Highly migratory neuroectodermal cells share a common embryonic origin with cells of the central nervous system (CNS). They include enteric, parasympathetic, sympathoadrenal, and sensory neurons of the peripheral nervous system, Schwann cells, melanocytes, endocrine cells, and cells forming connective tissue of the face and neck. Because of their common embryologic origin, these cells and the tumors that derive from them can share genetic and antigenic phenotypes with gliomas, tumors derived from CNS glia. We recently discovered that chlorotoxin (ClTx), a 4-kD peptide purified from Leiurus quinquestriatus scorpion, is a highly specific marker for glioma cells in biopsy tissues (Soroceanu et al. Cancer Res 58:4871-4879, 1998) that can target tumors in animal models. We report on the specificity of ClTx as a marker for tumors of neuroectodermal origin that include peripheral neuroectodermal tumors (PNET) and gliomas. Specifically, we histochemically stained frozen and paraffin tissue sections of human biopsy tissues from 262 patients with a synthetically manufactured and biologically active ClTx bearing an N-terminal biotin. The vast majority (74 of 79) of primary human brain tumors investigated showed abundant binding of ClTx with greater than 90% ClTx-positive cells in each section. By comparison, 32 biopsies of uninvolved brain used for comparison were largely ClTx-negative, with only a few isolated reactive astrocytes showing some ClTx binding. However, as with gliomas, the vast majority of PNETs examined showed specific ClTx binding (31 of 34). These include medulloblastomas (4 of 4), neuroblastomas (6 of 7), ganglioneuromas (4 of 4), melanomas (7 of 7), adrenal pheochromocytomas (5 of 6), primitive PNET (1), small cell lung carcinoma (2 of 3), and Ewing's sarcoma (2 of 2). Under identical staining conditions, normal tissues from brain, skin, kidney, and lung were consistently negative for ClTx. These results suggest that chlorotoxin is a reliable and specific histopathological marker for tumors of neuroectodermal origin and that chlorotoxin derivatives with cytolytic activity may have therapeutic potential for these cancers.

We used an in vitro model for glioma cell invasion (transwell migration assay) and patch-clamp techniques to investigate the role of volume-activated Cl(-) currents (I(Cl,Vol)) in glioma cell invasion. Hypotonic solutions ( approximately 230 mOsm) activated outwardly rectifying currents that reversed near the equilibrium potential for Cl(-) ions (E(Cl)). These currents (I(Cl,Vol)) were sensitive to several known Cl(-) channel inhibitors, including DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The IC(50) for NPPB inhibition of I(Cl,Vol) was 21 microm. Under isotonic conditions, NPPB (165 microm) blocked inward currents (at -40 mV) and increased input resistance in both standard whole-cell recordings and amphotericin perforated-patch recordings. Reducing [Cl(-)](o) under isotonic conditions positively shifted the reversal potential of whole-cell currents. These findings suggest a significant resting Cl(-) conductance in glioma cells. Under isotonic and hypotonic conditions, Cl(-) channels displayed voltage- and time-dependent inactivation and had an I(-) > Cl(-) permeability. To assess the potential role of these channels in cell migration, we studied the chemotactic migration of glioma cells toward laminin or vitronectin in a Boyden chamber containing transwell filters with 8 microm pores. Inhibition of I(Cl,Vol) with NPPB reduced chemotactic migration in a dose-dependent fashion with an IC(50) of 27 microm. Time-lapse video microscopy during patch-clamp recordings revealed visible changes in cell shape and/or movement that accompanied spontaneous activation of I(Cl,Vol), suggesting that I(Cl,Vol) is activated during cell movement, consistent with the effects of NPPB in migration assays. We propose that I(Cl,Vol) contributes to cell shape and volume changes required for glioma cell migration through brain tissue.

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