Growth of Human Lung Tumor in the Brain of the Nude Rat as ... · tumor-specific monoclonal antibodies have been developed to this neoplasm (36,37). The human small cell tumors were

[CANCER RESEARCH 45, 2827-2833, June 1985)

Growth of Human Lung Tumor in the Brain of the Nude Rat as a Model toEvaluate Antitumor Agent Delivery across the Blood-Brain Barrier1

Edward A. Neuwelt,2 Eugene P. Frenkel, Anthony N. D'Agostino, Desmond N. Carney, John D. Minna,

Peggy A. Barnett, and Christopher I. McCormick

Department ol Surgery. Division of Neurosurgery, Oregon Health Sciences University, Portland, Oregon 97201 [E. A. N., P. A. B., C. I. M.]; Department of InternalMedicine, Division of Hematology -Oncology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235 [E. P. F.Â¡;Department of Pathology, Good SamaritanHospital and Medical Center, Portland, Oregon 97210 [A. N. D.]; and NCI-Navy Medical Oncology Branch, Division of Cancer Treatment/NIH, Naval Hospital, Bethesda,

Maryland 20814 [D. N. C., J. D. M.]

ABSTRACT

We have developed a brain tumor model in the nude rat utilizingNCI-N417D human small cell carcinoma of the lung grown bothintracerebrally and s.c. The median latency period from the timeof intracerebral tumor inoculation to the onset of neurologicalsymptoms is 13 days with an intracerebral tumor take rate of91% (29 of 32). The median survival is 13 days, and all animalswere dead by Day 26. The tumor is discrete, well circumscribed,with occasional leptomeningeal spread and with minimal evidence of surrounding cerebral edema. Intracerebrally, this tumoris usually impermeable to Evan's blue:albumin (M, 68,500) but

not fluorescein (M, 376). Although variable, the intracerebraltumor is less permeable to methotrexate than is the same tumorgrown s.c. in the same animal (P < 0.005). The intraarterial andi.v. routes of methotrexate administration in the presence andabsence of blood-brain barrier opening were evaluated. Drug

delivery to the intracerebral tumor and ipsilateral brain wassignificantly (P < 0.025) greater when the methotrexate wasgiven intraarterially and was significantly (P < 0.0025) increasedafter osmotic blood-brain barrier opening. After barrier opening,methotrexate concentration was enhanced 3- to 4-fold in tumorand 10- to 20-fold in brain around tumor. Thus, the nude rat

provides a model to investigate the biology and therapeuticresponsiveness of human small cell carcinoma of the lung grownintracerebrally where it develops a blood-tumor barrier similar to

that seen in humans. This model further provides the uniqueopportunity to investigate the role of osmotic blood-brain barrieropening in the treatment of a tumor which is sensitive to chem-otherapeutic agents and for which tumor-specific monoclonal

antibodies are available.

INTRODUCTION

Although Greene (1, 2) reported heterotransplantation of human brain tumors in 1951, attempts to implant and grow spontaneous human tumors intracerebrally in laboratory animals havebeen inconsistent (3) and generally unsuccessful. Neither conditioning with immunosuppressive agents (4) nor utilization of thesubarachnoid space of the animal as a growth chamber (5)provides a satisfactory model for the study of gliomas or other

1This work was supported by the Oregon Medical Research Foundation,

National Cancer Institute Grant CA31770, Good Samaritan Hospital Foundation,NIH Grant CA 23115, Meadows Foundation, and the Southwestern Medical Foun-dation-Kinsler Williamson Brown Fund.

2To whom requests for reprints should be addressed, at Division of Neurosur

gery (L472), Oregon Health Sciences University, 3181 S.W. Sam Jackson ParkRoad, Portland, OR 97201.

Received 8/6/84; revised 11/14/84. 2/28/85; accepted 3/11/85.

human tumors as xenografts. For instance, intracerebral growthof human glioma in immunosuppressed rats results in a highlypermeable tumor (4) unlike the situation clinically (6, 7) or in avirally induced animal glioma model (8, 9).

The introduction of the athymic nude mouse (10) offered anew opportunity to study the growth and behavior of humantumor cells in vivo under controlled conditions (11). A variety ofhuman tumors have been established and serially passed in theathymic mouse (12). The histology and responsiveness for mosttumors in the nude mouse are similar to those in humans, andvalidation of this model for the study of a variety of biologicalfeatures of human tumors has been achieved (13). Tumors sogrown show a differential sensitivity to cytoreductive chemo-

therapeutic agents that reflects the clinical effectiveness of theseagents in the original patient. For instance, Giovanella et al. (14)evaluated the efficacy of 9 chemotherapeutic agents on 3 typesof human tumor xenografts implanted in the nude mouse (breastcancer, colon cancer, and melanoma); tumor regressions wereconsistent with the clinical experience in patients except for 2false-positive and one false-negative values. Histological evalu

ation of human tumor xenografts has been reported to maintainmorphological fidelity with the parent tumor (13). In addition,several reports (15-17) have shown that xenografts of human

tumors can be consistently transplanted intracerebrally into thenude mouse, and Chambers ef al. (18) have reported the intra-

cranial growth of cells from small cell carcinoma of the lung innude mice. One possible disadvantage of the nude mouse is thathuman tumors which have a high propensity to metastasize donot metastasize in nude animals.

Unfortunately, the small size of the nude mouse makes it avery difficult model for the study of complex multimodality therapeutic approaches to Â¡ntracerebral tumors. The ideal modelwould permit human xenografts to be examined at both intracerebral and systemic sites, and the intracerebral site shoulddevelop the biological characteristics of spontaneous primary ormetastatic tumors, i.e., to demonstrate at least a partial BBB3

(8, 9, 19, 20). In addition, animal size should permit a variety oftherapeutic manipulations, including i.a. (9, 21-23) infusions,opening of the BBB (6-8, 24, 25), and applicability for studies of"targeted" therapy, such as with monoclonal antibodies (26-29).

The nude rat provided the appropriate structural requirements,and human small cell (oat cell) xenografts provided the acceptable tumor for the development of a therapeutic model. Thenude rat has immunological features like those of the nudemouse (30, 31). Thus, the congenitally athymic nude rat acceptsallografts and xenografts, and their splenic lymphocytes respond

3The abbreviations used are: BBB, blood-brain barrier; MTX, methotrexate;

CNS, central nervous system; i.a., intraarterial(ly); i.e. intracarotid.

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NUDE RAT BRAIN TUMOR

poorly to T-cell mitogens. Unlike the nude mouse, the nude rat

is less susceptible to infections and is thereby easier to maintain.The nude rat does not provide as good a growth medium fortumor xenografts (31) as the mouse, perhaps as the result of ahigher level of natural killer cells (32, 33). However, a variety ofdifferent human cancers (e.g., carcinoma of the breast, gastrointestinal tract, melanoma, and rhabdomyosarcoma) have beengrown in the nude rat (34, 35).

The present studies focused on the small cell carcinoma (oatcell) of the humarj lung, since this carcinoma is a common tumorwhich frequently metastasizes to the CNS and is responsive toa variety of chemotherapeutic agents. In addition, a number oftumor-specific monoclonal antibodies have been developed tothis neoplasm (36,37). The human small cell tumors were grownin the nude rat, and histology, survival, and the delivery of achemotherapeutic agent (MTX) were examined. These studieshave shown the feasibility of the nude rat as a xenograft modelto examine this common human tumor and to evaluate strategiesof therapy by comparing the delivery of drugs to the tumor inthe brain with the tumor at s.c. sites. In particular, this modelallows an evaluation of the role of BBB modification in anti-tumor

agent delivery.

MATERIALS AND METHODS

Nude Rat Colony. The animals utilized were the athymic nude rat(rnu/rnu) which were bred from an outbred pair (rnu/+) furnished fromHarÃanSprague Dawley (Indianapolis, IN). The stock and breeding animals were kept in laminar flow contaminant hoods. Homozygote maleswere paired with hÃ©tÃ©rozygote(rnuÂ¡+) females which resulted in 50%homozygote nude (rnu/rnu) athymic animals. All animals received sterilized fat-enriched food and vitamin-supplemented water. Water and

bedding were changed twice weekly under sterile conditions. Animalswere examined daily.

Human Tumor Cell Line. The human tumor cell line used was theNCI-N417D (38) which originated in the laboratory of Dr. John D. Minna.

Cells were grown in 20 ml of RPMI Medium 1640 supplemented with10% heat-inactivated fetal calf serum, penicillin (10,000 units/ml), strep

tomycin (10,000 M9/ml), arid gentamicin (50 ng/m\). Complete mediumwas filtered (0.2-Mm Millipore) prior to use. All cells were maintained in5% CO2 at 37CC and passaged in culture twice weekly. The NCI-N417D

human tumor cells are free-floating cell suspensions that are easily

harvested by centrifugation at 250 x g (1200 rpm) for 3 to 5 min. Cellsuspensions for inoculation were made by resuspending the cell pellet inmedium to achieve a final concentration with a packed cell volume of 20Â±1% as measured in a microhematocrit tube. Cell viability was evaluatedby trypan blue exclusion; viability was greater than 85%.

Implantation of Human Tumor Cells into the Nude Rat. Implantations.c. was achieved by the s.c. injection into the right flank of 750 to 1000M! of tumor cell suspension with a packed cell volume of 20 Â± 1%(approximately 20 to 27 x 106 cells). Intracerebral implantation of tumor

cells was performed after the rats were anesthetized by i.p. injection ofsodium pentobarbital (50 mg/kg). The head was shaved, sterilized withiodine solution, and immobilized in a rat stereotaxic frame (David KopfInstruments, Tunjunga, Ã‡A).A 2-cm midline incision was made to exposethe frontal bone on the right, where a 2-mm burrhole with a Stryker drill

was placed just posterior to the coronal suture line and 4 mm lateral tothe sagittal suture. A micromanipulator (Brinkman Instruments, West-bury, NY) equipped with a 27-gauge needle and a 100-iil syringe was

used to inject 15 ^l of tumor cell suspension with a packed cell volumeof 20 Â±1% over 2 to 3 min at a depth of 1.5 mm. This concentration ofcells equals approximately 4 x 105 tumor cells. The wound was closed

in a single layer.

BBB Opening in the Nude Rat. BBB opening in the nude rat wasperformed and graded as previously described (8). Five min before BBBopening, Evans blue (2%, 2 ml/kg, i.v.) and/or fluorescein (10%, 0.12 to0.25 ml, i.v.) was administered.

MTX Delivery to Human Tumor in Nude Rats. Animals symptomaticfrom intracerebral tumor were randomly divided into 4 groups: Group I,i.v. MTX after i.e. 0.9% NaCI solution (saline) (BBB intact); Group II, i.e.MTX after i.e. saline (BBB intact): Group III, i.v. MTX after i.e. mannitol(BBB opened); or Group IV, i.e. MTX after i.e. mannitol (BBB opened). Inall groups, MTX (4 mg/kg) was administered immediately after i.e. salineor mannitol over 1 min. Thirty min after MTX administration, a serumsample was obtained, and the animals were sacrificed with an overdoseof sodium pentobarbital. Tumor, brain around tumor (1 to 2 mm of brainimmediately surrounding tumor), brain distant to tumor (coronal sectionof the occipital lobe) in the perfused hemisphere as well as in thecontralateral brain (coronal section of occipital lobe), s.c. tumor, andother organs and tissues were obtained for MTX determinations. MTXcontent in serum and tissue was determined by radioimmunoassay aspreviously described (22).

The effect of the intracerebral implantation technique on BBB integritywas examined in rats inoculated with lethally irradiated NCI-N417D cells(8000 to 9000 rads, using a 137Cssource). On Day 14 after intracerebral

inoculation, Evans blue, fluorescein, and MTX were administered i.v. asdescribed above.

Statistical Analysis. MTX levels are expressed in ng/g of tissue orng/ml of serum as the mean Â±SE. Comparisons of the log concentrationof tissues in the irradiated inoculum control group (Table 1) and withinthe 4 treatment groups (Table 2) were made by the one-way analysis ofvariance followed by the Neuman-Keuls multiple comparisons when theoverall difference was statistically significant. The 2-factor analysis of

variance was utilized to evaluate the effect of route of MTX administrationand/or osmotic BBB opening on delivery to brain tissues (Table 2, GroupsI to IV) (39, 40).

RESULTS

Survival and Onset of Neurological Symptoms. For the studyof survival characteristics, 4 x 105 human small cell carcinoma

of the lung cells (serially passaged in tissue culture) were injectedintracerebrally into the right parietal lobe of 32 nude rats. Themedian survival was 13 days (range, 10 to 25 days), and allanimals were dead by Day 25 (Chart 14). Because of the largevariation in time from death to autopsy, intracerebral tumorconfirmation was often not available in these rats.

Of 32 additional rats given injections of intracerebral NCI-

N417D human tumor cells, the median latency period fromintracerebral inoculation to the development of neurologicalsymptoms was also 13 days (range, 9 to 26 days) (Chart 1B).Symptoms identifying the presence of tumor included cachexia,weight loss, and dehydration. Animals died within 24 h of evidentsymptoms. Of these rats, 91% (29 of 32) developed verifiedintracerebral tumors. In the remaining 3 animals, intracerebraltumor could not be confirmed by visual inspection, but histologywas not performed.

Pathological Characteristics. When the animals become neu-

rologically symptomatic or at expiration, the tumors exhibited amass effect on the brain. The tumors were approximately 5 to 7mm in diameter with occasional leptomeningeal spread. No animal had multiple tumors. The tumors were usually subcorticaland often extended to the ependymal wall of the lateral ventricle.The intracerebral tumors were round and had neovascularity.The tumor margin was clearly defined, and little surroundingcerebral edema was present (Fig. 1-4). Systemic spread (other


2828




A100"5>

80'E

W60Â§

40

O;

1n=32

20

2 6 10 14 18 22 26

Days After Intracerebral Inoculation

n=32

2 6 10 14 18 22 26

Days After Intracerebral InoculationChart 1. A, survival curve of 32 rats inoculated intracerebrallywith NCI-N417D

humansmallcell carcinomaof the lung and followed until death. The medianlatencyperiod from intracerebral inoculation to death was 13 days (range, 10 to 25 days),and all animals were dead by Day 25. B, curve illustrating the development ofneurologicalsymptoms after intracerebral inoculationwith NCI-N417Dhumansmallcell carcinoma of the lung cells. The median latency period from intracerebralinoculation to onset of symptoms was 13 days (range, 9 to 26 days), and allanimals had symptoms by Day 26. Ninety-one % (29 of 32) had confirmedintracerebral tumor; the remaining 3 could not be confirmed by visual inspection,and histology was not performed.

than the simultaneously inoculated s.c. flank tumor) has not beenobserved. Microscopic examination of the tumor cells showsthem to be slightly larger than the characteristic human lungsmall cell tumor as identified in clinical material; mitoses werevery frequent (Fig. ÃŒB).

In the initial studies, the same size of tumor inoculum wasused intracerebrally and s.c. However, at the time the animalsbecame symptomatic from their intracerebral tumor, no s.c.tumor was detected when the tumor inoculum was the same atboth sites. When s.c. tumor inoculum was increased 50- to 65-

fold, the diameter of the s.c. tumor was 2 to 3 cm at the pointwhen the animals developed neurological symptoms from theintracerebral tumor. The s.c. tumor was 4 to 5 times larger thanthe intracerebral tumor. In addition, in almost half the animals,the s.c. tumors were partially cystic and contained serosangui-

neous fluid.Tumor Staining with Fluorescein and Evans Blue:Albumin.

During the preliminary studies, it was often difficult to identifydiscrete tumors at the time of death, even when Evans blue(which binds tightly to albumin, thereby identifying barrier integrity) had been given. Thus, there were several animals in whichthe intracerebral tumor was missed by visual inspection, but

confirmed histologically. This led to the use of i.v. fluorescein (M,376) when symptoms were evident, thus providing clearly demarcated, easily visible tumors identified by the yellow stain offluorescein (Fig. 2A). The relative integrity of the BBB wasevaluated by the concurrent administration of i.v. fluorescein (M,376) and Evans blue (Evans blue:albumin, M, 68,500). As shownin Fig. 2A, the intracerebral tumor usually stained with the low-molecular-weight moiety fluorescein, but not with the high-molecular-weight Evans blue:albumin, whereas both markers gen

erally penetrated s.c. tumor (Fig. 26). By contrast, when thesesame molecular markers (fluorescein and Evans blue:albumin)were given i.v. and osmotic barrier opening of the tumor-bearing

hemisphere was performed, both Evans blue and fluoresceinpenetrated the tumor as well as the surrounding brain. A greendiscoloration (due to the combination of yellow and blue) of thesetissues was seen in the area of brain where barrier opening wasperformed (Fig. 2, C and D). When only Evans blue was giveni.v. at the time of barrier opening, the tumor and the brain aroundthe tumor were stained blue.

MTX Delivery to Intracerebral and s.c. Tumor. To determinethe effect of the inoculation technique on BBB integrity, lethallyirradiated tumor inoculum was implanted intracerebrally. Therewas no evident staining with either i.v.-administered Evans blue

or fluorescein. Following i.v. MTX administration, MTX deliveryto the area of inoculation resulted in levels ranging between 0.5and 3% of serum levels, which was consistent with MTX levelsin the contralateral hemisphere of all inoculated animals (Table1). There was a statistical difference (P < 0.05) in MTX concentration between the inoculation site (94 Â±54) and brain distantto the inoculation (16 Â±3) site and contralateral brain (17 Â±3).Despite an equivalent dose of MTX, the serum drug levels intumor-bearing animals were higher than in animals receiving

irradiated tumor cells, which probably reflects cachexia andvolume depletion of tumor-bearing animals.

Drug delivery studies were begun when the animals manifested symptoms from their intracerebral tumor. Rats with bothintracerebral and s.c. tumor were given MTX either i.v. or Â¡.a.after either i.e. saline (Groups I and II) or mannitol infusion(Groups III and IV). After i.v. MTX, the intracerebral tumor inGroup I contained significantly (P < 0.005) less MTX than thes.c. tumor, suggesting limited entry of drug at the intracerebralsite (Table 2). The level of MTX in intracerebral tumor, althoughvariable, was approximately 8% of that in serum, whereas thelevel in the s.c. tumor was approximately 20% of the serum levelat the time of sacrifice. The intracerebral tumor, however, contained significantly more MTX than the brain around tumor, braindistant to tumor, and contralateral brain (P < 0.005).

Table1MTXconcentration in the intracerebral inoculation site of lethally irradiated small

cell carcinoma tumor cellsMTX (ng/g)"

Inoculation siteBrain around inoculation siteBrain distant to inoculation siteBrain contralateral to inoculation siteSerum (ng/ml)

94 Â±54"

25 Â±616Â±317Â±3

3033 Â±33"MTX, 4 mg/kg, was administered i.v. on Day 14 after the intracerebral

inoculation. Animals were sacrificed 30 min after MTX administration (n = 3)." MeanÂ±SE.0Significantly (P < 0.05) greater than brain distant to the inoculation site and

brain contralateral to the inoculation site.


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Table 2

MTX concentration in small cell lung carcinoma intracerebral and s.c. xenographs in the nude rat following i.v. or i.e.MTX administration with and without osmotic BBB opening

MTX, 4 mg/kg, was administered i.v. or i.e. to nude rats symptomatic with NCI-N417D human small cell lung carcinomaintracerebral xenograft immediately after i.e. saline or mannitol (BBB opened). Animals were sacrificed 30 min after MTXadministration. All animals infused with mannitol had 2+ or 3+ Evan's blueialbumin staining. Nonneural tissue values for

all groups were combined since there were no statistical differences among the 4 groups.MTX (ng/g)a

Control barrier OpenedbarrierGroup

III (i.v.Group I (i.v. MTX Group II (i.e. MTX MTX after manni-

Xenografts after saline, n = 6) after saline, n = 9) toi, n =8)Intracerebral

TumorBrainaroundtumorBraindistant totumorBraincontralateral totumorSerum

(ng/ml)Nonneural

tissues from all groups(n=31)s.c.tumorLiverKidneyMuscleSpleenHeartLung530

Â±116* 923 Â±299 843 Â±79138

Â±34 316 Â±99 784 Â±7897Â±26 123 Â±20 875 Â±8873Â±14 109 Â±21 113Â±166950Â±1972 9556 Â±1178 621 3 Â±2681378

+832000Â±48091

93Â±1395627Â±58934Â±95738Â±891922Â±162Group

IV (i.e. MTXafter mannitol, n =

8)2275

Â±6482631Â±8983725Â±1377206Â±668663Â±873

a MTX deliveries to tumor, brain around tumor, and brain distant to tumor were significantly affected by the route of

MTX administration (P < 0.025) and by osmotic BBB opening (P < 0.0025) (2-factor analysis of variance). For furtherdetails of statistical analysis, see "Results."

6 Mean Â±SE.

When the MTX was given i.a. after i.e. saline infusion (GroupII), there was a 2-fold increase of MTX delivery to the tumor and

brain around tumor as compared to Group I. Similarly, whenMTX was given i.a. after i.e. mannitol (Group IV), there was a 3-

fold increase to tumor and brain around tumor compared toGroup III. Thus, route of MTX administration significantly (P <0.025) altered drug uptake in tumor and surrounding brain.Osmotic BBB opening, independent of route of administration,also significantly (P < 0.0025) increased drug delivery to tumor,brain around tumor, and brain distant to tumor. MTX administered i.v. after osmotic opening (Group III) resulted in 2- to 9-fold

increases compared to Group I. When MTX was admnisteredi.a. after osmotic opening (Group IV), there was increased delivery of MTX, ranging from 2.5-fold in tumor to 30-fold in brain

distant to tumor as compared with Group II. The greatest MTXdelivery to tumor and ipsilateral brain was obtained after i.a.administration in the presence of BBB opening. By this route ofadministration, the MTX content in the intracerebral tumor wasdouble that in the tumor at the s.c. site. The MTX levels in thes.c. tumor and serum were not statistically different in any of thegroups.

Thus, less MTX entered this small cell carcinoma of lung grownintracerebrally in contrast to the tumor grown in s.c. tissues.Osmotic BBB opening combined with i.a. drug administrationachieved drug levels in intracerebral tumor and surrounding brainequal to or greater than that found in the systemic tissues ors.c. tumor.

DISCUSSION

The critical requirement of an adequate xenograft model forthe evaluation of complex multimodality therapeutic approachesto malignant intracerebral tumors is easily evident. For instance,

in previous studies with the avian sarcoma virus-induced glioma

in the rat, we have demonstrated that, by combining i.a. drugadministration with reversible osmotic BBB opening, the deliveryof MTX to the tumor and the brain around the tumor wasincreased over that achieved by routine (I.e., i.v.) routes of drugadministration (8). However, the avian sarcoma virus-induced

glioma model is not sensitive to most chemotherapeutic agents,and it is not amenable to an investigation of "targeted" therapeu

tic studies, since monoclonal antibodies have not been developedagainst these tumors.

The present studies utilized the immunologically athymic(nude) rat for the study of drug delivery to an intracerebral tumor.The rat was selected because its size permitted cannulation ofthe carotid artery necessary for i.a. drug administration and anexamination of the effect of osmotic BBB opening on drugdelivery. Although Colston ef a/. (31) reported that the nude ratwas less able than the nude mouse to sustain a human tumorxenograft, we have had little difficulty in growing either humancarcinoma of the breast or small cell carcinoma of the lung inthis model. The present studies focused on the characterizationof the growth of human small cell carcinoma of lung in the nuderat both intracerebrally and s.c.

This human small cell carcinoma of lung tumor model hasprovided several very important observations. The characteristics of this tumor model make it very suitable for the evaluationof antitumor agent (chemotherapeutic drug or monoclonal antibody) delivery in the presence or absence of BBB opening. Amost important finding demonstrated in this model is that tumorinoculated and grown in the brain almost totally excludes (viathe BBB) the uptake of a protein marker (Evans blue:albumin),even though there is partial uptake of smaller-molecular-weight

markers (i.e., MTX and fluorescein). Evidence that this is not aproperty of the tumor cells is the greater uptake of Evans


2830




blue:albumin or the cytotoxic agent MIX to the same tumorgrown at a s.c. site. As in most human primary and metastaticmalignant brain tumors, permeability to drug was variable. Asreported by Gazdar et al. (38), the dose of tumor cells necessaryto produce a s.c. tumor was much greater than the dose necessary to produce an intracerebral tumor. The reason for thisdifference is not apparent.

In spite of these observations, the significance of the BBB indrug delivery to CNS tumors is still a matter of some controversy.Hiesiger ef al. (24) recently stated that the blood tumor barrier inC6 rat gliomas is already open and cannot be further openedosmotically, thus explaining why MIX entry into such tumordoes not increase after i.e. hyperosmolar mannitol. These observations are in contrast to our tumor model, which has a differential permeability between the intracerebral and the s.c. tumor.Analogously, initial clinical trials of i.v.-administered "Tc-gluco-heptanate and, at a different time point, 131l-tumor-specific mono

clonal antibody resulted in positive scans with the low-molecular-weight "Tc-glucoheptanate but negative serial scans with themonoclonal antibody over a 10-day period. Thus, this patient's

glioma, as with our tumor model, displays more permeability tolow-molecular-weight than to high-molecular-weight markers.4 In

addition, BBB opening increases the concentration of MTX delivered to the intracerebral tumor in both this model and our virallyinduced glioma model (8, 9).

It must be emphasized that the current studies only examinethe question of drug delivery as a method to characterize thistumor, not drug efficacy. However, this model provides an exciting opportunity to evaluate therapeutic efficacy of osmotic BBBopening as a factor in therapy of cancers in the CNS, sincehuman small cell carcinoma of the lung is very sensitive tochemotherapy (41), and since these are tumors to which highlyspecific monoclonal antibodies have been produced (36, 37).Because a steep dose-response curve seems to characterize

most tumors (42) and often the proliferating edge of these tumorshas an intact BBB (43), the utilization of osmotic modification tomaximize antitumor agent delivery, be it drug or monoclonalantibody,56 seems very appropriate. The relatively brief and

consistent latency period from the time of tumor inoculation tothe onset of neurological symptoms further enhances the valueof this model.

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Fig. 2. A, coronal section of a nude rat brain 17 days after 4 x 105 NCI-N417D human small cell lung carcinoma cells were implanted in the right hemisphere. Prior to

sacrifice, Evans blue (2%, 2 ml/kg, i.v.) and fluorescein (10%, 0.12 ml, i.v.) were administered, followed by i.e. saline. Note the tumor is stained with fluorescein (M, 376)but not with Evans blue:albumin (M, 68,500). B, s.c. tumor in a nude rat 17 days after 27 x 10* NCI-N417D human small cell lung carcinoma cells were implanted.

Fluorescein and Evans blue were given as in A prior to i.e. saline. Note that the tumor is stained by both Evans blue albumin and fluorescein. C and D, intact (C) andcoronal (D) section of a nude rat brain 18 days after 4 x 105 NCI-N417D human small cell lung carcinoma cells were implanted in the right hemisphere. Prior to sacrifice,

Evans blue (2%, 2 ml/kg, i.v.) and fluorescein (10%, 0.12 ml, i.v.) were administered, followed by mannitol infusion into the right internal carotid artery. The tumor andsurrounding brain in the distribution of barrier opening are stained with Evans Wue:albumin and fluorescein (green due to blue and yellow).


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Fig. 1. A, brain tumor interface of nude rat brain 23 days after 4x10" NCI-N417D human small cell lung carcinoma cells were implanted in the right hemisphere.

There are neovascularity, a sharp tumor edge, and little surrounding cerebral edema. H & E, x 100. B, high magnification of section of Fig. 1/1. The tumor cells are largerthan the typical human small cell lung tumor, and mitotic figures are frequent. H & E, x 450.


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1985;45:2827-2833. Cancer Res Edward A. Neuwelt, Eugene P. Frenkel, Anthony N. D'Agostino, et al. Blood-Brain BarrierModel to Evaluate Antitumor Agent Delivery across the Growth of Human Lung Tumor in the Brain of the Nude Rat as a

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Growth of Human Lung Tumor in the Brain of the Nude Rat as ... · tumor-specific monoclonal antibodies have been developed to this neoplasm (36,37). The human small cell tumors were