/kg/inj.)
`
`40
`20
`
`500
`250
`
`30
`20
`15
`
`15
`10
`15
`10
`
`200
`100
`
`4
`
`40
`20
`
`500
`400
`
`30
`15
`30
`15
`
`Schedule
`
`Q4DX3
`
`Q4DX3
`
`single
`injection
`
`single
`injection
`
`Q4DX3
`
`Q4DX3
`
`Q4DX3
`
`Q4DX3
`
`single
`injection
`
`First day
`of
`treatment
`
`Increase in
`lile spant
`(%)
`
`Survivors/
`total
`
`3
`3
`
`3
`3
`
`3
`3
`3
`
`3
`3
`7
`7
`
`3
`3
`
`3
`
`3
`3
`
`3
`3
`
`I
`I
`7
`7
`
`0
`0
`
`23
`7
`
`0 (toxic)§
`49
`4
`
`0 (toxic)§
`44
`0 (toxic)§
`> 112
`
`12
`12
`
`2
`
`0 (toxic)§
`0 (toxic)§
`
`0
`0
`
`44
`>61
`8
`25
`
`016
`015
`
`115
`0/5
`
`2/5
`0/5
`015
`
`1/5
`2/5
`0/6
`3/6
`
`1/6
`015
`
`0/5
`
`01,,
`0/5
`
`115
`0/6
`
`2/5
`3/4
`0/6
`0/6
`
`• Five or six mice per group implanted with I ± 105 tumor cells in 0.025 ml volume.
`tDrugs injected i.p.
`Uncrf.'ase in life span-percentage median survival time of treated group divided by median survival time of umrcated tumor
`control group minus 100. Active if >25%.
`§Toxic-mice died with a significant decrease in median survival time compared to control mice.
`
`administered at 7 .5 mg/kg/injection every 4 days,
`starting on the third day after implantation. If
`treatment was delayed until
`IO days after
`implantation there was no increase in life span.
`No other drugs produced any significant increase
`in life span and there were no mice cured or that
`were long-term survivors.
`With the U-251 (Table 2) CCNU was very
`effective at the 20 mg/kg dose, producing a 49%
`ILS. At 10 mg/kg 3 days after tumor implant
`PCNU produced a 44% ILSand twosurvivorsout
`of five mice. When the treatment was delayed
`until day 7 50% of the mice were long-term
`survivors and the ILS was>! 12%. BCNU was also
`very effective when administered at 15 mg/kg l
`day aftt'r tumor implant and produced an ILS of
`:>61% with three survivors out of four mice, but
`was only minimally effective at the 15 mg/kg dose
`when treatment was delayed until day 7. The top
`doses of CCl\'U and PCNU were toxic and both
`
`doses of AZQ were toxic. No other drugs w<'r<'
`effective.
`In order to determine if late tumor stage
`treatment was as effective as early treatm<'nt and
`that, in fact, established tumor was being treated
`and the drug effects seen were not just tumor
`prevention, the experiment shown in Table 4 was
`performed. Mice were implanted with 2 X 10 5 ll-
`251 cells in order to reduce the time to death from
`that seen with
`I X 105 cells. After
`tumor
`inoculation the mice w1:re randomized and treated
`with BCNU at either 7, I 4 or 21 days at the dos<'
`indicated in the table. The BCNU for
`this
`experiment was a clinical formulation (Bristol).
`The mice were observed daily for death for a
`period of 88 days after
`tumor
`inoculation.
`Antitumor activity was seen with thc 40 mg/kg
`dose with treatment at both day 7 and day 14. At
`the 20 mg.ikg dose a greater increase in lift' span
`was seen on day 14 or 21 treatment than on day 7
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`Chemotherapy of Brain Tumor Xenografts
`
`803
`
`Table 4. Effect of BNCU on an intracrania/ly implanted
`human glioma (U-251) in nude mice*
`
`Group
`
`Trea1ment
`day
`
`Doset
`(mg/ kg)
`
`MSTl
`(days)
`
`!LS§
`(%)
`
`I
`2
`3
`4
`
`5
`6
`7
`8
`
`9
`10
`II
`12
`
`7
`
`14
`
`21
`
`40
`20
`10
`5
`
`40
`20
`10
`5
`
`20
`10
`5
`
`55
`42
`36
`38
`
`56
`48
`42
`38
`
`50
`41
`41
`31
`
`77
`35
`15
`22
`
`80
`55
`35
`22
`
`61
`32
`32
`
`*Seven mice per 1rea1ment group implanted wi1h 2 X IO' cells in
`0.025 ml volume. Group 12 (control) had 16 mice.
`tBCNU given i.p.
`lMST = median survival lime of group.
`§ !LS = increase in life span as defined in Tables 2 and 3.
`
`treatment. The IO mg/ kg dose was active only at
`day 14 and day 21 and the 5 mg/ kg dose showed
`activity only on the day 21 treatment schedule.
`A new drug, rapamycin, an inhibitor of DNA
`synthesis (Randall K. Johnson, personal com(cid:173)
`munication) which was shown to have efficacy
`against the Zimmerman ependymoblastoma by
`the Developmental Therapeutics Program of the
`NCI , was tested against the TE-671 and U-251
`tumors (Table 5). As can be seen, there was no
`effect with any dose of the drug on the TE-671
`medulloblastoma, while
`there was a very
`pronounced effect on the ILS in the U-251 glioma
`model. No toxicity was seen at any of the three
`doses of drug which were used in this study.
`
`Table 5. Effect of rapamycin (NSC-226080) on life
`span of nude mice inoculated intracranially with TE-
`67! medulloblastoma or U-25! glioma cells
`
`Tumor•
`
`TE-671
`
`U-251
`
`Drug doset
`(mg/ kg/inj.)
`
`MSTl
`(days)
`
`!LS§
`(%)
`
`800
`400
`200
`
`800
`400
`200
`
`17.5
`21.5
`21.5
`22.5
`49 .0
`40.5
`46.0
`27.5
`
`0
`0
`0
`
`78
`47
`67
`
`*Female nude miceinocula1edwi1h IX IO'cellsofTE-671 and
`2 X IO' cells of U-251 1umor.
`tDrugs given i.p. on days 2, 6 and 10 afler 1umor iilocula1ion.
`lMST=median survival lime.
`§!LS= increase in life span as defined in Tables 2 and 3.
`
`DISCUSSION
`The results presented in this study indicate the
`usefulness of such a model in the assessment of
`chemotherapy against human brain tumors. The
`results further confirm the report of Shapiro et al.
`[12] that drugs may be readily evaluated in this
`system and, further,
`that both dosage and
`schedule changes can be incorporated. Addition(cid:173)
`ally, the death time can be adjusted by changing
`the number of cells implanted in the i.e. model.
`Currently we are implanting the U-251 tumor at
`2 X I 05 cells and the median survival time is in the
`range of 27-32 days. Although the model reported
`in the present study would not be feasible for
`extensive screening of compounds because of the
`special care and costs associated with nude mice, it
`offers a real potential in the evaluation of high(cid:173)
`priority drugs for phase II or III clinical trials.
`Since large numbers of tumor cells can be grown
`in
`tissue culture with high viability and
`tumorigenicity maintained, the reproducibility of
`the system is very good. There is little variation
`from one experiment to another in the death time
`and the small differences are no more than those
`seen in solid murine tumor models.
`The fact that nitrosoureas were selected as
`active by the U-251
`tumor demonstrates that
`clinically useful drugs are selected by the system.
`Of course, gliomas from different patients may
`not have responded to this treatment. Bullard et
`al. [I 7] reported a study with the subcutaneous
`growth of U-251 and two other gliomas and their
`subsequent
`treatment with BCNU . BCNU
`produced consistent volume reduction of the U-
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`

`804
`
`D. P. Houchens et al.
`
`251 tumor in their study and somewhat less tumor
`reduction in the other tumors.
`The fact that late treatment of tumors, as seen in
`Table 4, was as effective as early treatment would
`indicate that this model is actually a measure of
`drug activity on existing tumor and not just
`prevention of tumor growth.
`The experiment with the new drug, rapamycin
`(Table 5 ), shows the possible usefulness of this
`model in developing new drugs. This drug is now
`being used in pre-clinical toxicology trials for the
`NCI and we are conducting combination
`radiation-chemotherapy studies with rapamycin
`in the U -251 model.
`The nude mouse-human brain tumor model
`described can also be used for other studies of
`combined modality therapy. Slagel et al. [18] used
`this model with the TE-671 and U-251 tumors to
`
`evaluate combination chemoradio1hcrapy and
`found marked synergism with combination
`· therapy compared to either modali1y alont' wht'n
`using procarbazine against TE-671 and BCNl J
`against U-251.
`In conclusion, the studies reported in this paper
`demonstrate the usdulncss of tht' human brain
`tumor xenograft model
`for cvalua1ion of
`antineoplastic effect of dwmothcrapcul ic agents.
`Degrees of response from negative to highly
`effective can be seen based on drug, dose and
`schedule. Such a model could aid
`in
`the
`development of therapy for clinical trials.
`
`Acknowledgement-:-Special thanks art' givm to Ms. Susan
`Toth for preparation of this ma11usnip1.
`
`5.
`
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`ependymoblastoma as an experimental model for screening potential antineoplasLic
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