the effects of fibronectin on the migration of human ... effects of fibronectin on cell migration...
Post on 22-Mar-2020
Embed Size (px)
J. Cell Set. 48, 3OI-3I4 (1981) 301 Printed in Great Britain © Company of Biologists Limited 1081
THE EFFECTS OF FIBRONECTIN ON THE
MIGRATION OF HUMAN FORESKIN
FIBROBLASTS AND SYRIAN HAMSTER
MELANOMA CELLS INTO THREE-DIMENSIONAL
GELS OF NATIVE COLLAGEN FIBRES
SETH L. SCHOR*, ANA M. SCHOR* AND GEORGE W. BAZILLf • Cancer Research Campaign Department of Medical Oncology, Christie Hospital and Holt Radium Institute, Wilmsloto Road, Manchester M50 gBX, U.K. and t Paterson Laboratories, Christie Hospital and Holt Radium Institute, Wihnslow Road, Manchester M20 gBX, U.K.
The effects of fibronectin on the migration of human skin fibroblasts and Syrian hamster melanoma cells into 3-dimensional gels of native collagen fibres have been examined. Cell migration into the 3-dimensional gel was measured by plating cells on the gel surface and then determining the percentage of cells within the gel at various times thereafter by direct micro- scopic examination. We find that fibronectin bound to collagen inhibits the migration of human skin fibrobroblasts and stimulates the migration of melanoma cells into the gel matrix. Fibronectin had no apparent effect on cell adhesion to the collagen gels, proliferation or morphology under the conditions studied.
The biochemical composition and structural orientation of the extracellular matrix play an important role in the control of cell migration under both normal and patho- logical conditions in vivo (Leighton, Kalla, Kline & Belkin, 1959; Trinkaus, 1969; Willis, 1973; Strauli & Weiss, 1971; Hay, 1978). Collagen is a major constituent of the extracellular matrix (Miller, 1977) and has been used as a substratum for the culture of a number of cell types in vitro (Ehrmann & Gey, 1956; Michalopoulos & Pitot, 1975; Emermann & Pitelka, 1977). Techniques for obtaining quantitative data regarding cell migration into 3-dimensional gels of native collagen fibres have been described in a previous communication (Schor, 1980); cell migration into the collagen gel is measured by plating cells on the gel surface and then determining the percentage of cells within the 3-dimensional collagen matrix at various times thereafter. In view of the important role played by the extracellular matrix in the control of cell migration in vivo, it is desirable that cell migratory behaviour in vitro be monitored on biologic- ally relevant macromolecular matrices, such as the 3-dimensional collagen gel, rather than on 2-dimensional artificial substrata. Our long-term objective is to study cell migration, especially in relation to the process of tumour cell invasion, on progressively more complex macromolecular matrices prepared by the stepwise addition of other
302 S. L. Schor, A. M, Schor and G. W. Bazill
matrix components to the collagen gel. We have chosen to begin with fibronectin because of its common association with collagen in the extracellular matrix (Linder, Stenman, Lehto & Vaheri, 1978) and at the cell surface (Bornstein & Ash,
Fibronectin is a high-molecular-weight glycoprotein found in an insoluble form at the surface of normal fibroblasts and other cell types (Mosher, Saksela, Keski-Oja & Vaheri, 1977; Hynes, Destree, Perkins & Wagner, 1979; Smith, Riggs & Mosesson, 1979) and in a soluble form in serum, where it has been referred to as cold insoluble globulin (Grinnell & Hays, 1978). Further information concerning the biochemistry of fibronectin and its role in mediating many aspects of cell-cell and cell-matrix interactions may be found in a number of excellent reviews (Vaheri & Mosher, 1978; Yamada & Olden, 1978; Hynes et al. 1979).
In this communication we report the effects of serum-derived fibronectin on cell migration into 3-dimensional gels of native collagen fibres, using normal human skin fibroblasts and the highly tumourigenic Syrian hamster melanoma cell line, RPMI- 3460 (Moore, 1964). Data are presented indicating that the presence of fibronectin in the collagen substratum stimulates the migration of the melanoma cells, but inhibits the migration of fibroblasts into the collagen gel.
MATERIALS AND METHODS
RPMI-3460 Syrian hamster melanoma cells were originally obtained from Dr M. Steinberg (Department of Pathology, New York University Medical Center) and human fibroblasts were isolated in this laboratory from foreskin specimens obtained from St Mary's Hospital, Man- chester. Stock cultures of both cell types were grown in plastic tissue culture dishes in Eagle's MEM supplemented with 10 % foetal calf serum, 2 min glutamine, 1 min sodium pyruvate, non-essential amino acids (Gibco-Biocult) and 100 units/ml of penicillin and streptomycin. Stock cultures were subcultured once a week and the medium changed 3 times a week. Cells to be used in the experiments were brought into suspension from stock cultures by exposure to 005 % trypsin (Sigma, Ltd, Cat. No. T-8253) in phosphate-buffered saline for 5 min at 37 °C, followed by the addition of equal volume of growth medium containing 10% foetal calf serum and collecting the cells by centrifugation for 5 min at 800 g.
Preparation of collagen substrata
Type I collagen was extracted from rat tail tendons as previously described (Schor, 1980). The concentration of collagen in the aqueous stock solution was adjusted to 23 mg/ml. Three- dimensional gels of native collagen fibres were prepared in 35-mm plastic tissue culture dishes (Gibco-Biocult, Ltd, Uxbridge, Cat. No. 53066) by rapidly mixing 85 ml of the collagen solution with 1 ml of 10 x concentrate MEM and 0-5 ml of 4'4% sodium bicarbonate and pipetting 2-ml aliquots into the dishes. Gels set within 5 min and were incubated at 37 °C for 24 h in a humidified COt incubator before use. These gels consist of a hydrated meshwork of native collagen fibres (Elsdale & Bard, 1972).
Determination of total cell number and migration of cells into collagen gel matrix
The total number of cells growing on the 3-dimensional collagen gels was determined as previously described (Schor, 1980) by dissolving the gel with bacterial collagenase (Sigma Ltd, Cat. No. C2139). The percentage of total cells within the collagen gel matrix was determined using the 'microscopic method' previously described (Schor, 1980). Accordingly, cultures were
Effects of fibronectin on cell migration 303
examined with phase-contrast optics using a Leitz Diavert microscope fitted with an SY2 photographic graticule defining an area of 09 x 065 cm1. The number of cells both on the gel surface and within the 3-dimensional collagen matrix was determined in approximately 20 regions of the gel surface selected at random moving across the diameter of the gel. Data collected from at least 3 such gels were used to calculate the mean ± S.D. of the percent of cells within the gel matrix for each result presented.
Isolation of fibronectin
Fibronectin was prepared from human serum by affinity chromatography on gelatin-Sepharose columns as described by Engvall & Rouslahti (1977). After application of the serum, the column (12 x 15 cm) was washed with 015 M NaCl in o-oi M potassium phosphate buffer, pH 7-4 (PBS), then with PBS containing i-6 M urea. Fibronectin was eluted with 6 M urea in PBS and the solution was dialysed overnight against 50 vol. of buffer containing 10 mM cyclohexylamino- propane sulphate, 015 M NaCl and 1 mM CaCls (CAPS buffer) (Yamada & Kennedy, 1979). Fibronectin was concentrated by precipitation with ammonium sulphate at 40 % saturation. The precipitate was dissolved in CAPS buffer and dialysed against this buffer to remove ammonium sulphate. The protein concentration was adjusted to 2-4 mg/ml (depending on the experiment) and SDS electrophoresis showed a major band (doublet) at mol.wt 220000 and minor bands (< 10 %) of low-molecular-weight components. Growth medium containing 15% fibronectin-depleted foetal calf serum was prepared by applying foetal calf serum to the gelatin- Sepharose column and washing with growth medium (Eagle's MEM containing 2 mM glut- amine, 1 mM sodium pyruvate, non-essential amino acids (Gibco-Biocult) and 100 units/ml of penicillin and streptomycin).
Fibronectin binds avidly to a specific region of the ai chain of type I collagen between amino acid residues 568 and 835 (Kleinman, McGoodwin & Klebe, 1976). In order to examine the effects of fibronectin on the migration of RPMI-3460 mela- noma cells, collagen gels containing bound fibronectin were prepared by incubating gels with 1 ml of serum-free growth medium containing either o, 1, 5 or 50 fig of fibronectin for 1 h at 37 °C and then washing the gels 5 times with serum-free medium to remove unbound material. The melanoma cells were detached from stock cultures by exposure to trypsin, resuspended in serum-free growth medium containing 1 mg/ml bovine serum albumin (Gibco-Biocult, Cat. No. 164) and 10% (v/v) lactalbumin hydrolysate (Sigma Ltd, Cat. No. A-4503) at 4X io4 cells/ml and 1 ml of this cell suspension plated on control and fibronectin-preincubated gels already over- laid with 1 ml of serum-free medium. The presence of bovine serum albumin and lactalbumin hydrolysate resulted in improved cell survival in serum-free medium during the course of the experiment. Cultures were then incubated at 37 °C and both total cell number and the percentage of cells within the gel were measured daily as previously described (Schor, 1980). As can be seen in Fig. IA, there was no increase in total cell number in serum-free medium during the 3-day incubation period on either control gels or gels preincubated with fibronectin. Cell viability was estimated at the end of the experiment by tryp