[CANCER RESEARCH 58. 4871-4879. November 1. 19981

Use of Chlorotoxin for Targeting of Primary Brain Tumors1

Liliana Soroceanu, Yancey Gillespie, M. B. Khazaeli, and Harald Sontheimer2

Dcptirlrnenls of Neurohiology. Brain Tumor Research ¡Mhoralories—Divisionof Ne iirosiirgery IL.S. H. S.I, Surgery [Y. Gì.ami Medicine (M. lì.K.J. University of Allibitimi ut

Binnin^hinn. Rirniingluiin. Aìnhumii.35294

ABSTRACT

Gliomas are primary brain tumors that arise from differentiated glialcells through a poorly understood malignant transformation. Althoughglioma cells retain some genetic and antigenic features common to glialcells, they show a remarkable degree of antigenic heterogeneity andvariable mutations in their genome. Glioma cells have recently beenshown to express a glioma-specific chloride ion channel (GCC) that is

sensitive to chlorotoxin (CTX), a small peptide purified from Leiurusquinquestriatus scorpion venom [N. Ullrich et al.. Neuroreport, 7: 1020-

1024, 1996; and N. Ullrich and H. Sontheimer, Am. J. Physiol. (CellPhysiol.), 270: C1511-C1521, 1996]. Using native and recombinant 125I-

labeled CTX, we show that toxin binding to glioma cells is specific andinvolves high affinity [dissociation constant (A',,i = 4.2 nM] and low affinity

(Ka = 660 nM) binding sites. In radioreceptor assays, I25l-labeled CTX

binds to a protein with M, = 72,000, presumably GCC or a receptor that

modulates GCC activity. In vivo targeting and biodistribution experimentswere obtained using I25I- and I3ll-labeled CTX injected into severe com

bined immunodeficient mice bearing xenografted gliomas. CTX selectivelyaccumulated in the brain of tumor-bearing mice with calculated brain:

muscle ratios of 36.4% of injected dose/g (ID/g), as compared to12.4%ID/g in control animals. In the tumor-bearing severe combinedimmunodeficient mice, the vast majority of the brain-associated radioac

tivity was localized within the tumor (tumor: muscle ratio, 39.13% ID/g;contralateral braimmuscle ratio, 6.68%ID/g). Moreover, 131I-labeled CTX

distribution, visualized through in vivo imaging by gamma ray camerascans, demonstrates specific and persistent intratumoral localization ofthe radioactive ligand.

Immunohistochemical studies using biotinylated and fluorescentlytagged CTX show highly selective staining of glioma cells in vitro, in situ,and in sections of patient biopsies. Comparison tissues including normalhuman brain, kidney, and colon were consistently negative for CTXimmunostaining. These data suggest that CTX and CTX-conjugated molecules may serve as glioma-specific markers with diagnostic and thera

peutic potential.

INTRODUCTION

Gliomas are among the most deadly forms of cancer for whicheffective treatment strategies are currently lacking. Indeed, despite theoverall advances in chemo and radiation therapy regimens, the mediansurvival of glioma patients has been unaltered in the last 20 years (1,2). This lack of success in treatment of gliomas seems to be due in partto their high resistance to radiation and chemotherapy but additionallyto their unusual ability to disperse and invade healthy brain tissue.Glioma cells display a remarkable level of heterogeneity within andamong gliomas, as manifested through clonal variation in growthpotential, differential genetic alterations, variable drug resistance, andexpression of antigens.

The molecular mechanisms that underlie the malignant transformation of glial cells to become gliomas are poorly understood. As withother cancers, a series of genetic alterations seem to be obligatory for

Received 6/23/98; accepted 9/2/98.The costs of publication of this anide were defrayed in pan by the payment of page

charges. This article must Ihereforc be hereby marked atlvenisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by NIH Grant ROI NS 36692 and American Cancer

Society Grant RPG-97-083." To whom requests for reprints should be addressed, at Department of Neurobiology.

CIRC 545. 1719 6th Avenue South. Birmingham. AL. 35294-0021.

this process. These include variable mutations of p53. pl6, cdk4. andRB protein, amplification or overexpression of epidermal growthfactor receptor or MDM2 genes (mostly found in glioblastomas thatarise "de novo").

Because of the lack of glioma specific markers, unequivocal diagnosis of gliomas requires tissue biopsy and relies primarily on his-

topathological criteria. Histopathologically, gliomas are highly cellular with marked pleomorphism and often contain multinucleate giantcells. Vascular proliferation throughout the lesion and the presence offocal areas of necrosis are histological features necessary for thediagnosis of GBM.1 the most malignant and prevalent form of glioma

in adults. Immunohistochemical studies show variable expression ofthe astrocyte-specific antigen glial fibrillary acidic protein; protein

levels were found to decrease with an increasing degree of malignancy. Vimentin expression is a common but not a reliable marker.Presently, no antigen or marker that could serve to selectively labelglioma cells is available. To date, two other reports describe proteinsspecifically expressed by glioma cells: brain-enriched hyaluronan

binding protein (3) and the novel interleukin 13 receptor a (4).We recently identified and characterized a chloride ion channel that

seems to be abundantly expressed in glioma cells but absent in normalbrain tissue (5. 6). GCC activity could be recorded biophysically incultured glioma cells (5. 6) and in freshly prepared vibratome sectionsfrom patient biopsy tissue (7). Channels were absent in nontumorbrain tissue and in tumor cells of nonglial origin. Because the activityof GCC seems to be modulated by rearrangements in the cells cy-

toskeleton (8). it has been proposed that these channels may facilitateshape changes during glioma cell migration and invasion. Interestingly, GCC expression in situ correlates with the histopathologicaltumor grade (7). Only 40-45% of low-grade astrocytomas (WHOgrade I-II) express GCC, whereas >90% of all of the high-grade

tumors (WHO grade III) and essentially all of the GBMs expressGCC. This correlation suggests that GCC may be a candidate proteinto serve as a glioma-specific marker and may be useful for diagnostic

and therapeutic purposes. Biophysical studies also demonstrated thatCTX, a 36-amino-acid peptide isolated from scorpion venom (Leiurus

quinquestriatus), effectively inhibits currents through GCC with-80% block at 600 nM CTX (5, 6). In the present study, we have

generated CTX in synthetic and recombinant forms and have attachedmoieties including I25I, ml, biotin, and various fluorophores to the

CTX molecule that subsequently allowed its detection. We have usedthese tagged CTX molecules to investigate specific binding of CTX toglioma cells in vitro and HI situ. These studies show that iodinatedCTX selectively binds malignant glioma cells and can be visualizedby in vivo imaging of animals bearing xenografted tumors. Moreover,biotinylated or fluorescently tagged CTX molecules reliably detectglioma cells in patient biopsies, which suggests that this moleculecould serve as a glioma-specific marker with diagnostic and thera

peutic potential.

1The abbreviations used are: GBM. gliohlastoma multiforme: CTX. chlorotoxin:

GCC, glioma chloride channel: HRP. horseradish peroxidase: Kd. dissociation constant:X-Gal. 5-bromo-4-chloro-3-mdolyl-b-n-galactopyranoside; TEPSA, 3-aminopropyltri-ethoxysilane: %ID/g. % (percentage) of injected dose/g: UAB. University of Alabama alBirmingham.

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CTX TARGETING OF HUMAN CHOMAS

MATERIALS AND METHODS

Cell Lines and Cell Culture. The following human glioma cell lines wereused: (a) D-54 MG; (bìU373MG; (r) U105MG; (dìU251MG (all from Dr.

D. D. Bigner, Duke University. Durham, NC); and (e) neuroglial derivedSK-MG-I (from G. Cairncross, University of Ontario, London. Ontario, Can

ada). Control cell lines were: (a) Te671, a rhabdomyosarcoma cell line fromAmerican Type Culture Collection: and (b) Balb 3T3 fibroblasts. In addition,we used primary cultured rat astrocytes as control glial cells (see below). Cellswere maintained in DMEM mixed 1:1 with Ham's F-12 medium, supple

mented with L-glulamine (2 mM) and 10% fetal bovine serum (Life Technologies). Cells were harvested from logarithmic phase-growth cultures by briefexposure to 0.5% trypsin and 0.53 HIMEDTA (Life Technologies). The D-54

MG cell line was transfected with the pCMVLticZll plasmid (American TypeCulture Collection) using the standard Lipofectamine method, according to themanufacturer's instructions (Life Technologies). Stably transfected clones

were selected by growth in media containing neomycin (G418, 5(X) /xg/ml).Verification of the incorporation of marker gene was made by immunohisto-

chemical staining for X-Gal (Promega Corp.. Madison, W[). Briefly, cells were

fixed in 4% paratbrmaldehyde. rinsed in PBS. and incubated overnight inX-Gal substrate solution (0.2% X-gal, 10 mM sodium phosphate (pH 7.0), 150mM NaCl, 3.3 mM K4Fe(CN),,'3H2O, and 3.3 mM K,Fe(CN)6], according to the

manufacturer's instructions (Promega Corp.).

Primary Astrocyte Cell Cultures. Cortical astrocytes were isolated fromSprague Dawley rat pups at postnatal day 0 or 1. Cortical tissues weredissected free and placed in ice-cold complete saline solution, consisting of

137 mM NaCl. 5.3 mM KC1, 2.0 mM CaCI2. 1.0 mM MgCU 25.0 mM glucose,and 10.0 mM HEPES acid (pH adjusted to 7.2 using NaOH). Meninges andblood vessels were then carefully removed, and the tissues were minced andincubated for 20 min at 37°Cin an enzyme solution consisting of complete

saline solution supplemented with 0.5 mM EDTA. 1.65 mM L-cysteine, and

30.0 units/ml papain (Worthington. Freehold. NJ). The enzyme solution wasremoved, and the cells were dissociated with a Pasteur pipette in completemedia |Earl's MEM (Life Technologies. Grand Island. NY), 10% PCS (v/v:

HyClone, Logan. UT). 5(X)unitsU/ml penicillin/streptomycin, and 20.0 mMglucose] supplemented with 7.5 mg/ml each. BSA and trypsin inhibitor. Cellswere then plated on polyornithine/laminin-coated coverslips at a density of1.2 X 104/cm2 or 8-well chamber slides and maintained in complete media inan atmosphere of 5% CO2:95% O2 at 37°C.The medium was replaced after

one day. Tissues treated in this manner produced cultures that were >95%positive for the glial-specific marker glial fibrillary acidic protein.

Intracranial Glioma Model. Female C.B.-17 SCID mice, ages 20-28

weeks, were anestheti/ed by i.p. administration of ketamine (20 mg/ml) plusxylazine (0.3 mg/ml) in saline at 0.07 ml/10 g of body weight. A midline scalpincision was made and an 0.5-mm burr hole was made 1.5-2 mm to the rightof the midline and 0.5-1.(X) mm posterior to the coronal suture. Tumor cells(IO6 cells/dose) resuspended in excipient solution were stereotactically in

jected using a 250-/xl Hamilton syringe with a 30-gauge needle mounted in a

Stoelting stereotaxic apparatus. The needle was advanced vertically throughthe burr hole to a depth of 2.5 mm and then slowly retracted 45-60 s afterinjection: the incision was then closed with a 9-mm Michel wound clip. Mice

were returned to sterile cages and were provided with sterile lab chow andsterile water ail libitum. Previous studies showed that the D-54 MG human

malignant glioma cells reliably grow in SCID mice brain and typically kill theanimals within 16-18 days (9). Thus, we elected an 8-9-day interval to allow

for tumor growth before reintervention through the same burr hole to deliverthe radiolabeled CTX (15 /¿Cil:sl-labeled CTX in 5 fil of PBS and 75 ¿iCi

'"[-labeled CTX/mouse) to both tumor-bearing and control (litter mates)

SCID mice. All of the animals received 0.8% Lugol solution in their drinkingwater 24 h before injection of the radiolabeled material to inhibit thyroiduptake of the iodine-labeled ligand.

In Vivo Localization and Biodistribution of Radiolabeled CTX. At 24,48, 72, and 96 h after tumor-bearing and control SCID mice were injected with'"l-labeled CTX, they were anesthetized and imaged using a large field-of-

view camera fitted with a pinhole collimator interfaced to a microcomputer toobtain a whole body scan. Each animal was imaged up to 1200 pixels(determined experimentally, as a value resulting in interprétablescintigramsabove background levels); scintigrams were analyzed using the Image Toolanalysis software. Both '"I- and I25l-labeled CTX-injected animals and their

control counterparts were killed at indicated time points for biodistributionstudies. Individual organs were removed, placed in preweighed tubes andprocessed for gamma scintillation counting. Radioactivity associated with eachorgan is expressed as %ID/g of tissue. In the case of tumor-bearing '"l-labeled

CTX-injected SCID mice, tumors were carefully removed from the right side

of the brain by dissection under the microscope and weighed and counted forassociated radioactivity. Results are presented as %ID/g of tissue and as ratiosof brain:muscle or livermuscle for the two groups of animals. Biodistributionexperiments were repeated three times using six to eight animals for each timepoint in each of the tumor-bearing and control groups.

Brain I2SI Autoradiography. I25l-labeled CTX-injected tumor-bearing

mice were killed at 48 h after injection of the radiolabeled material andperfused by intracardiac injection with PBS followed by 4% paraformaldehydein 10% sucrose. Brains were removed and fixed an additional 24 h at 4°C,

rinsed in PBS several times, embedded in OCT free/ing medium (Miles Inc.,Naperville, IL), and sectioned at 10 ¿¿mon a cryotome (Zeiss HM 505E):sections were mounted on TEPSA-coated slides. All of the subsequent steps

were performed under film safelight. Slides were coated with autoradiographyemulsion (Eastman Kodak) and exposed for 7 days in a light-tight slide boxcontaining Drierite at 4°C. For developing, we used the D-19 developer(Eastman Kodak); slides were dipped 3-5 min at 16°Cin D-19 solution,

washed for 3 min in distilled water, and placed in Rapid Fix Solution (EastmanKodak), prepared according to the manufacturer's instructions. Slides werethen washed thoroughly with water and counterstained with Mayer's hema-

toxylin.In Vitro Binding Assay. CTX was radiolabeled with ['~5I]sodium iodide at

the UAB Radiolabeling Facility using the chloramine-T method. Radioiodi-nated CTX was separated by reversed phase high-performance liquid chroma-tography. and radioactivity ranged from 3.6-5.8 Ci/mmol. Cells were grownfor 24 h in 24-well plates (2 X 10s cells/well). Subconfluent monolayers were

rinsed in ice-cold binding buffer (DMEM with 2% BSA) and then incubatedwith graded amounts of l:5I-labeled CTX diluted in binding buffer added to

duplicate wells in a total volume of 400 ¿il/wcll.For each concentration of125I-labeled CTX. duplicate wells received 100-fold molar excess "cold" CTX.

Four wells per plate received only binding buffer and were used to obtain cellcounts. Cells were maintained at 4°Cin a 10% CO2:90% O2 atmosphere on an

oscillating platform for 2-3 h. After incubation, supernatants were removed

and aliquots saved for counting; cells were then washed four times in bindingbuffer without BSA and incubated with solubilization buffer (1% TritonX-KX), 10% glycerol. and 20 mw HEPES (pH 7.2)1 for 30 min with agitation.

After solubilization, the contents of all of the wells were transferred tocentrifuge tubes, and insoluble debris was centrifuged in a microfuge (at13,000 X g for 20 min at 4°C).Aliquots from supernatants and cell pellets

were counted in a gamma scintillation counter to determine cell-associated andcell-"free" radioactivity. To the wells reserved for cell counting. 450 ¡i\of

1 x Trypsin-EDTA and 50 ¡uof trypan blue were added.I25l-labeled CTX Internalizaron Assay. D-54 MG cells were harvested

with trypsin/EDTA as described above and resuspended in binding buffer inpolypropylene tubes at a concentration of 6 x 10''cells/tube, 10 tubes for eachgroup. Cells were incubated (3 h at 4°C,95% O2:5% CO2) with variousdilutions (1.5-25 nM) of I25l-labeled CTX alone, with 100-fold molar excess"cold" CTX (each condition in duplicate), or with cell medium alone (for cell

counts) for 3-4 h at 4°Cwith agitation and 5% CO,:95% O2 atmosphere. After

incubation with the ligand. cells were washed three times with cold bindingbuffer without BSA to remove nonspecific binding and then resuspended in 2ml of medium, and one-half of each tube volume was transferred to new tubes."Old" tubes were maintained at 4°C,new tubes were transferred at 37°Cand

incubated for an additional 60 min, after which all of the tubes were washedonce with cold medium. Aliquots from washes were counted to determine"free" ligand. Cells were then resuspended in ice-cold 50 mM glycine-HCl (pH

2.5) in isotonic saline and incubated for 10 min to permit dissociation of boundligand. Afterward, cells were washed three times in 0.1 ml of ice-cold medium,and washes from each tube were pooled (membrane-associated radioactivity is

retrieved in acid washes). Aliquots from cells and washes were counted in agamma scintillator (Comprehensive Cancer Center, UAB). Cells incubatedwith medium alone were used to determine cell number.

Radioreceptor SDS-PAGE Analysis. Radioreceptor SDS-PAGE analysiswas performed using a modified version of the transforming growth factor-ßreceptor assay (10). Specifically, D-54 MG cells were plated overnight in

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CTX TARGETING OF HUMAN GLIOMAS

Fig. I. A, Scatchard plot analysis of '~5l-labeledCTX binding to D-54 MG human glioma cell line.

Data are from one representative experiment; bestfit indicates two binding sites receptor kinetics(Kd = 4.3 and 660 nM). This assay was repeated

four times with similar results. B, autoradiographyrepresenting SDS-PAGE analysis of lysates obtained after cross-linking of human glioma cellmembranes to I25l-labeled CTX; the blot demonstrates the presence of a single band (Mt —¿�72,000),corresponding to the CTX-binding receptor with theintensity considerably decreased by competitivebinding of "cold" CTX (Lanes I, and 3).

B

72kDa.

O 20 40 60 80 100 120 140 160 34BoundCTX(pMxIO2)

6-well plates at 2 x IO5 cells/ml. Cell monolayers were rinsed in bindingbuffer without BSA, then incubated for 3-4 h with 25 nM 12SI-labeled CTXalone or together with 100-fold molar excess "cold" CTX on an oscillating

platform at 120 rpm. Cells were washed in cold binding buffer (without BSA):5 ju.1of 27 mM disuccinimidyl suberate solution was added to each well andagitated for 15 min. Cells were rinsed briefly in detachment buffer [0.25 Msucrose, 10 mM Tris, 1 mM EDTA (pH 7.4), and 0.3 mM phenylmethanesul-

fonyl fluoride] and then scraped using a disposable teflon scraper. Aftercentrifugation ( 12,000 X g for 2 min) the supernatants were removed, and cellswere solubilized in 100 jul of solubilization buffer [125 mM NaCl. lOmM Tris,l mM EDTA (pH 7), 0.1% volume/volume Triton X-100] supplemented with10 /xl/ml protease inhibitor cocktails I and II (cocktail I = 1 mg/ml leupeptin,

1 mg/ml antipain, 5 mg/ml aprotinin, and 10 mg/ml soybean trypsin inhibitor,cocktail II = 1 mg/ml pepstatin, 1 mg/ml bestatin, and 30 mM phenylmeth-

anesulfonyl fluoride in dymethyl sulfoxide]. Cells were incubated (40 min,4°C)in solubilization buffer with end-over-end mixing for 40 min at 4°C.Cells

were centrifuged at 12.000 X g for 15 min, cell debris was removed, and thesupernatant was mixed with 1 volume of electrophoresis sample buffer [100mM Tris (pH 6.8), 20% glycerol, 2% SDS, 0.05% bromphenol blue, and 100mM DTT] and boiled for 3 min. A 10-/xg sample of each reaction (determinedby the Bio-Rad protein assay using the Coomassie Brilliant Blue G-250 dye)were loaded on precasted 4-20% gradient Tris-acrylamide minigels (Bio-Rad)

and electrophoresed at constant voltage (90 mV). After electrophoresis, gelswere fixed in 7% acetic acid and 25% methanol solution for 20 min, dried ina vacuum-operated gel dryer (Bio-Rad, model 583). and exposed to X-ray filmfor 7-10 days at -70°C.

Biotinylation of CTX. EZ-Link"»-Sulfo-NHS-LC-Biotin (Pierce Chemi

cals) was used to biotinylate CTX. The peptide was resuspended in 50 mMsodium bicarbonate buffer (pH 8.5) at a concentration of 1 mg/ml; 200 /xl ofCTX was incubated with 15 /xl of EZ-Link-Biotin reconstituted in distilledwater at 1 mg/ml (2-3 h at 4°C)with end-over-end mixing, according to themanufacturer's instructions. To remove excess biotin, the biotinylated peptide

was dialyzed against PBS using Slide-A-Lyzer® (Pierce Chemicals) membrane (M, cut-off, 3000) for 3 h in three changes of PBS at 4°C.Biotinylated,

dialyzed CTX-biotin was stored in aliquots in the freezer and used for immu-

nohistochemical studies at concentrations ranging between 0.5 and 1 ¡J.M.Cytochemical and Immiinohistochemical Studies. Cells were stained

using standard immunohistochemical techniques, as previously described (9).Glioma cells and control cell lines were cultured overnight in eight-wellchamber slide trays or on glass coverslips (2 X IO5 cells/ml): semiconfluent

cultures were fixed in 4% paraformaldehyde for 30-60 min at room temper

ature. After fixation, cells were washed with PBS, blocked for l h with PBScontaining 2% BSA and then incubated overnight with primary antibodies(biotinylated CTX or fluorescent-tagged CTX). Control wells were incubatedwith blocking buffer alone; for competition experiments, cells were preincu-bated with a 100-fold molar excess of native CTX for 2 h at room temperature

before immunostaining. Cells were visualized using various secondary agents.HRP-linked streptavidin (Pierce) at 0.1 mg/ml in conjunction with 3,3'-

diaminobenzidine-peroxidase substrate (Vector Laboratories), or rhodamine-conjugated streptavidin (Molecular Probes), according to manufacturer's in

structions. For double-staining experiments, cells were first reacted with theX-Gal substrate, as described in the "Cell Lines and Cell Cultures" section,

then processed for immunoperoxidase staining as described above. Slides werecovered using Aquamount (Fisher) or Fluoromount (Vector Laboratories) andviewed under epifluorescence or light microscope using standard procedures.Images were captured with a Leica microscope equipped with digital camera.

Staining of SCID Mouse Brain Sections. For demonstration of CTXbinding to glioma cells in .vim. intracranial tumors were induced in SCID miceusing D-54LacZ transfected cells. At 10-15 days after tumor induction,

animals were killed by intracardial perfusion of PBS. followed by 4%paraformaldehyde. Brains were removed and additionally fixed overnight inparaformaldehyde and 12% sucrose. After fixation, brains were rinsed severaltimes with PBS, sectioned coronally, and reacted overnight for X-Gal enzyme

immunochemistry, as described below; brain sections were then embedded inOCT freezing medium and sectioned. Sections, 8-10 /xm thick, were preparedon a cryotome, mounted on TEPSA-coated slides, and reacted for biotinylatedCTX immunoperoxidase reaction, as described below. Sections were counter-stained with Mayer's hematoxylin. dehydrated through increasing concentra

tions of ethanol and coverslipped (using Aquamount) for microscopical analysis. CTX binding was also observed by direct or indirectimmunofluorescence, using brain sections that had been incubated (at 4°C

overnight in the dark) with Oregon-Green5>0-tagged CTX or biotinylated CTX

in a humidified chamber. For direct detection of Oregon Green-labeled CTX,

slides were washed two times with PBS and coverslipped. To visualizebiotinylated CTX, slides were incubated (2 h, room temperature, in the dark)with rhodamine-linked streptavidin (Molecular Probes), washed, and cover-slipped. Fluorescence-tagged localization of CTX was visualized as described

above.Staining of Human Tissue Sections. Freshly resected brain biopsy sam

ples (obtained as waste tissues from UAB Neurosurgery, under an InstitutionalReview Board approved protocol) were embedded in OCT. sectioned (8-10jam) on a cryotome (Zeiss HM505E). and mounted on TEPSA-coated slides.

Sections were fixed in 4% paraformaldehyde for 30 min at room temperature,rinsed twice in PBS, and then blocked in 0.04% H,O2 in methanol for 10 minto inhibit the activity of endogenous peroxidase. After PBS washes, slides wereblocked with the avidin-streptavidin blocking reagent (Vector Laboratories,Burlingame) according to the manufacturer's instructions or with normal goat

serum (Vector Laboratories; 20% in PBS). Slides were then incubated withbiotinylated CTX (4 /¿g/ml)overnight, washed with PBS (three times, 10 mineach), then incubated (2 h, room temperature) with streptavidin-HRP (Pierce;0.2 mg/ml). After PBS washes (three times, 10 min each), we used a 3,3'-

diaminobenzidine substrate kit (Vector Laboratories) to develop the enzymatic-reaction. Counterstaining was done using Mayer's hematoxylin; slides were

subsequently dehydrated, coverslipped with Aquamount, and viewed under thelight microscope. Control sections were either incubated with secondary antibody alone or pre-treated with CTX for 30-45 min. washed, and then incu-

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CTX TARGETING OF HUMAN GLIOMAS

Control

Tumor 24h 48h

240,220200160160140-120-100-80-60-40-20-

0-n m240,220-72h

200.180-160.140-120-M0>Õ6o:60-1

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Fig. 2. A. hiodislrihulion of 1:'I-labeled CTX al four time points (upper panels} after intracrani.il administration in tumor-hearing and contro! SCID mice. Dala represent mean valuesfrom three experiments. The results (expressed as %ID/g tissuel show persistent accumulation of '''l-labclcd CTX within the xenografted tumor tissue in the right hrain of the animals.Stomach accumulation of iodine is also visihle and is most likely a result of the dchalogenaticn ol the radioligand (see also "Results"), /fiirs. SI) ; lesi analysis was performed, and

results for the righl-hrain values are shown: for all of the time points. P < 0.001 (»*».extremely significant). B. autoradiograms: coronal brain sections from glioma-hearing mice wereobtained at 4K h alter intracranial injection of 15 ¿¿Cii;ífil-labeledCTX. The four panels demonstrate selective accumulation of silver grains (arrows) within the human transplanted

tumor cells, whereas the host tissue is virtually radioactivity free. Burs. 1.3 mm and 400 /xm (upper left and right photomicrographs, respectively) and 100 u.m (liwerphotomicrographs).

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CTX TARGETING OF HUMAN GLIOMAS

Fig. 3. Tissue distribution of ml-labeled CTX at

24 and 72 h after intracranial injection of 50 ¿tCiradiolabeled toxin. The biodistribution results(mean values from two experiments) demonstratetumor accumulation of the radiolabeled toxin (lower two panels', bars, SD). Accumulation of ml-

labelcd CTX in other organs (e.g., the stomach) isalso noticeable (see comment in "Results"). The

upper four panels show representative whole-body

mouse scintigrams obtained by gamma ray cameraimaging at the indicated time points. Radioactivityis seen as dark areas in the brain (h}. stomach (si).and urinary bladder (a.b.). Note the difference inintensity of the brain-accumulated radioactivity between the control and tumor-bearing animals.

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bated with biotinylated CTX as described in the "Cytochemical and Immuno-histochemical Studies" section.

Reagents and Chemicals. Native CTX was purchased from AlomoneLaboratories (Jerusalem, Israel). Synthetic CTX was synthesized and high-

performance liquid chromatography purified (>98% purity) at the PeptideSynthesis Core Facility of Louisiana State University. All of the other chemicals were purchased from Sigma, unless otherwise noted.

Statistical Analysis. Statistical analysis was performed on results frombinding assays and I2il-labeled CTX biodistribution experiments, using the

Instai software program (GraphPad, version 1.1 la, 1990). The unpaired t testwas used to obtain two-tailed />s and to establish SDs in the accumulation of

radioactive CTX in organs between the tumor-bearing and control animals.

RESULTS

In Vitro Binding of CTX. As a first step to characterize thespecific CTX binding to glioma cells, we used I25l-labeled CTX and

measured binding to cultured glioma cell lines. A representativeScatchard plot analysis of 125I-labeled CTX binding to D-54 MG

human glioma cells is shown in Fig. \A. The data are best fit with twolines, indicating the presence of two binding sites; the apparent Kd forthis experiment is 4.3 nM for the high affinity site and 660 nM for thelow affinity site. From these data, the total number of CTX bindingsites were calculated to be 1,300 high affinity and 13,300 low affinitysites per cell. Because these experiments were carried out at 4°C,

internalization of the peptide and its receptor was negligible. Bindingassays using the D-54 MG cell line were repeated four times, and the

results were consistent with the example presented in Fig. \A: K¿of4.8, 3.9, and 5.1 nM for the high affinity site (mean Ka, 4.5 nM;SD = 0.57) and 590. 550, and 640 nM, respectively, for the lowaffinity site (mean K¿¡,610 nM; SD = 49.66). We obtained similar

binding curves for six other human malignant glioma cell lines,namely SK-l-MG, U87-MG, U105-MG, U251-MG, U373-MG, andD-54 MGLacZ. Apparent CTX affinities for these cell lines ranged

between 3.8 and 9.8 nM for the high affinity and 490 and 960 nM forlow affinity binding sites. Using the same assay, we studied the

binding of CTX to normal rat astrocytes and Te671, a human rhab-domyosarcoma cell line. In agreement with our previous electrophysi-

ological characterization, these cells lacked CTX receptor sites, andspecific binding of radiolabeled toxin could not be demonstrated (datanot shown). We also assessed the ability of glioma cells to internalizebound CTX. Glioma cells in suspension were treated with I2<"l-labeled

CTX at 4°Cand 37°C,respectively. We counted radioactivity in themembrane-associated pool of the I25l-bound CTX [stripped off with a

glycine buffer (pH = 2.5)]; the membrane-bound fraction was 88.2%of the total specific bound material at 4°C.in contrast to only 36.8%

of the total cell-associated radioactivity after incubation for 60 min at37°C(data not shown). The remainder of the cell-associated radioac

tivity was acid resistant and presumably located within the cell. Thesedata suggest that, at physiological temperatures, significant internalization of CTX occurs.

Radioreceptor SDS-PAGE Analysis. In an effort to begin the

characterization of the receptor molecule for CTX, radioreceptor blotanalysis was performed on U-251 MG and D-54 MG cell lines (Fig.IB). 125I-labeled CTX (25 DM)was bound to cultured glioma cells in

the presence (Lanes I and 3) or absence (Lanes 2 and 4) of 100-foldmolar excess cold material and cross-linked using the cross-linking

agent, disuccinimidyl suberate (Pierce). Cells were lysed in the presence of proteinase inhibitors, and membrane fractions were preparedby serial centrifugation: aliquots containing equivalent amounts ofprotein were separated on SDS-PAGE and exposed to X-ray film.125I-labeled CTX detected a single band in the membrane preparation,

which was significantly reduced in intensity by coincubation withexcess unlabeled ligand. Using the Bio-Rad Kaleidoscope®

prestained molecular weight marker, we estimated the size of thisband to be Mr —¿�72.000.These data indicate that human glioma cells

express a receptor molecule of A/r 72,000 for CTX. The molecularweight of this molecule is similar to that of members of the CLCfamily of chloride channels recently cloned from muscle and kidney(11).

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CTX TARGETING OF HUMAN GL1OMAS

Fig. 4. Fluorescence photomicrographs ol brainsections from glioma-hearing SCID mouse. The intensely labeled cells (ii/rmv.v) represent human tumorcells stained with hiotinylated CTX and avidin-rho-damine (li and l)\ or with Oregon green-tagged CTX

M and O. demonstrating specific in sint CTX labeling of human glioma cells. Bars. 100 jim (A and O.200 /xm </i). and 50 ^im (I)}.

In Vivo Biodistribution of I25l-labeled CTX and Brain Autora-

diography. Because our results demonstrated specific, high-affinity

binding of CTX to glioma cells in culture, we investigated the bindingcharacteristics of this molecule in vivo, using an animal model ofhuman malignant glioma cells stereotactically implanted in SCIDmice brains (9). Control animals were injected with saline. Nine daysafter intracerebral injection both groups (tumor and control mice)received 15 /tCi I2sl-labeled CTX (in 6 /j.1 of PBS total volume/

animal) intracranially administrated through the same burr hole usedfor tumor-cell or saline injection. Subsequently, the distribution ofI25l-labeled CTX was determined by scintillation counting of tissue

and fluid samples. As shown in Fig. 2A (four upper panels), theaccumulation of radioactive CTX in the brain of glioma-hearing micewas approximately 3-fold higher than in the corresponding side of thebrain in sham-injected animals at all of the time points studied (24-96

h). The presence of radioactivity in the left hemisphere is likely dueto the dispersion of the injected radioligand via the ventricles; in manyinstances, there was a variable (albeit smaller) amount of tumor massseen in the left side of the brain, presumably because of ventriculardissemination (seen also in Fig. 5B); ratios of accumulated I25I-

labeled CTX between the right and left hemisphere ranged between1.5 and 3.1. In the tumor-bearing mice, retention of CTX at 24 h afterradioactive ligand injection was 190<7rID/g tissue in the right side of

the brain and oSVrlD/g tissue in the left hemisphere. In contrast, inage- and gender-matched saline-injected mice, accumulation was

25%ID/g in the right hemisphere and 18%ID/g in the left side of thebrain. At subsequent time points, the relative accumulation of I25I-

labeled CTX in the glioma-hearing mice was consistently higherwithin the right brain (212%ID/g, SD = 24.8 at 48 h; 76%ID/g,SD = 10.1 at 72 h; 46%ID/g, SD = 8.2 at 96 h) compared to the lefthemisphere (64, 28, and 21<7rID/g at 48, 72, and 96 h, respectively).

In all of the cases, the differences in the right-brain accumulatedradioactivity between the tumor-bearing and the control groups weresignificant (two-tailed P < 0.001).

To gain additional insight into the intratumoral distribution of CTX,

we performed autoradiography on the tumor-bearing animals at 24,

48, and 72 h after injection of the radiolabeled ligand. Fig. 2B (fourlower panels) shows examples of autoradiograms obtained at 48 hafter administration of the radioligand. Silver grains (indicating localization of 125I-labeled CTX) are found mostly within the implanted

tumor cells and not the host brain cells, supporting the notion thatradiolabeled CTX can be efficiently targeted to the tumor implants.

In Vivo Radioimaging with 131I-labeled CTX. To obtain an in

vivo estimate of the ability of CTX to localize to the tumor tissue weused '"l-labeled CTX intracranially delivered (50 piCi/mouse) to

glioma-hearing and control SCID mice. Because of its higher yemittance, "'I affords the opportunity to obtain gamma-ray scinti-

gram scans in vivo. In Fig. 3 (upper panels) representative examplesof whole-body scintigrams obtained from tumor-bearing and control

animals are shown (dorsal view, head of animal facing up). Accumulation of the radioactive ligand was significantly higher in the brain oftumor-bearing animals compared with their age- and gender-matched

control. There was some visible accumulation in the stomach (whichis observed routinely as a result of dehalogenation) as well as in theurinary bladder. Tumor-bearing animals were less active and probablyreduced their food and water intake after injection of the '"[-labeled

CTX, which could account for the persistently higher radioactivityseen within their gastrointestinal track compared with that of controlanimals. Gamma camera imaging of '"l-labeled CTX was used to

obtain qualitative information about selective retention of CTX withintumor tissue. In addition, biodistribution of "'l-labeled CTX was

analyzed, and data for the 24- and 72-h time points are shown in Fig.

3 (lower panels). In this case, we dissected the tumor separately fromthe right hemisphere (designated "right brain"); we could thus estab

lish that most of the right-brain associated radioactivity was due to the

accumulation of radiolabeled CTX within the tumor tissue. At 24 hafter the injection of '3'l-labeled CTX, calculated tumor:muscle ratio

of accumulated radioactivity (mean value, 39.13; SD = 4.6) was

significantly higher than the liver: muscle ratio (mean value, 1.12;

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CTX TARGETING OF HUMAN GLIOMAS

Fig. 5. A. ¿íjcZ-transfectedD-54 MG human glioma cells demonstrate labeling with biotinylatedCTX. B, coronal section of SCID mouse brain induced with D-54 MGlMcZ glioma; mice were sacrificed at 9 days after tumor injection and processedfor LticZ enzyme histochemistry. C-F. double labeling of xenografted human glioma cells: panels C-Fillustrate immune staining of LttcZ D-54 cells with

biotinylated CTX (arrows), indicating that most ofthe human glioma cells are CTX-positive after hav

ing formed tumors in SCID mice brain, whereas noCTX labeling is seen within the host tissue. Bars. 3mm(ß). 1.5 mm (C). 100 pirn (D), 50 /im (Eand Fl.

jr.«!«

t.

•¿�

V

,•¿�

SD = 0.08; P = 0.072). These values had changed to 11.6 and 1.3,respectively, 72 h after administration of "'I-labeled CTX.

Immunohistochemical Localization of CTX. Immunohistochem-

ical analysis of CTX binding to glioma cells in culture and humanglioma xenografts in SCID mice was pursued using two differentapproaches. Direct labeling of CTX was obtained by conjugation ofCTX to the FITC-like fluorochrome, Oregon Green® (Molecular

Probes, Inc.). This compound was tested for its ability to label gliomacells in xenografted tumors in situ (Fig. 4, A and C). Glioma cells arereadily identified by fluorescent-labeled CTX within the host tissue.As a second approach, we used rhodamine-labeled streptavidin to

recognize biotinylated CTX. Examples that demonstrate selectivelabeling of glioma cells with this two-step immunohistochemicalapproach are shown in Fig. 4 (panels B and D, again staining of D-54

MG cells xenografted intracranially into SCID mice brains). To unequivocally identify the implanted human tumor cells and differentiate them from mouse host tissue, we used D-54 glioma cells that were

transfected with the reponer gene (Lac Z) allowing identification oftransfected cells by the blue X-gal reaction product. TransfectedD-54LacZ cells stained positive with biotinylated CTX followed byHRP-linked streptavidin before injection into SCID mice (Fig. 5/4).

Once implanted, the tumors could be readily visuali/.ed by the blueX-Gal label (Fig. 5B). Frozen sections of such tumors were stainedwith CTX-biotin and showed that most of the transplanted tumor cells

were also positive for CTX. Representative fields of sectioned humanxenografts in SCID mouse brain are shown in Fig. 5, panels C-F.

Specificity of staining was tested in all of the immunohistochemicalexperiments by pretreating control cells or brain sections with "cold"

(untagged) CTX at 100-fold molar excess. These were consistently

negative (data not shown).To show that CTX is an effective immunohistochemical marker for

human glioma cells, we also analyzed a variety of human biopsy andautopsy tissues obtained from patients with various pathologies. Fig.6/\ illustrates a representative example of CTX labeling of a humanGBM tissue section; control sections preincubated with native CTX(100-fold molar excess) showed no positive peroxidase immuno-

staining (not shown). Fig. 6C shows that the staining of brain tissuethat was unaffected by malignancy exhibits no significant CTX labeling. We have analyzed lower-grade astrocytoma (WHO grade II)tissues for CTX immunostaining (Fig. 60). The number of CTX-

positive cells was found to increase with the malignancy grade of thetumors. This is in excellent agreement with our previous electrophysi-

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CTX TARGETINGOF HUMAN GLIOMAS

Fig. 6. Imniunohistochcmical analysis of CTXstaining of human brain tissue. Upper ¡eftpanelsdemonstrate labeling with biotinylatcd CTX andstreptavidin-HRP of GBM (A) and low-grade astro-cyloma (C}. Significantly fewer CTX-positive cells

are found in the nonmalignant brain tissue (E). Righipanels (B, I), and F) represent H&E staining of thesame tissues to illustrate characteristic cellularity foreach pathology. Kur. 20 firn.

'f,**

«t*

.A,

*

ological demonstration of enhanced expression of CTX-sensitive

channels with enhanced malignancy grade (7).

DISCUSSION

The data presented show specific and selective binding of CTX toglioma cells in vitro and in vivo. CTX (12, 13) is a 36-amino acid

peptide that, because of four disulfide bridges, is tightly folded (14).Its small size and compact shape are convenient for intracranialdelivery. Other favorable characteristics include its capacity to berecepted after radioiodination of its accessible tyrosine residues andafter conjugation of its free amine group in formation of sulfo-NH-ester bonds (for biotinylation). Because CTX inhibits GCCs in elec-

trophysiological studies (5, 6), we assume that it binds directly tochloride channels. However, it is possible that CTX binds to anindependent receptor and indirectly modulates the activity of GCCs.For purposes of targeting glioma cells using CTX, this difference isnot relevant.

The function of the GCC targeted by CTX is not clear. Evidence isaccumulating that these channels may control glioma cell proliferation(6), and may facilitate volume and cell-shape changes (8) that are

necessary during glioma cell migration and the invasion of surrounding normal brain tissue. It is thus likely that GCC is of functionalimportance in the malignant transformation of gliomas or in regulating their highly invasive nature.

On the basis of our binding studies, CTX specifically binds malignant glioma cells in culture, exhibiting high-affinity (K¿= 4.2 nM,1300 sites/cell) and low-affinity (K¿= 660 nw, 13,300 sites/cell)

binding sites. Given the high degree of heterogeneity typical ofgliomas, it is remarkable that CTX binding and expression of GCCwere observed across all of the malignancy grades of human gliomasstudied. The high affinity for glioma cells with little or no binding tonormal brain cells, together with its favorable physical properties,makes this molecule an attractive candidate for the targeting ofgliomas. Feasibility of such an approach is supported by our in vivoexperiments that show selective retention of radiolabeled CTX withinxenografted gliomas in SCID mice. Moreover, the imaging of '•"!-

labeled CTX injected intracranially demonstrated specific in vivoaccumulation of the radiolabeled toxin in the tumors of the glioma-

bearing mice, thus supporting its potential use for diagnosis or therapyof brain tumors. Additional pharmacokinetics studies after the admin-

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CTX TARGETING OF HUMAN GLIOMAS

istration of radiolabeled CTX intracranially and i.v. will preciselyestablish its circulatory half-life and whether multiple injections may

be necessary for persistent retention within the tumor. To date, theresults of our studies using '25I-labeled CTX compare favorably with

those obtained by others (15-17) using monoclonal antitumor anti

bodies tagged with various radioactive molecules (18). We have notyet obtained complete toxicity data, but preliminary results indicatethat CTX (40 mg/kg) injected into SCID mice (i.p. or i.v.) had notoxic effect for 60 days after administration of the drug.

Our immunohistochemical data suggest that CTX-like molecules

with fluorescent or biotin labels can be successfully used for immunohistochemical labeling of gliomas. The potential correlation between the CTX receptor expression levels and malignancy gradeoffers an attractive perspective for using CTX in the diagnosis andgrading of primary human malignant gliomas. We are presently evaluating a larger sample of human gliomas (WHO grades I-IV) as well

as metastatic brain tumors and nontumor patient biopsy tissues forspecific binding of CTX to gather comprehensive data on the relativeexpression of CTX receptor and its relationship to malignancy grade.

In addition to being useful glioma-specific markers, molecules that

use CTX as a targeting moiety have the potential to be developed intocell-type-specific cytotoxic drugs. Previous approaches have used

radiolabeling or linkage of monoclonal antibodies to cytotoxic proteins for delivery to cancerous cells (15, 19-21). Findings described

in this report form the basis for our ongoing studies toward linkingCTX to Saporin officinalis. a ribosomal inactivating toxin (22, 23).For such approaches to be effective, internalization of the ligand isessential. Results presented herein (from experiments using I25I-

labeled CTX) suggest that >60% of the toxin is ultimately internalized at physiological temperatures, such that the aforementioned targeting strategy seems feasible.

Given the high affinity of interaction between CTX and GCC, thespecificity of binding of CTX-based moieties to human glioma cells,

the small size of the CTX molecule, and its potential for chemical andgenetic manipulation, this molecule seems to be an ideal candidate toengineer additional chimerical reagents to be used as diagnosis toolsand therapeutic modalities. These approaches may offer novel strategies in addressing this uniformly fatal disease.

ACKNOWLEDGMENTS

We thank Mark Campbell for assistance with the radiolabeling of CTX.Gayle Elliot for help with the use of the gamma ray camera, Katherine Mercerfor technical help in immunostaining. and Katherine Nobles and James Robinson for technical assistance.

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1998;58:4871-4879. Cancer Res   Liliana Soroceanu, Yancey Gillespie, M. B. Khazaeli, et al.   Use of Chlorotoxin for Targeting of Primary Brain Tumors

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