Use of chlorotoxin for targeting of primary brain tumors.

Gliomas are primary brain tumors that arise from differentiated glial cells through a poorly understood malignant transformation. Although glioma cells retain some genetic and antigenic features common to glial cells, they show a remarkable degree of antigenic heterogeneity and variable mutations in their genome. Glioma cells have recently been shown to express a glioma-specific chloride ion channel (GCC) that is sensitive to chlorotoxin (CTX), a small peptide purified from Leiurus quinquestriatus scorpion venom [N. Ullrich et al, Neuroreport, 7: 1020-1024, 1996; and N. Ullrich and H. Sontheimer, Am. J. Physiol. (Cell Physiol.), 270: C1511-C1521, 1996]. Using native and recombinant 125I-labeled CTX, we show that toxin binding to glioma cells is specific and involves high affinity [dissociation constant (Kd)=4.2 nM] and low affinity (Kd=660 nml) binding sites. In radioreceptor assays, 125I-labeled CTX binds to a protein with Mr=72,000, presumably GCC or a receptor that modulates GCC activity. In vivo targeting and biodistribution experiments were obtained using 125I- and (131)I-labeled CTX injected into severe combined immunodeficient mice bearing xenografted gliomas. CTX selectively accumulated in the brain of tumor-bearing mice with calculated brain: 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 immunodeficient mice, the vast majority of the brain-associated radioactivity was localized within the tumor (tumor:muscle ratio, 39.13% ID/g; contralateral brain:muscle ratio, 6.68%ID/g). Moreover, (131)I-labeled CTX distribution, visualized through in vivo imaging by gamma ray camera scans, demonstrates specific and persistent intratumoral localization of the radioactive ligand. Immunohistochemical studies using biotinylated and fluorescently tagged CTX show highly selective staining of glioma cells in vitro, in situ, and in sections of patient biopsies. Comparison tissues including normal human brain, kidney, and colon were consistently negative for CTX immunostaining. These data suggest that CTX and CTX-conjugated molecules may serve as glioma-specific markers with diagnostic and therapeutic potential.

Tendon synovial cells secrete fibronectin in vivo and in vitro.

The chemistry and cell biology of the tendon have been largely overlooked due to the emphasis on collagen, the principle structural component of the tendon. The tendon must not only transmit the force of muscle contraction to bone to effect movement, but it must also glide simultaneously over extratendonous tissues. Fibronectin is classified as a cell attachment molecule that induces cell spreading and adhesion to substratum. The external surface of intact avian flexor tendon stained positively with antibody to cellular fibronectin. However, if the surface synovial cells were first removed with collagenase, no positive reaction with antifibronectin antibody was detected. Analysis of immunologically stained frozen sections of tendon also revealed fibronectin at the tendon synovium, but little was associated with cells internal in tendon. The staining pattern with isolated, cultured synovial cells and fibroblasts from the tendon interior substantiated the histological observations. Analysis of polyacrylamide gel profiles of 35S-methionine-labeled proteins synthesized by synovial cells and internal fibroblasts indicated that fibronectin was synthesized principally by synovial cells. Fibronectin at the tendon surface may play a role in cell attachment to prevent cell removal by the friction of gliding. Alternatively, fibronectin, with its binding sites for hyaluronic acid and collagen, may act as a complex for boundary lubrication.

Cell populations of tendon: a simplified method for isolation of synovial cells and internal fibroblasts: confirmation of origin and biologic properties.

Tendons transmit the force of muscle contraction to bone to effect limb movement. Special structural and biological properties of tendon have developed to facilitate force transmission. The tendon has a complex organization of cells surrounding the collagen bundles inside tendon as well as at the tendon surface. Internal cells may act to maintain the bulk of the collagen in tendon. External cells in the epitenon may provide lubrication for tendon gliding. To develop better understanding of these processes and the roles the cell populations play, we isolated cells from the surface and interior of tendon and studied them in vitro. Flexor tendons from 8-week-old white Leghorn chickens were separated into two distinct cell populations: the outer synovial cells and the fibroblasts more internal in tendon. These cell populations were discernible by their locations in the intact tendon, determined by sequential enzymatic and physical release from their substrata. Initially, some cells eluted in Hanks' salt solution (HSS) (population 1); then synovial cells were released after a 2-min treatment with 0.5% collagenase (population 2). Next, a population of synovial cells was released in high yield by treatment with 0.25% trypsin (step III, population 3). Step III, population 3 cells were used as synovial cells (SCs). Next, a population of SCs and fibroblasts were released by scraping with a rubber policeman (population 4). Subsequently, fibroblasts were released after incubation with 0.5% collagenase (population 5). A more direct procedure (procedure 2) to isolate the synovial and internal tendon cells involved treatment in 0.5% collagenase followed by sedimentation at 900 g. Cells that sedimented were largely fibroblasts, whereas the cells that remained at the top of the tube were largely SCs. Cells designated as SCs, isolated by procedure 2, most likely contained surface cells from epitenon and internal interfascicular cells from endotenon and paratenon. Surface tendon cells separated by sequential enzymatic and physical release from their substrata (by procedure 1) had all the following characteristics: distinct subpopulations of cells based on morphology; presence of cytoplasmic, lipid-containing vesicles; decreased sensitivity to trypsin; and reduced generation time as compared with that of internal fibroblasts. Conversely, the internal fibroblasts (IFs) appeared to represent a more uniform population based on morphological characteristics.

Response of glioma cells to interferon-gamma: increase in class II RNA, protein and mixed lymphocyte reaction-stimulating ability.

Previous results by ourselves and others demonstrated that brain cells and cell lines express major histocompatibility complex class II antigens. We examined interferon-gamma (IFN-gamma)-mediated induction of human class II antigen expression on the glioma cells. Purified IFN-gamma induced the expression of HLA-DR antigens on the surface of the glioma cell lines U-373 MG and U-105 MG. Concomitant increase of HLA-DR alpha- and HLA-DC beta-specific RNA in the cytoplasm was also observed after treatment with IFN-gamma. Increases of class II antigen paralleled the increased level of class II-specific RNA. The effect of IFN-gamma on the induction of human class II antigen expression was dose and time dependent. A marked induction of human class II antigen expression was observed when glioma cells were cultured with more than 100 U/ml of IFN-gamma. Little or no induction was observed with less than 50 U/ml of IFN-gamma. Compared to human blood monocytes, glioma cells needed higher concentrations of IFN-gamma for the induction of class II antigen expression. In allogenic mixed lymphocyte cultures, the glioma cell line U-373 MG stimulated a mixed lymphocyte response (MLR). MLR-stimulating capacity was augmented by IFN-gamma. The concomitant augmentation of class II antigen levels and MLR-stimulating capacity suggests that the most relevant factor for MLR stimulation may be antigen density. This is the first report of MLR stimulation by a glioma cell line.