Invading C6 glioma cells maintaining tumorigenicity

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✓ To characterize rat glioma cell invasion, 2 × 106 fluorophore-labeled or transfection-labeled C6 rat glioma cells were implanted in the rat frontal lobe. Eighty percent of the rats implanted formed bulk tumors (3–4 mm in diameter). Two weeks after implantation, fluorescence microscopy revealed single tumor cells in sites over 16 mm from the bulk brain tumor. Tumor cells distant from the bulk tumor remained single without mass formation and invaded primarily along white matter tracts. Two weeks after tumor implantation, three cell lines were created from each brain by disaggregation and initiation in culture of 1) bulk tumor, 2) contralateral hemisphere, and 3) cerebellum; all disaggregated specimens generated viable cultures. Cells cultured from the contralateral hemisphere were morphologically indistinguishable from cells from the bulk tumor and from the original C6 cell line. Cells cultured from the cerebellum were morphologically quite distinct from the C6 cell line. Cells from disaggregated specimens obtained from the tumor, contralateral hemisphere, and cerebellum were implanted in the frontal lobe of naive rats to test tumorgenicity. Bulk tumor formed in 58% of the rats implanted with specimens from tumor, in 75% of the rats implanted with specimens from contralateral hemisphere, and in only 12.5% of the rats implanted with specimens from the cerebellar hemispheres. Experiments using C6 cells labeled by transfection with the p3′ss DNA vector prior to implantation confirmed that the cells cultured from the contralateral hemisphere were derived from the implanted C6 cells. Experiments with C6 cells anchored in agar served to verify that movement to the contralateral hemisphere was secondary to parenchymal invasion rather than dispersion in the cerebrospinal fluid.

Article Information

Address reprint requests to: Daniel L. Silbergeld, M.D., Department of Neurological Surgery, Washington University School of Medicine, Campus Box 8057, 660 South Euclid Avenue, St. Louis, Missouri 63110–1093.

© AANS, except where prohibited by US copyright law.

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    Schematic drawing summarizing glioma implantation experiments in Sprague-Dawley rats. Part I: C6 cells in culture were labeled with fluorophore (Fast blue), and implanted in rat frontal lobes. Part II: Two weeks after implantation of labeled C6 cells, rat brains were removed and divided into three sections: tumor-bearing cerebral hemisphere, cerebral hemisphere contralateral to tumor, and cerebellum. Part III: Each section of brain from Part II was disaggregated into a single-cell suspension for implantation into naive rats, and for reinitiation in culture. Two to 3 weeks after implantation, the rat brains were removed for histopathological evaluation.

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    Schematic drawing summarizing glioma implantation experiments in Wistar rats. Part I: C6 cell in culture were transfected with the p3′ss DNA vector and implanted in rat frontal lobes in an agar suspension. Part II: Two weeks after implantation of p3′ss-transfected C6 cells, rat brains were removed and divided into two sections: tumor-bearing cerebral hemisphere and cerebral hemisphere contralateral to tumor. Part III: Each section of brain from Part II was disaggregated into a single-cell suspension for reinitiation in culture in hygromycin B—containing medium and for implantation into naive rats. After establishment of hygromycin B-resistant cultures from each hemisphere, cells were reimplanted into naive rats. No cells survived from the disaggregated cerebellar specimens cultured in hygromycin B. Two to 3 weeks after implantation, the rat brains were removed for histopathological evaluation.

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    Fixed gross specimen of Sprague-Dawley rat brain demonstrated well-circumscribed tumor 2 weeks after injection of labeled C6 cells in the right frontal lobe.

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    Indirect immunofluorescence staining for the Lac I gene product expressed by p3′ss-transfected cells viewed with fluorescence microscopy (λ = 470 nm). Upper: These cells from the cerebral hemisphere of a rat implanted 10 days prior with p3′ss-transfected C6 cells suspended in agar in the contralateral frontal lobe were cultured in medium containing hygromycin B. Growth in hygromycin B confirmed that these cells, cultured from the hemisphere contralateral to the site of bulk tumor growth, were derived from the implanted tumor cells and not from the native brain cells. Original magnification × 20. Lower: A coronal section of brain harvested from a Wistar rat that was implanted, at the site shown, 2 weeks prior with p3′ss-transfected C6 cells suspended in agar. Original magnification × 4.

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