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Cell surface gangliosides are involved in the control of human glioma cell invasion in vitro
Neuroscience Letters 177 (1994) 44-46
ELSEVIER
HEUROSCIENC[ lETTERS
Cell surface gangliosides are involved in the control of human glioma cell invasion in vitro Abderrahim Merzak*, Shahriar Koochekpour, Geoffrey J. Pilkington Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK
Received 19 May 1994; Accepted 22 June 1994
Abstract The involvement of cell surface gangliosides in glioma cell invasion in vitro was examined using the ganglioside-specific antibody A2B5 and Matrigel-coated 8-/1m porosity polycarbonate filters. Invasion of six cell lines derived from glial tumours of different histological grades was found to be markedly inhibited by A2B5 (50-96% inhibition) in a dose-dependent manner. Furthermore, exogenous gangliosides were found to prevent cell invasion when they were incubated with cells during the invasion assay. These results suggest that cell surface gangliosides are involved in glioma cell invasion in vitro, probably because of their adhesionpromoting action to basement membrane components. Key words." Glioma; Ganglioside; Invasion, Matrigel
Gliomas, the major form of primary'brain tumour in man [20], are characterized by their local invasive infiltration of the brain, irrespective of their histological grade of malignancy. The factors which control this particular behaviour are poorly documented. Gangliosides, a class of sialic acid-containing glycosphingolipids, are ubiquitously present in the body but exist in high concentration in the brain [5]. Among the multiple functions proposed for these molecules is their role in adhesion [6] which is a crucial step in migration. In agreement with this, ganglioside-expressing glial progenitor cells in the developing mammalian nervous system have been shown to be highly migratory [22]. We proposed, therefore, that these molecules may be involved in the invasive behaviour of glioma [19]. Moreover, we demonstrated recently that exogenously added gangliosides promote glioma cell adhesion, migration and invasion in vitro [12,13]. However, the role of intrinsic cell surface gangliosides in invasion remains to be investigated. To study this potential role, we assessed the effect of
*Corresponding author. Fax: (44) (71) 708-3895. 0304-3940/94/$7.00© 1994 Elsevier ScienceIreland Ltd. All rights reserved S S D I 0304-3940(94)00490-2
A2B5 antibody on the in vitro invasion activity of six cell lines derived from human glioma of different histological grades (Table 1). A2B5 antibody was initially reported as specific for G Q l c ganglioside [3] but has been, subsequently, demonstrated to react with several other gangliosides [4,8]. Invasiveness was assessed by the method of Albini et al. [1] with modifications. 8-ktm porosity polycarbonate filters in 24-well transwells were coated with 20 p g of the reconstituted basement membrane composite Matrigel (Collaborative Research, Lexington, MA) in cold Dulbecco's modified Eagle's medium, to form a thin continuous layer on top of the filter. The Matrigel was left to air-dry overnight. Before addition of the cells, excess medium was removed from the upper compartment. 5 x 104 cells from each cell line were resuspended in 100pl of serum-free medium or undiluted A2B5 supernatant and plated on coated filters for 18 h at 37°C in a humidified 95% air-5% CO, atmosphere. The lower compartment contained 0.5 ml of serum-free medium supplemented with P D G F (10 ng/ml) as a chemoattractant. Cells were then fixed with acetic acid-alcohol and stained with Giemsa. Cells on the upper surface of the filter were removed by wiping with a cotton swab and invasion was determined by counting the cells that had
A. Merzak et al./Neuroscience Letters 177 (1994) 44~16 200 •
[]
== l= 3 z = ® 0
Control +A2B5
100
o
o o
Fig. 1. Effect of A2B5 on invasion of h u m a n glioma cell lines. 13 fields were counted in each assay. Each sample was assayed in triplicate and assays were repeated twice. Statistical significance was assessed by Student's t test (P < 0.01).
migrated to the lower side of the filter with a phasecontrast microscope at 200 × magnification. This system has proved very useful in identifying the factors involved in glioma cell invasion in vitro [10,11,14]. The invasion activity of all six glioma cell lines studied was strongly (50-96%) inhibited by A2B5 antibody (Fig. 1). Furthermore, we found that this effect is dose-dependent by incubating the cells in the presence of increasing amounts of A2B5 antibody during the invasion assay (Fig. 2). As a control, an epidermal growth factor receptor antibody proved to have no effect on cell invasion (data not shown). These results suggest that cell membrane gangliosides play an important role in the invasive behaviour of glioma cells in vitro. We have demonstrated recently that exogenously added gangliosides stimulate glioma cell invasion in vitro [13]. In addition, we reported that this effect may be explained by their adhesion-promoting action to basement membrane proteins, including laminin [12]. Although the extracellular space of the brain is devoid of a well-defined ECM [21], basement membrane does exist around blood vessels, in the glia limitans externa and underlying choroid plexus epithelial cells [18,21]. Laminin has been demonstrated to be intensly
45
expressed in the basement membranes of blood vessels and leptomeninges in low-grade gliomas [15] and may be synthesized by reactive and neoplastic astrocytes [9]. Since 60% of Matrigel is laminin [7], this commercially available ECM composite was felt to provide a good model for our adhesion and invasion studies. We postulated that exogenous gangliosides, added during the assay, may compete with cell surface gangliosides in binding to Matrigel and, therefore, prevent cell invasion. To verify this hypothesis in the present study, IPNT-H cells were incubated, during the invasion assay, in the presence or absence of 100 ng/ml of either G D l a or GD3 (Sigma, UK). The number of invasive cells dropped dramatically by 86 and 98% by GD3 and GDla, respectively (Fig. 3), confirming the role of cell surface gangliosides in glioma cell invasion in vitro. The data presented here not only confirm our previous findings with exogenous gangliosides but also demonstrate, for the first time, the involvement of cell membrane gangliosides in glioma cell invasion in vitro. It has been shown that expression of gangliosides correlates well with migration ([22]; Martin et al., in prep.). Dramatic changes in ganglioside expression have also been reported in ontogenesis and oncogenesis [5]. It is also likely that gangliosides may be involved in migration and invasion via cell-extracellular matrix (ECM) interactions. In agreement with that, we have demonstrated, recently, that gangliosides enhance adhesion of human glioma cell lines to fibronectin, laminin, vitronectin and collagen I [12]. This is supported by previous reports of the involvement of gangliosides in fibronectinmediated cell adhesion and of the identification of a 200 •
C
[]
1/100 UIO 1/1
•
._e IL.
== E
100
Z o 0
Table 1 Designation
Passage (n)
Histological type
IPTR-6 IPNT-H IPSB-18 GO-G-CCM GO-G-UVW IPRM-5
6 9 32 < 100 < 100 15
Ganglioglioma Pilocytic astrocytoma Anaplastic astrocytoma Anaplastic astrocytoma Anaplastic astrocytoma Glioblastoma multiforme
Z
~
~
~
~,
o
@
Fig. 2. Effect of undiluted (1/1) and 1/10- and 1/100-diluted A2B5 supernatant on invasion of h u m a n glioma cell lines. Assays were carried out as in Fig. 1. Statistical significance was assessed by Student's t test (P < 0.01).
46
A. Merzak et al. / Neuroscience Letters 177 (1994) 44-46
60
IPNT-H
50
•o
40
gI iz I,,.
e ,,¢1
E 30 3
Z Q
o
20
10
Control
GD3
GDla
Fig. 3. Effect of exogenous G D l a and GD3 on invasion of IPNT-H cells. Assays were carried out as in Fig. 1. Statistical significance was assessed by Student's t test (P < 0.01).
ganglioside-binding site on fibronectin [24]. Furthermore, integrins, a major class of cell surface receptors for ECM proteins, are subjected to regulation by gangliosides [2,16,23]. We have demonstrated recently the role of these molecules in glioma cell adhesion to basement membrane proteins in vitro (Merzak et al., in prep.). In addition, these molecules have also been shown to be involved in glioma cell invasion in vitro [17]. A study of the mechanisms involved in ganglioside function may help to gain valuable insights into the invasive properties of neoplastic glia. This work was supported by a grant from the Leverhulme Trust. The A2B5 monoclonal antibody-secreting clone was a gift from Professor Frank Walsh. [1] Albini, A., ]wamoto, Y., Kleinman, H.K., Martin, G.R., Aaronson, S.A., Kozlowski, J.M. and McEwan, R.N., A rapid in vitro assay for quantitating the invasive potential of tumour cells, Cancer Res., 47 (1987) 3239 3245. [2] Cheresh, D.A., Pierschbacher, M.D., Herzig, M.A. and Mujoo, K., Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins, J. Cell Biol., 105 (1987) 1163-1173. [3] Fishman, P.H. and Brady, R.O., Biosynthesis and function of gangliosides, Science. 194 (1976) 90(~915. [4] Fredman, P., Magnani, G.L., Nirenberg, M. and Ginsberg, V., Monoclonal antibody A2B5 reacts with many gangliosides in neuronal tissue, Arch. Biochem. Biophys., 233 (19841 661 666. [5] Hakomori, S.I., Glycosphingolipids in cellular interaction, differ-
entiation and oncogenesis, Annu. Rev. Biochem., 50 (1981) 733 764. [6] Hakomori, S.I., Bifunctional role of glycosphigolipids, J. Biol. Ch9m., 265 (1990) 18713 18716. [7] Kleinman, H.K., McGarvey, M.L., Hassell, J.R., Star, V.L., Cannon, F.B., Laurie, G.W. and Martin, G.R., Basement membrane complexes with biological activity, Biochemistry, 25 (1986) 312 318. [8] Kundu, S., Pleetman, M., Redwine, W., Boyd, A. and Marcus, D., Binding of monoclonal antibody A2B5 to gangliosides, Biochem. Biophys. Res. Commun., 116 (1983) 83(~842. [9] McComb, R.D. and Bigner, D.D., Immunolocalization oflaminin in neoplasms of the central and peripheral nervous system, J. Neuropathol. Exp. Neurol., 44 (1985) 242-253. [10] Merzak, A., McCrea, S., Koochekpour, S. and Pilkington, G.J., Control of human glioma cell growth, migration, and invasion in vitro by transforming growth factor-betal, Br. J. Cancer, in press. [I 1] Merzak, A., Koochekpour, S. and Pilkington, G.J., CD44 mediates human glioma cell adhesion and invasion in vitro, Cancer Res., in press. [12] Merzak, A., Koochekpour, S. and Pilkington, G.J., Gangliosides modulate adhesion of human glioma cell lines to basement membrane proteins in vitro, submitted. [13] Merzak, A., Koochekpour, S., McCrea, S., Roxanis, I. and Pilkington, GJ., Gangliosides modulate proliferation, migration, and invasiveness of human brain tumour cells in vitro, submitted. [14] Merzak, A., Parker, C., Koochekpour, S., Sherbet, G.V. and Pilkington, G.J., Overexpression of the 18A2/mtsl gene and downregulation of the TIMP-2 gene in invasive human glioma cell lines in vitro, Neuropathol. Appl. Neurobiol., in press. [15] Morris, C.S and Esiri, M.M., Immunocytochemical study of macrophages and microglial cells and extracellular matrix components in human CNS disease. 1: Gliomas, J. Neurol. Sci., 101 (1991) 47 58. [16] Mugnai, G., Lewandowska, K., Choi, H.U., Rosenberg, L.C. and Culp, L.A., Ganglioside-dependent adhesion events of human neuroblastoma cells regulated by the RGDS-dependent fibronectin receptor and proteoglycans, Exp. Cell Res., 175 (1988) 229247. [17] Paulus, W. and Tonn, J.C., Basement membrane invasion of glioma cells mediated by integrin receptors, J. Neurosurg., 80 (1994) 515 519. [18] Peters, A., Palay, S.L. and Webster, H.F., Neurons and Their Supporting Cells. The Fine Structure of the Nervous System, Oxford University Press, New York, NY, 1991. [19] Pilkington, G.J., Glioma heterogeneity in vitro: the significance of growth factors and gangliosides, Neuropathol. Appl. Neurobiol., 18 (1992) 434 442. [20] Russell, D.S. and Rubinstein, L.J., Pathology of Tumours of the Nervous System, 5th Ed., Williams and Wilkens, Baltimore, MD, 1989, pp. 83-289. [21] Rutka, T.J., Apodaca, G., Stern, R. and Rosenblum, M., The extracellular matrix of the central and peripheral nervous system: structutre and function, J. Neurosurg., 69 11988) 155 170. [22] Small, R.K., Riddle, R and Noble, M., Evidence for migration of oligodendrocyte-type 2 astrocyte progenitor cells into developing rat optic nerve, Nature (London), 328 (1987) 155 157. [23] Stallcup, W.B., Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin, Exp. Cell Res., 177 (1988) 9(~102. [24] Thompson, L.K., Horowitz, P.M., Bentley, K.L., Thomas, D.D., Alderete, J.F. and Klebe, R.J.. Localization of the gangliosidebinding site of fibronectin. J. Biol. Chem., 261 (1986) 5209 5214.
ELSEVIER
HEUROSCIENC[ lETTERS
Cell surface gangliosides are involved in the control of human glioma cell invasion in vitro Abderrahim Merzak*, Shahriar Koochekpour, Geoffrey J. Pilkington Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, UK
Received 19 May 1994; Accepted 22 June 1994
Abstract The involvement of cell surface gangliosides in glioma cell invasion in vitro was examined using the ganglioside-specific antibody A2B5 and Matrigel-coated 8-/1m porosity polycarbonate filters. Invasion of six cell lines derived from glial tumours of different histological grades was found to be markedly inhibited by A2B5 (50-96% inhibition) in a dose-dependent manner. Furthermore, exogenous gangliosides were found to prevent cell invasion when they were incubated with cells during the invasion assay. These results suggest that cell surface gangliosides are involved in glioma cell invasion in vitro, probably because of their adhesionpromoting action to basement membrane components. Key words." Glioma; Ganglioside; Invasion, Matrigel
Gliomas, the major form of primary'brain tumour in man [20], are characterized by their local invasive infiltration of the brain, irrespective of their histological grade of malignancy. The factors which control this particular behaviour are poorly documented. Gangliosides, a class of sialic acid-containing glycosphingolipids, are ubiquitously present in the body but exist in high concentration in the brain [5]. Among the multiple functions proposed for these molecules is their role in adhesion [6] which is a crucial step in migration. In agreement with this, ganglioside-expressing glial progenitor cells in the developing mammalian nervous system have been shown to be highly migratory [22]. We proposed, therefore, that these molecules may be involved in the invasive behaviour of glioma [19]. Moreover, we demonstrated recently that exogenously added gangliosides promote glioma cell adhesion, migration and invasion in vitro [12,13]. However, the role of intrinsic cell surface gangliosides in invasion remains to be investigated. To study this potential role, we assessed the effect of
*Corresponding author. Fax: (44) (71) 708-3895. 0304-3940/94/$7.00© 1994 Elsevier ScienceIreland Ltd. All rights reserved S S D I 0304-3940(94)00490-2
A2B5 antibody on the in vitro invasion activity of six cell lines derived from human glioma of different histological grades (Table 1). A2B5 antibody was initially reported as specific for G Q l c ganglioside [3] but has been, subsequently, demonstrated to react with several other gangliosides [4,8]. Invasiveness was assessed by the method of Albini et al. [1] with modifications. 8-ktm porosity polycarbonate filters in 24-well transwells were coated with 20 p g of the reconstituted basement membrane composite Matrigel (Collaborative Research, Lexington, MA) in cold Dulbecco's modified Eagle's medium, to form a thin continuous layer on top of the filter. The Matrigel was left to air-dry overnight. Before addition of the cells, excess medium was removed from the upper compartment. 5 x 104 cells from each cell line were resuspended in 100pl of serum-free medium or undiluted A2B5 supernatant and plated on coated filters for 18 h at 37°C in a humidified 95% air-5% CO, atmosphere. The lower compartment contained 0.5 ml of serum-free medium supplemented with P D G F (10 ng/ml) as a chemoattractant. Cells were then fixed with acetic acid-alcohol and stained with Giemsa. Cells on the upper surface of the filter were removed by wiping with a cotton swab and invasion was determined by counting the cells that had
A. Merzak et al./Neuroscience Letters 177 (1994) 44~16 200 •
[]
== l= 3 z = ® 0
Control +A2B5
100
o
o o
Fig. 1. Effect of A2B5 on invasion of h u m a n glioma cell lines. 13 fields were counted in each assay. Each sample was assayed in triplicate and assays were repeated twice. Statistical significance was assessed by Student's t test (P < 0.01).
migrated to the lower side of the filter with a phasecontrast microscope at 200 × magnification. This system has proved very useful in identifying the factors involved in glioma cell invasion in vitro [10,11,14]. The invasion activity of all six glioma cell lines studied was strongly (50-96%) inhibited by A2B5 antibody (Fig. 1). Furthermore, we found that this effect is dose-dependent by incubating the cells in the presence of increasing amounts of A2B5 antibody during the invasion assay (Fig. 2). As a control, an epidermal growth factor receptor antibody proved to have no effect on cell invasion (data not shown). These results suggest that cell membrane gangliosides play an important role in the invasive behaviour of glioma cells in vitro. We have demonstrated recently that exogenously added gangliosides stimulate glioma cell invasion in vitro [13]. In addition, we reported that this effect may be explained by their adhesion-promoting action to basement membrane proteins, including laminin [12]. Although the extracellular space of the brain is devoid of a well-defined ECM [21], basement membrane does exist around blood vessels, in the glia limitans externa and underlying choroid plexus epithelial cells [18,21]. Laminin has been demonstrated to be intensly
45
expressed in the basement membranes of blood vessels and leptomeninges in low-grade gliomas [15] and may be synthesized by reactive and neoplastic astrocytes [9]. Since 60% of Matrigel is laminin [7], this commercially available ECM composite was felt to provide a good model for our adhesion and invasion studies. We postulated that exogenous gangliosides, added during the assay, may compete with cell surface gangliosides in binding to Matrigel and, therefore, prevent cell invasion. To verify this hypothesis in the present study, IPNT-H cells were incubated, during the invasion assay, in the presence or absence of 100 ng/ml of either G D l a or GD3 (Sigma, UK). The number of invasive cells dropped dramatically by 86 and 98% by GD3 and GDla, respectively (Fig. 3), confirming the role of cell surface gangliosides in glioma cell invasion in vitro. The data presented here not only confirm our previous findings with exogenous gangliosides but also demonstrate, for the first time, the involvement of cell membrane gangliosides in glioma cell invasion in vitro. It has been shown that expression of gangliosides correlates well with migration ([22]; Martin et al., in prep.). Dramatic changes in ganglioside expression have also been reported in ontogenesis and oncogenesis [5]. It is also likely that gangliosides may be involved in migration and invasion via cell-extracellular matrix (ECM) interactions. In agreement with that, we have demonstrated, recently, that gangliosides enhance adhesion of human glioma cell lines to fibronectin, laminin, vitronectin and collagen I [12]. This is supported by previous reports of the involvement of gangliosides in fibronectinmediated cell adhesion and of the identification of a 200 •
C
[]
1/100 UIO 1/1
•
._e IL.
== E
100
Z o 0
Table 1 Designation
Passage (n)
Histological type
IPTR-6 IPNT-H IPSB-18 GO-G-CCM GO-G-UVW IPRM-5
6 9 32 < 100 < 100 15
Ganglioglioma Pilocytic astrocytoma Anaplastic astrocytoma Anaplastic astrocytoma Anaplastic astrocytoma Glioblastoma multiforme
Z
~
~
~
~,
o
@
Fig. 2. Effect of undiluted (1/1) and 1/10- and 1/100-diluted A2B5 supernatant on invasion of h u m a n glioma cell lines. Assays were carried out as in Fig. 1. Statistical significance was assessed by Student's t test (P < 0.01).
46
A. Merzak et al. / Neuroscience Letters 177 (1994) 44-46
60
IPNT-H
50
•o
40
gI iz I,,.
e ,,¢1
E 30 3
Z Q
o
20
10
Control
GD3
GDla
Fig. 3. Effect of exogenous G D l a and GD3 on invasion of IPNT-H cells. Assays were carried out as in Fig. 1. Statistical significance was assessed by Student's t test (P < 0.01).
ganglioside-binding site on fibronectin [24]. Furthermore, integrins, a major class of cell surface receptors for ECM proteins, are subjected to regulation by gangliosides [2,16,23]. We have demonstrated recently the role of these molecules in glioma cell adhesion to basement membrane proteins in vitro (Merzak et al., in prep.). In addition, these molecules have also been shown to be involved in glioma cell invasion in vitro [17]. A study of the mechanisms involved in ganglioside function may help to gain valuable insights into the invasive properties of neoplastic glia. This work was supported by a grant from the Leverhulme Trust. The A2B5 monoclonal antibody-secreting clone was a gift from Professor Frank Walsh. [1] Albini, A., ]wamoto, Y., Kleinman, H.K., Martin, G.R., Aaronson, S.A., Kozlowski, J.M. and McEwan, R.N., A rapid in vitro assay for quantitating the invasive potential of tumour cells, Cancer Res., 47 (1987) 3239 3245. [2] Cheresh, D.A., Pierschbacher, M.D., Herzig, M.A. and Mujoo, K., Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins, J. Cell Biol., 105 (1987) 1163-1173. [3] Fishman, P.H. and Brady, R.O., Biosynthesis and function of gangliosides, Science. 194 (1976) 90(~915. [4] Fredman, P., Magnani, G.L., Nirenberg, M. and Ginsberg, V., Monoclonal antibody A2B5 reacts with many gangliosides in neuronal tissue, Arch. Biochem. Biophys., 233 (19841 661 666. [5] Hakomori, S.I., Glycosphingolipids in cellular interaction, differ-
entiation and oncogenesis, Annu. Rev. Biochem., 50 (1981) 733 764. [6] Hakomori, S.I., Bifunctional role of glycosphigolipids, J. Biol. Ch9m., 265 (1990) 18713 18716. [7] Kleinman, H.K., McGarvey, M.L., Hassell, J.R., Star, V.L., Cannon, F.B., Laurie, G.W. and Martin, G.R., Basement membrane complexes with biological activity, Biochemistry, 25 (1986) 312 318. [8] Kundu, S., Pleetman, M., Redwine, W., Boyd, A. and Marcus, D., Binding of monoclonal antibody A2B5 to gangliosides, Biochem. Biophys. Res. Commun., 116 (1983) 83(~842. [9] McComb, R.D. and Bigner, D.D., Immunolocalization oflaminin in neoplasms of the central and peripheral nervous system, J. Neuropathol. Exp. Neurol., 44 (1985) 242-253. [10] Merzak, A., McCrea, S., Koochekpour, S. and Pilkington, G.J., Control of human glioma cell growth, migration, and invasion in vitro by transforming growth factor-betal, Br. J. Cancer, in press. [I 1] Merzak, A., Koochekpour, S. and Pilkington, G.J., CD44 mediates human glioma cell adhesion and invasion in vitro, Cancer Res., in press. [12] Merzak, A., Koochekpour, S. and Pilkington, G.J., Gangliosides modulate adhesion of human glioma cell lines to basement membrane proteins in vitro, submitted. [13] Merzak, A., Koochekpour, S., McCrea, S., Roxanis, I. and Pilkington, GJ., Gangliosides modulate proliferation, migration, and invasiveness of human brain tumour cells in vitro, submitted. [14] Merzak, A., Parker, C., Koochekpour, S., Sherbet, G.V. and Pilkington, G.J., Overexpression of the 18A2/mtsl gene and downregulation of the TIMP-2 gene in invasive human glioma cell lines in vitro, Neuropathol. Appl. Neurobiol., in press. [15] Morris, C.S and Esiri, M.M., Immunocytochemical study of macrophages and microglial cells and extracellular matrix components in human CNS disease. 1: Gliomas, J. Neurol. Sci., 101 (1991) 47 58. [16] Mugnai, G., Lewandowska, K., Choi, H.U., Rosenberg, L.C. and Culp, L.A., Ganglioside-dependent adhesion events of human neuroblastoma cells regulated by the RGDS-dependent fibronectin receptor and proteoglycans, Exp. Cell Res., 175 (1988) 229247. [17] Paulus, W. and Tonn, J.C., Basement membrane invasion of glioma cells mediated by integrin receptors, J. Neurosurg., 80 (1994) 515 519. [18] Peters, A., Palay, S.L. and Webster, H.F., Neurons and Their Supporting Cells. The Fine Structure of the Nervous System, Oxford University Press, New York, NY, 1991. [19] Pilkington, G.J., Glioma heterogeneity in vitro: the significance of growth factors and gangliosides, Neuropathol. Appl. Neurobiol., 18 (1992) 434 442. [20] Russell, D.S. and Rubinstein, L.J., Pathology of Tumours of the Nervous System, 5th Ed., Williams and Wilkens, Baltimore, MD, 1989, pp. 83-289. [21] Rutka, T.J., Apodaca, G., Stern, R. and Rosenblum, M., The extracellular matrix of the central and peripheral nervous system: structutre and function, J. Neurosurg., 69 11988) 155 170. [22] Small, R.K., Riddle, R and Noble, M., Evidence for migration of oligodendrocyte-type 2 astrocyte progenitor cells into developing rat optic nerve, Nature (London), 328 (1987) 155 157. [23] Stallcup, W.B., Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin, Exp. Cell Res., 177 (1988) 9(~102. [24] Thompson, L.K., Horowitz, P.M., Bentley, K.L., Thomas, D.D., Alderete, J.F. and Klebe, R.J.. Localization of the gangliosidebinding site of fibronectin. J. Biol. Chem., 261 (1986) 5209 5214.