Use of Chlorotoxin for Targeting of Primary Brain Tumors1
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[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 this process. These include variable mutations of p53. pl6, cdk4. and RB protein, amplification or overexpression of epidermal growth Gliomas are primary brain tumors that arise from differentiated glial factor receptor or MDM2 genes (mostly found in glioblastomas that cells through a poorly understood malignant transformation. Although arise "de novo"). glioma cells retain some genetic and antigenic features common to glial cells, they show a remarkable degree of antigenic heterogeneity and Because of the lack of glioma specific markers, unequivocal diag variable mutations in their genome. Glioma cells have recently been nosis of gliomas requires tissue biopsy and relies primarily on his- shown to express a glioma-specific chloride ion channel (GCC) that is topathological criteria. Histopathologically, gliomas are highly cellu sensitive to chlorotoxin (CTX), a small peptide purified from Leiurus lar with marked pleomorphism and often contain multinucleate giant quinquestriatus scorpion venom [N. Ullrich et al.. Neuroreport, 7: 1020- cells. Vascular proliferation throughout the lesion and the presence of 1024, 1996; and N. Ullrich and H. Sontheimer, Am. J. Physiol. (Cell Physiol.), 270: C1511-C1521, 1996]. Using native and recombinant 125I- focal areas of necrosis are histological features necessary for the diagnosis of GBM.1 the most malignant and prevalent form of glioma labeled CTX, we show that toxin binding to glioma cells is specific and involves high affinity [dissociation constant (A',,i = 4.2 nM] and low affinity in adults. Immunohistochemical studies show variable expression of (Ka = 660 nM) binding sites. In radioreceptor assays, I25l-labeled CTX the astrocyte-specific antigen glial fibrillary acidic protein; protein binds to a protein with M, = 72,000, presumably GCC or a receptor that levels were found to decrease with an increasing degree of malig modulates GCC activity. In vivo targeting and biodistribution experiments nancy. Vimentin expression is a common but not a reliable marker. were obtained using I25I- and I3ll-labeled CTX injected into severe com Presently, no antigen or marker that could serve to selectively label bined immunodeficient mice bearing xenografted gliomas. CTX selectively accumulated in the brain of tumor-bearing mice with calculated brain: glioma cells is available. To date, two other reports describe proteins specifically expressed by glioma cells: brain-enriched hyaluronan muscle ratios of 36.4% of injected dose/g (ID/g), as compared to 12.4%ID/g in control animals. In the tumor-bearing severe combined binding protein (3) and the novel interleukin 13 receptor a (4). immunodeficient mice, the vast majority of the brain-associated radioac We recently identified and characterized a chloride ion channel that tivity was localized within the tumor (tumor: muscle ratio, 39.13% ID/g; seems to be abundantly expressed in glioma cells but absent in normal contralateral braimmuscle ratio, 6.68%ID/g). Moreover, 131I-labeled CTX brain tissue (5. 6). GCC activity could be recorded biophysically in distribution, visualized through in vivo imaging by gamma ray camera cultured glioma cells (5. 6) and in freshly prepared vibratome sections scans, demonstrates specific and persistent intratumoral localization of from patient biopsy tissue (7). Channels were absent in nontumor the radioactive ligand. Immunohistochemical studies using biotinylated and fluorescently brain tissue and in tumor cells of nonglial origin. Because the activity tagged CTX show highly selective staining of glioma cells in vitro, in situ, of GCC seems to be modulated by rearrangements in the cells cy- and in sections of patient biopsies. Comparison tissues including normal toskeleton (8). it has been proposed that these channels may facilitate human brain, kidney, and colon were consistently negative for CTX shape changes during glioma cell migration and invasion. Interest immunostaining. These data suggest that CTX and CTX-conjugated mol ingly, GCC expression in situ correlates with the histopathological ecules may serve as glioma-specific markers with diagnostic and thera tumor grade (7). Only 40-45% of low-grade astrocytomas (WHO peutic potential. grade I-II) express GCC, whereas >90% of all of the high-grade tumors (WHO grade III) and essentially all of the GBMs express INTRODUCTION GCC. This correlation suggests that GCC may be a candidate protein to serve as a glioma-specific marker and may be useful for diagnostic Gliomas are among the most deadly forms of cancer for which and therapeutic purposes. Biophysical studies also demonstrated that effective treatment strategies are currently lacking. Indeed, despite the CTX, a 36-amino-acid peptide isolated from scorpion venom (Leiurus overall advances in chemo and radiation therapy regimens, the median survival of glioma patients has been unaltered in the last 20 years (1, quinquestriatus), effectively inhibits currents through GCC with -80% block at 600 nM CTX (5, 6). In the present study, we have 2). This lack of success in treatment of gliomas seems to be due in part to their high resistance to radiation and chemotherapy but additionally generated CTX in synthetic and recombinant forms and have attached moieties including I25I, ml, biotin, and various fluorophores to the to their unusual ability to disperse and invade healthy brain tissue. Glioma cells display a remarkable level of heterogeneity within and CTX molecule that subsequently allowed its detection. We have used among gliomas, as manifested through clonal variation in growth these tagged CTX molecules to investigate specific binding of CTX to potential, differential genetic alterations, variable drug resistance, and glioma cells in vitro and HI situ. These studies show that iodinated expression of antigens. CTX selectively binds malignant glioma cells and can be visualized The molecular mechanisms that underlie the malignant transforma by in vivo imaging of animals bearing xenografted tumors. Moreover, tion of glial cells to become gliomas are poorly understood. As with biotinylated or fluorescently tagged CTX molecules reliably detect other cancers, a series of genetic alterations seem to be obligatory for glioma cells in patient biopsies, which suggests that this molecule could serve as a glioma-specific marker with diagnostic and thera Received 6/23/98; accepted 9/2/98. peutic potential. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1The abbreviations used are: GBM. gliohlastoma multiforme: CTX. chlorotoxin: ' This work was supported by NIH Grant ROI NS 36692 and American Cancer GCC, glioma chloride channel: HRP. horseradish peroxidase: Kd. dissociation constant: Society Grant RPG-97-083. X-Gal. 5-bromo-4-chloro-3-mdolyl-b-n-galactopyranoside; TEPSA, 3-aminopropyltri- " To whom requests for reprints should be addressed, at Department of Neurobiology. ethoxysilane: %ID/g. % (percentage) of injected dose/g: UAB. University of Alabama al CIRC 545. 1719 6th Avenue South. Birmingham. AL. 35294-0021. Birmingham. 4871 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1998 American Association for Cancer Research. CTX TARGETING OF HUMAN CHOMAS MATERIALS AND METHODS control counterparts were killed at indicated time points for biodistribution studies. Individual organs were removed, placed in preweighed tubes and Cell Lines and Cell Culture. The following human glioma cell lines were processed for gamma scintillation counting. Radioactivity associated with each used: (a) D-54 MG; (bìU373MG; (r) U105MG; (dìU251MG (all from Dr. organ is expressed as %ID/g of tissue. In the case of tumor-bearing '"l-labeled D. D. Bigner, Duke University. Durham, NC); and (e) neuroglial derived CTX-injected SCID mice, tumors were carefully removed from the right side SK-MG-I (from G. Cairncross, University of Ontario, London. Ontario, Can of the brain by dissection under the microscope and weighed and counted for ada). Control cell lines were: (a) Te671, a rhabdomyosarcoma cell line from associated radioactivity. Results are presented as %ID/g of tissue and as ratios American Type Culture Collection: and (b) Balb 3T3 fibroblasts. In addition, of brain:muscle or livermuscle for the two groups of animals. Biodistribution we used primary cultured rat astrocytes as control glial cells (see below). Cells experiments were repeated three times using six to eight animals for each time were maintained in DMEM mixed 1:1 with Ham's F-12 medium, supple point in each of the tumor-bearing and control groups. mented with L-glulamine (2 mM) and 10% fetal bovine serum (Life Technol Brain I2SI Autoradiography. I25l-labeled CTX-injected tumor-bearing ogies). Cells were harvested from logarithmic phase-growth cultures by brief mice were killed at 48 h after injection of the radiolabeled material and exposure to 0.5% trypsin and 0.53 HIMEDTA (Life Technologies). The D-54 perfused by intracardiac injection with PBS followed by 4% paraformaldehyde MG cell line