Proc. Natl. Acad. Sci. USAVol. 84, pp. 891-895, February 1987Neurobiology

Imaging of a glioma using peripheral benzodiazepinereceptor ligands

(brain tumor/positron emission tomography/flunitrazepam/autoradiography)

SIMON STAROSTA-RUBINSTEIN*, BRIAN J. CILIAXt, JOHN B. PENNEY*, PAUL MCKEEVERt,AND ANNE B. YOUNG*§Departments of *Neurology, 1Pathology, and tPharmacology, University of Michigan, Ann Arbor, MI 48109

Communicated by Stanley M. Garn, October 20, 1986

ABSTRACT Two types of benzodiazepine receptors havebeen demonstrated in mammalian tissues, one which is local-ized on neuronal elements in brain and the other, on glial cellsand in peripheral tissues such as kidney. In vivo administrationof 3H-labeled PK 11195 [1-(2-chlorophenyl-N-methyl-N-(l-methylpropyl)-3-isoquinoline carboxamide] or [3H]flunitraze-pam with 5 mg of clonazepam per kg to rats with intracranialC6 gliomas resulted in high levels of tritiated-drug binding tothe tumor as shown by quantitative autoradiography. Phar-macological studies indicated that the bound drugs labeled theperipheral benzodiazepine binding site. Binding to the periph-eral benzodiazepine site was confined primarily to malignantcells with little binding to adjacent normal brain tissue or tonecrotic tissue. Tumor cell binding was completely inhibited bypreadministration of the peripheral benzodiazepine blockingagent PK 11195 at 5 mg/kg. The centrally selective benzodi-azepine ligand clonazepam had no effect on PK 11195 bindingto the tumor cells. When binding to other tumor cell linesgrown in nude mice and nude athymic rats was evaluated, littleor no peripheral benzodiazepine binding was detected onhuman pheochromocytoma (RN1) and neuroblastoma (SK-N-MC, SK-N-SH) tumor cells, respectively. However, high den-sities of peripheral benzodiazepine binding sites were observedon tumors derived from a human glioma cell line (ATCC HITB14, U-87 MG). The presence of high concentrations of specificperipheral benzodiazepine receptors on glial tumors suggeststhat human primary central nervous system tumors could beimaged and diagnosed using peripheral benzodiazepine ligandslabeled with positron- or y-emitting isotopes,

Primary tumors of the central nervous system account for1.2% of all autopsied deaths (1-3). Each year in the UnitedStates 17,000 new cases of primary intracranial neoplasm arediagnosed to give an annual rate of 8.2 persons with newlydiagnosed brain tumors per 100,000 population. Gliomas arethe most common type of primary central nervous systemtumor and account for about one-half of confirmed primaryintracranial neoplasms (1-3). The current methods for imag-ing central nervous system tumors depend either on agentsthat image the disruption of the blood-brain barrier by thetumor or on agents that image edema and density changeswithin the tumor and surrounding brain tissue (4-8). Thecurrent methods, however, cannot distinguish betweenhealthy brain and brain infiltrated with tumor cells. Further-more, the methods rarely show changes that are specific tothe tissue of tumor origin and do not measure tumor viability.A vigorous interest in imaging and delivering radionuclides

and antineoplastic agents to gliomas has emerged from thedevelopment of hybridoma and monoclonal antibody tech-nology (9-12). Xenographs using 131I-labeled anti-glioma

monoclonal antibodies have successfully imaged subcutane-ous gliomas in athymic mice and intracranial gliomas inathymic rats (9-12). These new technologies are unlikely toreplace computerized tomography or magnetic resonanceimaging for examining brain tumors but are preparing theground for innovative forms of tumor detection and therapy.We report here the successful imaging of a central nervoussystem glioma using in vivo ligands for the peripheral ben-zodiazepine binding site.Two classes of benzodiazepine binding sites have been

identified in mammalian tissues. One class of sites is locatedon neurons and represents the sites at which benzodiazepineligands are thought to exert their antianxiety, anticonvulsant,and muscle relaxant effects (13-15). High-affinity ligands forthe second class of benzodiazepine binding sites (the "pe-ripheral" binding site) have been shown to bind to astrocytesand rodent glial tumor lines in homogenate studies but bindvery little to normal brain (16-20). In a search for an agentthat could actually image infiltrating glioma cells in vivo, weinvestigated the use of peripheral benzodiazepine ligands forimaging glial tumor cells directly. Specifically, we haveinvestigated the ability of intravenously administered[3H]flunitrazepam and 3H-labeled PK 11195 [1-(2-chlorophen-yl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxam-ide] to label C6 glioma tumors in rat brain.

MATERIALS AND METHODS

[3H]Flunitrazepam (78.5 Ci/mmol; 1 Ci = 37 GBq) wasobtained from Amersham. 3H-labeled PK 11195 (74.5Ci/mmol) was obtained from New England Nuclear.Clonazepam, flunitrazepam, chlordiazepoxide, and Ro5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine-2-one or 4'-chlordiazepam] weregenerously donated by P. F. Sorter of Hoffmann-La Roche.PK 11195 was kindly donated by G. Le Fur of PharmukaLaboratoires. C6 glioma and U-87 MG glioma cell lines wereobtained from the American Type Culture Collection. Pheo-chromocytoma (RN1) and neuroblastoma (SK-N-MC andSK-N-SH) cell lines were donated by M. Tobes and S.Jaques, University of Michigan. Wistar rats, nude athymicrats, and nude athymic mice were purchased from SpartanResearch.

Wistar rats, 150-200 g were anesthetized with ketamine at1 mg/kg and xylazine at 0.4 mg/kg i.m. and then injectedintracerebrally into the right hemisphere with 5 x 105 C6glioma cells in 2.5 a1 of minimal essential medium plus 10%(vol/vol) fetal calf serum (21-23). After 2 weeks, the animals

Abbreviations: PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide; Ro 5-4864, 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine-2-oneor 4'-chlordiazepam.§To whom reprint requests should be addressed at: NeuroscienceLaboratory Building, 1103 East Huron, Ann Arbor, MI 48104.

891

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. In vitro benzodiaze-pine binding to C6 glioma in ratbrain. [3H]Flunitrazepam binding(at 4.5 nM) was determined invitro in the presence and absenceof 1 AM clonazepam and/or 1 ,uMPK 11195. (A) Autoradiogram of[3H]flunitrazepam binding in pres-ence of 1 ,M PK 11195. Binding isalmost exclusively to normal brainand avoids areas of tumor. (B)Autoradiogram of [3H]flunitra-zepam in presence of 1 ,uM clona-zepam,. Binding is predominantlyto tumor and choroid plexus. (C)Autoradiogram of [3H]flunitra-zepam binding in presnce of both1 ,uM clonazepam and 1 ,uM PK11195. Virtually no binding re-mains in normal brain or the brain

*s!' tumor. (D) Cresyl violet stain ofsame section that produced auto-radiogram in A. Note the largetumor in the right hippocampusand diencephalon. (x2.2.)

were anesthetized with ether, catheterized with arterial andvenous lines, and then allowed to recover for 3 hr from theanesthesia. All animals were then given clonazepam i.v. (5mg/kg) [vehicle, dimethyl sulfoxide/ethanol, 1:1 (vol/vol),0.1 ml/kg] and half were also given PK 11195 (5 mg/kg) 30min prior to administration of [3H]flunitrazepam (250 ,Ci).Additional animals were given 3H-labeled PK 11195 (125 ,Ci)i.v. with no pretreatment. In all animals, arterial bloodsamples were withdrawn at 2-min intervals up to 10 min. Tenminutes after the radioligand injection, animals were decap-itated. Their brains were then rapidly removed, frozen in dryice, and mounted on tissue pedestals. Twenty-micrometersections were cut on a cryotome, mounted on gelatin-coatedglass slides, and dried at 600C. Sections of brain were alsosolubilized, and the radioactivity was extracted and analyzedfor metabolites by TLC.

In parallel with the in vivo studies, a series of mice and ratswere inoculated intracerebrally with C6 glioma cells (Wistarrats), human gliom4 (U-87 MG) cells (nude athymic rats), andhuman neuroblastoma cells (nude athymic mice). After 2-6weeks, the rodents were decapitated, and the brains wereremoved and evaluated for in vitro benzodiazepine binding(24, 25). Dose-response curves for [3H]flunitrazepam binding(5 nM to 1.5 ,M) were determined by incubating 20-gmsections in 50 mM Tris!HCl, pH 7.4, in the presence andabsence of 1 ,uM clonazepam or 1 AM PK 11195. After a

60-min incubation, sections were rinsed for 5 min in coldbuffer and dried under a stream of cool air.

Sections from both the in vivo and in vitro experimentswere then mounted in x-ray cassettes along with appropriatestandards, apposed to Ultrofilm 3H (LKB) and exposed for2-4 weeks. Resultant autoradiograms were analyzed asdescribed (24-26). After exposure, the sections were

postfixed over paraformaldehyde vapors for 1 hr at 60°C andstained with cresyl violet or hematoxylin and eosin forhistological verification of tumor type and location.Comparisons between the distribution of tumor cells and

localization of receptor binding were made using a Leitzmicroscope fitted with a camera lucida. Each region of the

stained tissue was compared to the corresponding region ofa photographic enlargement of the autoradiogram generatedby that section.

RESULTSHistology of Tumors. All tumors could be distinguished

easily from normal brain. Neuroblastoma and pheochromo-cytoma tumors showed the typical histologic features ofsmall, dark lymphocyte-like cells and basophilic, granularcells, respectively. Human U-87 MG gliomas were smallcompact tumors of pleomorphic cells with dense, hyperchro-matic nuclei. C6 glioma tumors were very hypercellular withup to two mitotic figures per 20x fieldt The nuclei weremoderately pleomorphic with many round and oval nuclei.Many nuclei were hyperchromatic with clumped chromatin.Some nuclei had marginated chromatin. Within the tumorwere occasional microcysts. There were also areas ofcoagulative necrosis with central cavitary necrosis in ageographic pattern. There were rims of pseudopalisadingaround the necrotic areas. The borders of the tumor were

Table 1. Central and peripheral benzodiazepine binding tovarious tissues of normal rat and to human tumors

[3H]Flunitrazepam,pmol/mg of protein

Central neuronal PeripheralTissue receptor binding site

Rat kidney <0.01 1.33Rat cerebral cortex 1.04 <0.01Rat C6 glioma <0.01 2.02Human pheochromocytoma (RN1) 0.05 0.07Human neuroblastoma (SK-N-SH) <0.01 <0.01Human neuroblastoma (SK-N-MC) <0.01 <0.01Human glioma (U-87 MG) <0.01 0.80

Binding in vitro was done at 4.5 nM [3H]flunitrazepam. Values arethe mean of binding in three animals and varied less than 15%.

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Proc. Natl. Acad Sci. USA 84 (1987)

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Proc. Natl. Acad. Sci. USA 84 (1987) 893

FIG. 2. [3H]Flunitrazepam binding (at 4.5 nM) in the presence of1 AM clonazepam to a section of rat brain with a human U-87 MGglioma. The U-87 MG cells were innoculated into the brain of a nudeathymic rat 4 weeks prior to death. The animal was decapitated, thebrain was removed rapidly and frozen on dry ice. Twenty-microm-eter sections were mounted onto gelatin-coated slides, and thesections were assayed for peripheral binding sites in vitro. The singlearrow indicates the tumor, and the double arrows point to the choroidplexus. (x 2.3.)

moderately diffuse with numerous short fingers or rests oftumor cells extending beyond the margin of the tumor.

In Vitro Autoradiographic Characterization of PeripheralBenzodiazepine Binding Sites on Tumors. Initial studies fo-cused on characterizing the types of binding sites on tumorlines using in vitro autoradiographic techniques. Incubationof sections in [3H]flunitrazepam in the presence and absenceof 1 ,M clonazepam was used to distinguish peripheral andcentral benzodiazepine binding sites, respectively. Bindingto central sites was inhibited preferentially by classicalbenzodiazepines including clonazepam, diazepam, flunitraz-epam, and chlordiazepoxide as well as by certain nonbenz-odiazepines including the 1-carbolines (25). Binding to theperipheral sites was inhibited selectively by PK 11195 and Ro5-4864 but not by clonazepam. [Clonazepam binds predom-inantly to the central receptor but has no actions at theperipheral binding site (15-17, 20). Conversely, Ro 5-4864

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and PK 11195 have selective actions at the peripheral bindingsite but not at the central receptor (16, 17, 20, 27-29).Flunitrazepam and diazepam act at both sites (17, 18)].

[3H]Flunitrazepam binding to peripheral sites on brainsections from rats with C6 gliomas indicated a high density ofthese sites on the C6 glioma cells (Fig. 1). Binding to thecentral sites, however, completely avoided areas of tumorcell growth. Binding to the tumor cells was affected only bythose drugs known to be active at the peripheral site-i.e.,flunitrazepam, PK 11195, and Ro 5-4864, but not clonaze-pam. The [3H]flunitrazepam binding to C6 glioma cells had aKd of 44 nM and a Bma,, of 17 pmol/mg of protein, which issimilar to the binding of [3H]flunitrazepam to kidney (10pmol/mg of protein). The Bmax of binding to C6 glioma cellswas 7 times the Bmax of binding to central benzodiazepinereceptors in dentate gyrus of rat hippocampus (2.50 pmol/mgof protein) and 20 times that in rat striatum (0.86 pmol/mg ofprotein).

Sections assayed for peripheral and central benzodi-azepine binding were counterstained with cresyl violet.Direct comparisons could be made between areas with intacttumor cells and those in which the tumor had undergonenecrotic changes. Binding was present in all nonnecroticareas. In areas where there was necrosis, binding was absentor greatly reduced.

Brains from nude athymic rats and nude athymic mice withtumors derived from human U-87 MG glioma, neuroblasto-ma, and pheochromocytoma cell lines were examined for[3H]flunitrazepam binding (Table 1). Peripheral benzodi-azepine binding sites were present in high density on U-87MG glioma tumors, but were very low on the pheochromo-cytoma tumors, and absent on the neuroblastoma tumors. Nocentral benzodiazepine binding sites were detectable on U-87MG glioma or neuroblastoma tumors and were minimallydetectable on pheochromocytoma tumors. The density ofperipheral binding sites on U-87 MG gliomas (Fig. 2) wasabout half that observed in kidney and C6 gliomas.

In Vivo Imaging of Brain Tumors by Autoradiography. Invivo administration of [3H]flunitrazepam to animals with C6

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FIG. 3. In vivo autoradiogramsand cresyl violet sections of C6gliomas in rats. (A) Autoradio-gram of a 20-,um section of ratbrain through a C6 glioma of a rattreated with clonazepam at 5mg/kg i.v. 30 min before an i.v.bolus of 250 ,Ci of [3H]flunitraze-pam. Note the dense binding overthe right hemisphere with a cleararea in the center (arrow). Thebackground binding to brain isminimal. (B) Cresyl violet stain ofthe section in A that produced theautoradiogram. Microscopic ex-amination of the area outlined bythe arrows indicated necrotic de-bris. (C) Autoradiogram of a sec-tion from another animal with atumor treated identically to that inA except that the rat receivedpretreatment with both clonaze-pam at 5 mg/kg and PK 11195 at 5mg/kg. Note presence of onlybackground binding in brain withno evidence of label in the tumor(see D). (D) Cresyl violet stain ofsection that produced autoradio-gram in C. Note presence of largeright hemisphere tumor. These re-sults have been replicated in fiveadditional animals. (x2.1.)

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FIG. 4. In vivo labeling ofC6 glioma with 3H-labeled PK 11195. (A) Autoradiogram of a section of rat brain from an animal given an i.v. bolusof 125 ,uCi of 3H-labeled PK 11195 10 min before death. Note absence of binding to normal brain tissue but dense binding over clusters of tumorcells seen on cresyl violet of same section in B. The densely stained area in B (single arrow) displays only moderate 3H-labeled PK 11195 bindingbut many of the cells in this region appear to be undergoing autolysis when examined at higher magnification. Double arrows indicate braininfiltrated with tumor cells. (C) Higher magnification of outlined area of autoradiogram shown in A. Note correspondence of heavy 3H-labeledPK 11195 binding seen on autoradiogram (arrows) to tumor cells seen in corresponding enlargement of cresyl violet stain (arrows) (D). The resultspresented in this figure have been replicated in 10 additional animals. (A and B, x2.3; C and D, x7.2.)

gliomas pretreated with clonazepam at 5 mg/kg demonstrat-ed striking images of the tumors on a background of minimalbinding to normal brain (Fig. 3 A and B). Under theseconditions, 99% of binding to the central receptors wasblocked with clonazepam but [3H]flunitrazepam binding tothe peripheral receptor was unaffected. Binding was veryhigh in the tumor but appeared to avoid necrotic areas.Inspection of the stained sections indicated a high correspon-dence between clusters of tumor cells and high concentra-tions of [3H]flunitrazepam binding. Metabolism studies indi-cated that >90% of radioactivity represented authentic[3H]flunitrazepam in normal brain at 10 min after injection. Itis unlikely that the image was secondary to a local break-down of the blood-brain barrier since pretreatment of ratswith both clonazepam (to block central receptors) and PK11195 (to block peripheral receptors) completely blockedthe binding of [3H]flunitrazepam to the tumor (Fig. 3 C andD).The tumors were also imaged after in vivo intravenous

administration of 3H-labeled PK 11195 (Fig. 4 A and B).3H-labeled PK 11195 binding correlated with the presence ofclusters of tumor cells (Fig. 4 C and D). Labeling by both[3H]flunitrazepam and 3H-labeled PK 11195 indicated thatbinding to the tumor was to peripheral type of benzodi-azepine receptors. Both [3H]flunitrazepam and 3H-labeledPK 11195 entered brain rapidly although preliminary evi-dence suggests that the latter enters somewhat more slowlythan the former.When cresyl-violet-stained sections were inspected and

compared to the autoradiograms, a striking correspondencebetween receptor binding and intact cells could be seen.

Rests of infiltrating tumor cells were imaged autoradiographi-cally. In areas of necrosis, ligand binding was minimal.

DISCUSSIONComputerized x-ray tomography scan of the head can estab-lish with high probability the presence of an intracranialneoplasm (4-8). The diagnosis is based on the presence ofbrain mass, abnormality of the blood-brain barrier, andedema. In many cases, however, computerized x-ray tomog-raphy scanning cannot establish tumor histology nor the fullextent of the tumor borders. Viable tumor can often be foundto extend considerably beyond an enhancing tumor ring andto form islets in the central area of low attenuation (4-8).There are limitations in defining tumor extent within edem-atous areas or areas of brain with infiltrating tumor cells. Inpatients who have hemorrhaged into a tumor, it is oftenimpossible to identify the tumor or determine its extent (4-8).The ability to image the actual abnormal cells within aneoplasm would provide a sensitive test for the diagnosis andmanagement ofbrain tumors. The demonstration in this studythat the in vivo administration of peripheral benzodiazepineligands can image rodent and human gliomas suggests thatthis technique could be useful in subsequent studies inhumans with brain tumors. This technique would be partic-ularly useful, if as suggested in these animal studies, theimaging of tumor cells is not dependent on the breakdown ofthe blood-brain barrier and if only viable tumor cells but notnecrotic ones are imaged.

In contrast to central benzodiazepine binding sites that arepresent on a subunit of the neuronal membrane receptor for

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Proc. Natl. Acad. Sci. USA 84 (1987) 895

y-aminobutyric acid, peripheral binding sites appear to belocalized in cell nuclei and/or in mitochondria (16, 30, 31).Nevertheless, the role of peripheral benzodiazepine bindingsites on tumor cells is unknown. The sites have beenidentified on neuroblastoma (NB41A3) (19), thymoma (AKRmouse thymoma line) (32), the Friend erythroleukemia line(20, 33), and mouse melanoma lines (34). In some of the lines,peripheral benzodiazepine ligands alter tumor cell growthand differentiation (32-34). Whether the binding sites on theC6 glioma and U-87 MG glioma lines control aspects ofgrowth and differentiations has yet to be determined.The presence of receptors for neurotransmitters and

neuromodulators on astrocytes and certain cultured tumorcell lines has been observed. These receptors include y-aminobutyric acid, ,-adrenergic, muscarinic cholinergic, andother receptors (35-42). Most of these receptors, however,are also present on nerve cells. Thus, ligands for imagingthese receptors would be unlikely to serve as agents thatcould easily distinguish normal from abnormal brain. Periph-eral benzodiazepine ligands on the other hand, bind verypoorly to normal rat and human brain with the highestconcentration of sites (Bmax) approaching 300 fmol/mg ofprotein (29). These same ligands label C6 and U-87 MGgliomas very densely. The presence of these binding sites onhuman glioma tumor cell lines suggests several areas forfuture research. These ligands could be labeled with positron-or Ry-emitting isotopes and used for imaging brain tumors inman by positron or single-photon emission tomography. Theclinical advantages of determining ligand binding in vivo inman is that binding could define regions of viable tumor forbiopsy and for surgery, help the clinician determine thelocation of the tumor borders and the presence infiltratingcells, and aid in assessing response to therapy. The reactionof highly specific ligands with glial tumors may also make itpossible to design cytotoxic agents that could be used to treatspecific brain tumors. Furthermore, if tumors are shown tohave specific profiles of different receptor binding sitesdepending on their tissue of origin and degree of malignancy,it might be possible to diagnose tumors based on the resultsof noninvasive imaging.

We thank Zane Hollingsworth and Darrell Debowey for technicalassistance and Jan Pappas for secretarial assistance. Ro 5-4864 wasgraciously donated by Dr. P. F. Sorter of Hoffmann-La Roche andPK 11195 by Dr. G. Le Fur of Pharmuka Laboratoires. We thankDrs. Sanford Jaques and Michael Tobes for donating the neuroblas-toma and pheochromocytoma cell lines. This work was supported byGrant NS 15655 from the National Institute of Neurological andCommunicative Disorders and Stroke (J.B.P. and A.B.Y.).

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