Studies on the Chemotherapy of Experimental Brain Tumors ... · Studies on the Chemotherapy of Experimental Brain Tumors: Development of an Experimental Model James I. Ausman,1 William

(CANCER RESEARCH 30, 2394-2400, September 1970]

Studies on the Chemotherapy of Experimental Brain Tumors:Development of an Experimental Model

James I. Ausman,1 William R. Shapiro,2 and David P. Rail

Office of the Associate Scientific Director for Experimental Therapeutics, National Cancer Institute, NIH, Bethesda, Maryland 20014

SUMMARY

A method of rapidly implanting tumors intracerebrally inlarge numbers of mice is described. Four tumors previouslyinduced by carcinogen implantation into brains of mice wereused. Histologically and biologically, they were gliomas(ependymoblastoma). The pathology of the tumors, theirpathogenesis in the host brain, and the clinical course oftumor-bearing animals was evaluated. The average median dayof death and the number of animals surviving longer than 60days from failure of the tumor to take were determined andare presented. The advantages and disadvantages in the use ofthese animal systems in intracerebral tumor models are discussed.

INTRODUCTION

Prognosis for the patient with malignant brain tumor hasnot improved despite the advent of modern neurosurgical techniques and radiation therapy (25). Over the past decade,numerous attempts to prolong the survival of such patients byuse of chemotherapeutic agents have had little success (19). Atthe present time, there seems to be little possibility of improving the surgical or radiation therapies of malignant braintumors; however, as more knowledge is gained about thepharmacology of chemotherapeutic agents, the biology of thetumor cell, and the reaction of the host, chemotherapyappears as a more promising approach in increasing the survivaltime of patients with cancer (11).

In a recent review on the chemotherapy of brain tumors,Shapiro and Ausman (19) noted that the potential of chemotherapy against brain tumors has not been fully explored.They suggested that the conduct of the clinical drug evaluationstudies could be improved and that useful basic informationcould be gained from experimental model systems. In reviewingthe clinical studies, they noted that, although a variety ofdrugs, routes, and dosage schedules had been used, no adequately controlled study had been performed which confirmedor rejected the value of a drug against brain tumors.

The screening of effective therapeutic agents by clinical trialis not an efficient method of drug evaluation. Drugs used in

1 Present address: Department of Neurosurgery, University ofMinnesota Hospitals, Minneapolis, Minn. 55455.

2 Present address: Neuropsychiatrie Service, Memorial Sloan-Kettering Cancer Center, New York, N. Y. 10021.

Received January 23, 1970; accepted May 29, 1970.

the past in brain tumor chemotherapy were chosen for trialbecause of their efficacy against extracerebral tumors, becauseneurological side effects suggested drug entry into brain, or onan empirical basis. Rail has suggested that a small animal braintumor model system would be advantageous in screening drugsfor potentially effective agents which might then be testedclinically (3). Several animal systems are in use as brain tumormodels in which glial tumors are grown s.c., or nonglial tumorsintracerebrally, for drug testing (5, 17, 22). Correlation of theresults obtained from these experimental models with theavailable clinical data is not possible at the present timebecause adequate clinical studies with these same drugs havenot been performed. Since such model systems may notrepresent faithfully the intracerebrally growing solid glialtumor, it seemed reasonable to develop a model system inwhich a stable glial tumor could be grown intracerebrally inlarge numbers of easily maintained animals to select drugswhich might be clinically tested. Of the available tumors, thecarcinogen-induced murine ependymoblastoma, a glial tumorwhich is easily transplanted, histologically stable, and widelystudied, was the-most satisfactory choice. This neoplasm couldbe propagated easily in the mouse, a suitable animal for large-

scale testing. The present work describes the development andstandardization of this tumor in the mouse as an intracerebralbrain tumor model. Reports on the use of this model systemfor the evaluation of various cancer chemotherapeutic agentsand for the determination of the quantitative distribution ofdrugs in various compartments of the tumor and brain tissuehave been made briefly (1, 2, 20) and will be followed bymore detailed publications (21).

MATERIALS AND METHODS

Tumors. Four mouse gliomas were used in these experiments: ependymoblastoma, Glioma 261, Glioma 26, andEpendymoblastoma A. Ependymoblastoma was obtained fromDr. Labe C. Scheinberg of the Albert Einstein College of Medicine, Bronx, N. Y., and was induced by carcinogen implantation in the brains of mice by Zimmerman and Arnold (27).Glioma 261, induced by Seligman and Shear (18), and Glioma26, induced by Sugiura (23), were both obtained from thefrozen stock maintained by the National Cancer Institutefacilities of Microbiological Associates, Bethesda, Md. Ependymoblastoma A was derived accidentally from ependymoblastoma during routine s.c. passage. For reasons described

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Brain Tumor Chemotherapy: Experimental Model

below, Ependymoblastoma A was considered a mutant sublineof ependymoblastoma.

Animals. The mice used in these experiments were C57BL/6males. They weighed from 18 to 24 g when used. The animalswere maintained on routine diets and were housed in cageswith hardwood chip bedding of a type known not to inducemicrosomal drug-metabolizing enzymes (24).

Technique of Tumor Implantation. The tumors were maintained for use in the experiments by s.c. passage to the flanksof mice every 14 days. Four to 5 tumor-bearing animals weresacrificed at the time of transplantation. The tumors wereremoved aseptically and placed in a sterile Petri dish containing sterile Locke's solution or Eagle's balanced salt solu

tion at room temperature (14). The necrotic material andconnective tissue capsules were separated from the tumors,and the remainder of the tumor tissue was cut into 1-cu mmpieces. One such fragment was then placed in the bevel at thetip of a no. 19 3-inch spinal needle and the stylet was withdrawn, sucking the tumor into the barrel lumen. In thismanner, the needle was loaded with a piece of tumor ready forimplantation. An animal was anesthetized with diethyl ether,the right frontoparietal scalp was washed with 70% alcohol,and the needle was inserted through the skull. The stylet wasadvanced so that it became flush with the needle hub, thusdischarging the tumor fragment into the brain of the animal.For assurance that the needle entered each brain to a constantdepth, a guard was placed on the outside of the barrel 1.5 mmfrom the top of the bevel (Fig. 1). This distance was calculatedto place the discharged fragment in the cortical-subcorticalregion. Animals observed to have improper tumor implantation were discarded. A few minutes after the implantation, theanimals recovered from anesthesia and resumed their usualactivities. Usually, about 80 animals could be successfullygiven tumor implants in 1.5 hr.

As a result of the diethyl ether anesthesia and tumorimplantation, about 2 to 3% of the animals died immediately.An additional 3 to 4% were discarded because of extrusion ofthe tumor fragment from the implantation site after withdrawal of the needle or because of hemorrhage from thecraniotomy site. Thus, as a result of the operative procedure, amaximum of 7% of the animals failed to survive or were discarded. No death attributable to the operative procedure occurred beyond 24 hr after tumor implantation.

In a similar manner, a tumor fragment could be placed inthe s.c. tissue of the flank of a mouse for tumor propagation;however, a no. 13 trochar containing larger tumor fragmentswas used for implantation in this site.

Histology and Pathogenesis of Tumor Growth. Over 100generations of each tumor were transplanted. Samples of eachtumor were fixed at regular intervals, stained with hemo-toxylin and eosin, and examined to check for histolÃ³gica!stability.

In a series of experiments, 2 or 3 animals were also sacrificed at 3-day intervals after implantation to determine thecourse of tumor growth in the intracerebral site.

Clinical Course and Animal Weights. In several experiments,animals in the control group which received only an intracerebral tumor implant were weighed immediately prior to and

daily after implantation to establish the course of the averageanimal weight change from inoculation to death. In order topermit a comparison of animal weights at equivalent times inthe life-span, animal weights on the day prior to death to 16days prior to death were averaged and plotted.

The clinical source of tumor growth was followed daily byobserving the animals for signs of altered behavior, and thetime of such appearance was noted.

Sham Tumor Inoculation. In a study of the effects ofanesthesia and intracerebral needle insertion on animal mortality, 50 mice were subjected to the usual anesthesia andintracerebral tumor implantation procedure, but no tumorfragment was introduced into the brain. The animals werefollowed for 60 days, and their survival or mortality wasrecorded.

Determination of Animal Survival Times. In drug studies tobe reported (21), 125 animals were used in each drug evaluation experiment. Several experiments were necessary to determine the efficacy of each drug. In each experiment, theanimals were divided into 3 groups: a control group of 25untreated tumor-bearing mice; a drug-treated, tumor-bearinggroup; and a nontumor-bearing drug control group used toevaluate drug toxicity. The median day of death of tumor-bearing control animals was recorded in each of the experiments, performed with the 4 gliomas. The mean and standarddeviation of the medians was calculated. The number and percentage of mice surviving beyond 60 days was also noted.

RESULTS

Histology and Pathogenesis of Tumor Growth. The 4tumors were histologically the same (Fig. 2), consisting ofsmall uniform polygonal cells with oval, darkly stainingnuclear chromatin and scanty cytoplasm; the cells werearranged in sheets with little connective tissue stroma. Pseudo-rosettes were present, and mitotic figures occurred predominantly at the periphery of the tumor. The appearance wastypical of an ependymoblastoma. Glioma 26 tended to bemore vascular than ependymoblastoma or Glioma 261 and notinfrequently contained gross hemorrhage. Areas of microscopic necrosis were evident in all tumors following intracerebral growth. Gliomas 26 and 261 remained histologicallystable for over 100 generations in our laboratory. Ependymoblastoma, after repeated transplantations, grew histologicallyas the same tumor but developed 2 distinct biological growthrates, as noted below.

The growth of ependymoblastoma in the brain was followedserially. Tumor cells were initially seen in the brain tissue atthe base of a large needle tract with some associated hemorrhage. The cells proliferated and developed as a mass in thebrain and eventually grew over the surface of the hemispherebetween the skull and the brain as a thick sheet of cells.Tumor cells also grew out through the cranial defect as anarrow stalk of cells which then produce a dome-shaped s.c.tumor nodule. The tumor could replace almost the entire righthemisphere with growth into the left hemisphere and yet becompatible with life (Fig. 3). Occasionally, tumor growth

SEPTEMBER 1970 2395



/. /. Amman, W. R. Shapiro, and D. P. Rail

22

2l

20

19

18

17

16

15

14

13

12

I I

IO -

Iâ€”

o--oGliomo 261â€¢â€”â€¢Glioma26

16 15 14 13 12 II IO 9 8 7 6 5DAYS PRIOR TO DEATH

In the s.c. tissue, the tumor was surrounded by a connectivetissue capsule which was invaded by inflammatory cells; thelatter were not present around intracerebral tumors. Subcutaneous tumors grew as 1 nodule or as multiple nodules in aline apparently along the site of inoculation.

Animal Weights after Tumor Inoculation. The weight ofanimals in the control groups declined following implantationof the tumor; after a 2-day period, the weight resumed anincremental rate which paralleled that of the nontumor-bearing control animals. As shown in Chart 1, the average ofthe weight of the animal fell as members of the group becameill and approached death. Animal weight losses of up to 33%of the initial weight were often seen prior to death. In general,animal weight loss began about 11 days prior to death.

Table 2Glioma 261: control group median survival,

range, and >60-day survivors

Chart 1. Effect of intracerebral murine gliomas on animal weight.Each point is the mean Â±1 S.E. of the weight of 25 animals. The daysof death have been normalized to maintain the full number of animals.

Table 1Ependymoblastoma: control group median survival,


Experiment1234567891011121314151617181920212223242526Total

26No.

ofanimals2526202125252525252525252525252525252525252525252525642Median

dayofdeath28.53627283131262835273330322930.529313129332826232826.527JPÂ±S.D.

=29Â±3.0Range

ofdays ofdeath19-4918-4120-3122-3720-4411-4120-3422-3723-19-5524-3519-24-3716-4122-3822-3422-5221-3422-18-4020-3420-4016-3217-3718-4016-Survivors> 60days000000001002000000100000015

(0.8%)


14No.


dayofdeath2022.526272423242423272423.52526JT+S.D.

=24Â±1.9Range

ofdays ofdeath12-2716-317-16-15-4117-4821-3318-4016-22-3116-2720-2818-3916-Survivors> 60days002100000000003

(0.9%)

Table 3Glioma 26: control group median survival,


involved only the brain stem. At other times, tumor growingalong the ventricular surfaces produced hydrocephalus. Serialgrowth of Gliomas 26 and 261 was not evaluated. With all ofthe tumors, an increase in the size of the skull was seen inmany animals as tumor growth proceeded.


13No.


dayofdeath22.522.52025252025262925272527JTÂ±S.D.

=24Â±3.0Range

ofdays ofdeath16-3911-2513-2817-3418-4514-2619-3319-3122-16-2718-3618-5915-Survivors> 60days00000000000000

(0.0%)





Table 4Ependymoblastoma A : control group median survival,


Experiment1

2345678No.

ofanimals25

25252525252525Median

dayof death17

19192021181919Range

ofdays ofdeath14-21

15-2215-5118-2615-3516-3214-2015-22Survivors

> 60 days0

1100101

Total 8 200 19Â±1.2 4 (2%)

Clinical Course of the Tumor-bearing Animals. Followingtumor implantation, the animals recovered from the anesthesiaand generally resumed their previous activity in the cage. Someanimals had a transient ataxia and others remained lethargicfor a brief period; however, all eventually returned to normalactivities. No physical signs of disease following tumor implantation were seen until approximately the 15th posttransplantation day. At that time, s.c. tumor nodules becamepalpable over the frontoparietal skull. Greater than 90% of theanimals developed such s.c. nodules before death. Approximately 5 days prior to death, the animals demonstratedclinical signs of illness, when malaise was first seen, with theanimals assuming a hunchback posture and exhibiting a dull,ruffled coat. This was followed by signs of diminished activity,including lethargy, gait disturbance, spinning, and/or focalneurological deficits with upper or lower limb paresis. All ofthese signs were not necessarily present prior to death. Thehead size also increased in most of the animals, indicatingincreased intracranial pressure.

Sham Intracerebral Injections. In the 50 animals which hada sham intracerebral injection without tumor implantation, theanesthetic and operative mortality were the same as that intumor-inoculated animals, but no deaths occurred in the60-day period of observation which followed. Thus, theimplantation procedure did not contribute to the animalmortality.

Animal Mortality. In Tables 1 to 4, the median survivaltimes of brain tumor control groups bearing each of the 4gliomas are listed. Also included are the ranges of individualdays of death and the number of animals surviving longer than60 days.

For the 642 animals bearing ependymoblastoma, theaverage of the median days of death of the 26 control groupswas 29 Â±3 days (Table 1). Only 0.8% of the animals survivedlonger than 60 days and were considered as animals in whichthe tumor failed to grow, or "no-takes." The average of the

median days of death of 340 animals bearing Glioma 261 was24 Â±1.9 days (Table 2). Only 0.9% no-takes were found. Inthe animals with Glioma 26, the average of the median days ofdeath of the 13 groups of animals was 24 Â±3 days (Table 3).All of the animals with intracerebral implantation of this

tumor died before the 60-day observation period terminated.The 8 groups of animals bearing Ependymoblastoma A had anaverage median survival of 19 Â±1.2 days with 2% of theanimals surviving longer than 60 days (Table 4).

DISCUSSION

Shapiro and Ausman (19) in their review listed 16 experimental intracerebral animal tumor models which were "in useor of potential value" in research. The need for a small animal

model which could be maintained inexpensively and whichcould be used in large numbers for drug screening eliminatedfrom consideration tumors propagated in large animals such asthe human choriocarcinoma (4), the hepatocarcinoma in themonkey (7), and the Rous sarcoma virus tumor in the dog(15). The need for a model with intracerebrally propagatedglial tumor further eliminated the L1210 (5, 22), Sarcoma 180(17), Ehrlich carcinoma (17), uterine epithelioma (17), mammary carcinoma (10). and melanoma (9) animal systems. Thetendency for the mouse glioblastoma (8) to be histologicallyunstable made it unsuitable for use in this model. The inconsistent location, histology, and number of tumors developingfrom the Rous sarcoma virus injection in hamsters (16) madethis system unattractive for large scale testing. The humanoligodendroglioma was maintained in tissue culture with somedifficulty and was not practical to use (26). Judging from thereports of the investigators (6, 13), the ependymoma of Pereseappeared to be 2 different tumors with respect to biologicalgrowth. In contrast to all of these, the carcinogen-inducedependymoblastomas of Zimmerman and Arnold (27), Selig-man and Shear (18), and Sugiura (23) best fulfilled the criteriafor a model tumor.

Although they were histologically similar, the 4 ependymoblastomas did not all produce the same mortality time whenimplanted intracerebrally. To see if such differences extendedto sensitivity to drugs, all of the tumors were used in chemotherapy trials. Such experiments confirmed quantitative differences in tumor sensitivity to individual chemotherapeuticagents (21).

The occurrence of the new tumor line, EpendymoblastomaA, which produced earlier animal deaths raised the problem ofstability of ependymoblastoma. In fact, ependymoblastomawas very stable during the 2 years of experience in our hands.The standard deviation of the average median day of death ofependymoblastoma-bearing animals was 3 days. The differencebetween 29 days and 19 days in the median survival timerepresents >2 S.D. variation, the chances for which would beless than 1 in 20. Since the Ependymoblastoma A line continued true and stable, was histologically an ependymoblastoma, and responded to one test drug [ 1,3-bis(2-chloro-ethyl)-l-nitrosourea] in the same way as the original tumor(21), we considered Ependymoblastoma A to be a truemutation.

The use of the intracerebral ependymoblastoma as a braintumor model is subject to several criticisms. First, the tumor istraumatically implanted in the animal brain and disruption ofthe blood-brain barrier may be produced, making the modelunlike a primary brain tumor. However, the exact nature of

SEPTEMBER 1970 2397



y. /. Amman, W. R. Shapiro, and D. P. Rail

the blood-brain barrier in any human brain tumor is unknownin any quantitative aspect (19). Using quantitative steady-statephysiological techniques, Ausman and Levin (1) found thatthe permeability of the intracerebral murine ependymo-blastoma is significantly greater than that of samples of braindistant from the tumor. If a human brain tumor is proved tohave a restricted permeability approaching that of normalbrain, then it can be conceded that the permeabilities of thetumor model system and the human tumor differ. Such adifference must be correlated further with a variation inresponse to chemotherapeutic agents before the model may berejected as not representative of that particular tumor. Experiments with monkeys with intracerebrally implanted chorio-carcinoma suggested that such procedures do not necessarilyalter the permeability of the brain (J. I. Ausman, V. A. Levin,and W. E. Brown, Jr., unpublished observations). Brain immediately adjacent to the implanted choriocarcinoma had thepermeability characteristics of normal brain. Therefore, thecriticism that the model is not analogous to human tumorsmust await substantiation by correlation between man andanimal of comparable physiological and chemotherapeuticdata. The results obtained with the models, however, applyonly to the model system, and any extrapolation to clinicalsituations must await the appropriate correlating clinicalstudies.

Secondly, the implantation procedure can be described asimprecise and nonquantitative. The discovery of the tumor inareas of the brain beyond the site of implantation is consistentwith the blind method by which the tumor fragment isinserted. Rupture of the tumor into the ventricles on implantation or with growth could permit spread through the ventricular system with obstruction resulting in hydrocephalus. Theangles and needle placement on tumor implantation and thestructures disrupted may not be consistent from animal toanimal. Although the needle has a protective guard to ensureuniform depth of placement, variation in the thickness of theskull and scalp of the mouse or slight extrusion of fragmentcould alter the final placement of the tumor fragment. In spiteof the variability in technique, greater than 99% of the animalsso inoculated died within a consistent time and range, a factwhich permitted the system to be utilized for large-scaletesting. Wilson et al. (26) achieved a narrow range of mortalitywith stereotaxic implantation of a quantitatively determinedcell suspension from culture, but the technique is not practicalfor large-scale screening.

The growth of the tumor in the scalp can result from proliferation of cells left in the s.c. site at the time of implantationor from extension of the intracranial growth through thecranial defect remaining after needle placement. In a fewexperiments, wax was placed over the defect in an effort toprevent the extracranial extension of tumor, but s.c. tumorstill appeared, and it was not certain whether the bone waxhad remained in position. A concern in the chemotherapyexperiments was that the blood supply to the intracranialtumor might come from the s.c. nodule. However, chemotherapy was usually instituted on the 2nd day after implantation before any s.c. growth was present, and drug could reachthe tumor only through the intracerebral circulation. Finally,even with therapy begun on the 14th day after implantation,

at the time when s.c. nodules were present, no significantdifference in the response of the tumor as compared to earlierdrug therapy was noted (21).

The presence of multiple tumor nodules after s.c. transplantation was probably the result of the growth of tumorcells which were deposited along the needle tract during inoculation. These tumors have not been noted to metastasize (12).The multiple s.c. growth pattern presents a severe limitation inthe use of the s.c. tumor as an indicator of response to chemotherapy. It is difficult to evaluate the inhibition of multiplesmall nodules. We did not use the s.c. tumor as a model forchemotherapy because (a) the size of the tumor nodules s.c.changed little even in the presence of considerable necrosis, (b)the measurement of tumor size was not consistent in ourhands, and (c) great variation in tumor "take" and growth was

seen with the s.c. implants.The average of the median survival times of animals with

Gliomas 26 and 261 were the same, but both were less thanthat found with the ependymoblastoma and greater than thatof Ependymoblastoma A. Gliomas 26 and 261 differed in thatthe former tumor was more hemorrhagic and necrotic than thelatter. Thus, although all the tumors were histologically thesame, they differed in their growth characteristics.

The range of days of death found with all 4 tumors can beexplained by the variability inherent in the transplantationtechnique, by the quantity of tumor implanted, by the location of the tumor, by the expansion of the skull, and by thedecompression of the tumor through the trephine hole. Also,the neurological deficit produced and the coincident alterationin the functional state of the animals, such as feeding,drinking, and ambulating activities, may influence the time ofdeath. In spite of these variables, the consistency of themedian day of death from group to group was remarkable. Thesham inoculation experiment showed that the trauma of theimplantation procedure did not influence the mortality timesof the animals.

Although no autopsies were performed on the animals atdeath, the presence of tumor growth in the scalp above theimplantation site, the development of focal neurological signs,the confirmation of the presence of tumor in sacrificedanimals, the failure of sham-inoculated controls to die within60 days, and the survival of only 2% or less of the tumor-inoculated controls beyond 60 days all indicated that deathoccurred in these animals from tumor growth.

ACKNOWLEDGMENTS

The technical assistance of Mr. Sidney T. Yancey is gratefullyacknowledged.

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1. Ausman, J. I., and Levin, V. A. Intra- and Extravascular Distribution of Standard Drug Molecules in Brain Tumor and Brain. In:C. G. Drake and R. Duvoisin (eds.), Fourth International Congressof Neurological Surgery, 1969, p. 41. New York: Excerpta Medica,1969.

2. Ausman, J. I., Shapiro, W. R.. and Slivka, J. J. The Effect of





Chemotherapeutic Agents on an Experimental Mouse Brain Tumor.Proc. Am. Assoc. Cancer Res. 9: 4, 1968.

3. Bering, E. A., Jr., Wilson, C. B., and Norrell, H. A., Jr. The Kentucky Conference on Brain Tumor Chemotherapy. J. Neurosurg.,27: 1-10,1967.

4. Brown, W. E., Jr. Transplantation of Human Choriocarcinoma tothe Brains of Rhesus Monkeys. In: C. ÃŒ.Lund and J. W. Shoate(eds.), Transcript of the 4th Rochester Trophoblast Conference,University of Rochester School of Medicine and Dentistry, 1967,pp. 224-233. Rochester, N. Y.: University of Rochester Press,1967.

5. Chingos, M. A., Humphreys. S. R., and Goldin, A. Effectiveness ofCytoxan against Intracerebrally and Subcutaneously InoculatedMouse Lymphoid Leukemia L1210. Cancer Res., 22: 187-195,1962.

6. Hatanaka, H., Soloway, A. N.. and Sweet, H. Incorporation ofPyrimidines and Boron Analogues into Brain Tumor and BrainTissue of Mice. Neurochirurgia, 10: 87-95, 1967.

7. Kelly, M. G., O'Gara, R. W.. Walker, M. D.. Dawe, C. J.. Morgan.

W. D., and Kerber. W. T. Intracerebral Transplantation into RhesusMonkeys of Cell Cultures (NCLP-6) Derived from a ChemicallyInduced Primate Hepatocarcinoma. J. Nati. Cancer Inst. 39:153-169, 1967.

8. Kirsch, W. M., Schulz, D., and Leitner, J. W. The QuantitativeHistochemistry of Experimental Glioblastoma: Glycolysis andGrowth. Acta Histochem, 28: 51-85, 1967.

9. Kotsilimbas, D. G., Karpf, R.. Merideth. S.. and Scheinberg. L. C.Evaluation of Parenteral 5-FU on Experimental Brain Tumors.Neurology, 16: 916-918, 1966.

10. Kotsilimbas, D. G., Levy, W. A. and Scheinberg, L. C. Chloro-merodrin Hg2Â°3 and Electrolyte Distribution in Murine BrainTumors. Arch. Neurol., 13: 525-532, 1965.

11. Luce, S. K., Bodey, G. R.. Jr., and Frei, E., III. The SystemicApproach to Cancer Therapy. Hosp. Pract.. 2(10): 42-55, 1967.

12. Netsky, M. G. Experimental Induction and Transplantation ofBrain Tumors in Animals. Acta Neurochir., 10 (Suppl.).' 46-55,

1964.13. Nystrom, S. H. M. Effects of High Energy Protons on Brain and

Glioma of Mice. Acta Radiol. Therapy Phys. Biol., 5: 133-148,

1966.14. Paul, J. Cell and Tissue Culture. Baltimore: The Williams & Wilkins

Co., 1961.15. Rabotti, G. F., Grove, A. S., Jr.. Sellers, R. L., and Anderson, W.

R. Induction of Multiple Brain Tumors (Gliomata and Leptomenin-geal Sarcomata) in Dogs by Rous Sarcoma Virus. Nature, 209.'

884-885, 1966.16. Rabotti, G. F., and Raine, W. A. Brain Tumors Induced in Ham

sters Inoculated Intracerebrally at Birth with Rous Sarcoma Virus.Nature, 204: 898-899, 1964.

17. Rosso, R., Donelli, M. G., Innocenti, I. R.. and Garattini, S.Chemotherapy of Tumors Transplanted Intracerebrally. EuropeanJ. Cancer, 3: 125-137, 1967.

18. Seligman, A. M., and Shear, M. J. Studies in Carcinogenesis. Vili.Experimental Production of Brain Tumors in Mice with Methyl-cholanthrene. Am. J. Cancer, 37: 364-395, 1939.

19. Shapiro, W. R., and Ausman, J. I. Chemotherapy of Brain Tumors:Clinical and Experimental Review. In: F. Plum (ed.). RecentAdvances in Neurology, pp. 149-235. Philadelphia: F. A. DavisCompany, 1969.

20. Shapiro, W. R., and Ausman, J. I. Effect of ChemotherapeuticAgents on Experimental Brain Tumors. Proc. Am. Assoc. CancerRes., 10: 79, 1969.

21. Shapiro, W. R., Ausman, J. I., and Rail. D. P. Studies on theChemotherapy of Experimental Brain Tumors: Evaluation of1,3-Bis(2-chloroethyl)-l-nitrosourea Cyclophosphamide,Mithramycin, and Methotrexate. Cancer Res., 30: 2401-2413,1970.

22. Skipper, H. E., Schabet, F. M., Jr.. Trader. M. W., and Thompson,J. R. Experimental Evaluation of Potential Anticancer Agents. VI.Anatomical Distribution of Leukemic Cells and Failure of Chemotherapy. Cancer Res.. 21: 1154^ 1164, 1961.

23. Sugiura, K. Tumor Transplantation. In: W. I. Gay (ed.), Methods ofAnimal Experimentation Vol. 2. pp. 171-222. New York:Academic Press, Inc., 1969.

24. Vessel, E. S. Genetic and Environmental Factors Affecting Hexo-barbital Metabolism in Mice. Ann. N. Y. Acad. Sci., 151: 900-912,1968.

25. Walker, A. E. (ed.) A History of Neurological Surgery. New York:Hafner Publishing Co., 1967.

26. Wilson, C. B., Norrell, H., Jr.. and Barker, M. Intrathecal Injectionof Methotrexate (NSC-740) in Transplanted Brain Tumors. CancerChemotherapy Rept.. 51: 1-6, 1967.

27. Zimmerman, H. M., and Arnold, H. Experimental Brain Tumors. I.Tumors Produced with Methylcholanthrene. Cancer Res.. 1:919-924, 1941.

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J. I. Amman, W. R. Shapiro, and D. P. Rail

1C tumor

Distant brain Adjacent brain

Fig. 1. Photograph of an intracerebral implanting needle showing the stylet. The guard, placed 3 mm from the tip of the needle, permitsimplantation of the tumor fragment into the cortical-subcortical region.

Fig. 2. Microscopic appearance of ependymoblastoma.Fig. 3. Coronal section of head of mouse with ependymoblastoma, demonstrating an intravitally trypan blue-stained tumor in s.c. tissue above

the skull and the brain.




1970;30:2394-2400. Cancer Res James I. Ausman, William R. Shapiro and David P. Rall Development of an Experimental ModelStudies on the Chemotherapy of Experimental Brain Tumors:

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Studies on the Chemotherapy of Experimental Brain Tumors ... · Studies on the Chemotherapy of Experimental Brain Tumors: Development of an Experimental Model James I. Ausman,1 William