the effects of fibronectin on the migration...

J. Cell Set. 48, 3OI-3I4 (1981) 301Printed 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 andHolt Radium Institute, Wilmsloto Road, Manchester M50 gBX, U.K. andt Paterson Laboratories, Christie Hospital and Holt Radium Institute, Wihnslow Road,Manchester M20 gBX, U.K.

SUMMARY

The effects of fibronectin on the migration of human skin fibroblasts and Syrian hamstermelanoma cells into 3-dimensional gels of native collagen fibres have been examined. Cellmigration into the 3-dimensional gel was measured by plating cells on the gel surface and thendetermining 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 humanskin fibrobroblasts and stimulates the migration of melanoma cells into the gel matrix. Fibronectinhad no apparent effect on cell adhesion to the collagen gels, proliferation or morphology underthe conditions studied.

INTRODUCTION

The biochemical composition and structural orientation of the extracellular matrixplay 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 theextracellular matrix (Miller, 1977) and has been used as a substratum for the cultureof a number of cell types in vitro (Ehrmann & Gey, 1956; Michalopoulos & Pitot,1975; Emermann & Pitelka, 1977). Techniques for obtaining quantitative dataregarding cell migration into 3-dimensional gels of native collagen fibres have beendescribed in a previous communication (Schor, 1980); cell migration into the collagengel is measured by plating cells on the gel surface and then determining the percentageof cells within the 3-dimensional collagen matrix at various times thereafter. In viewof the important role played by the extracellular matrix in the control of cell migrationin 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, ratherthan on 2-dimensional artificial substrata. Our long-term objective is to study cellmigration, especially in relation to the process of tumour cell invasion, on progressivelymore 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 fibronectinbecause 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 atthe 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 insolubleglobulin (Grinnell & Hays, 1978). Further information concerning the biochemistryof fibronectin and its role in mediating many aspects of cell-cell and cell-matrixinteractions 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 cellmigration into 3-dimensional gels of native collagen fibres, using normal human skinfibroblasts and the highly tumourigenic Syrian hamster melanoma cell line, RPMI-3460 (Moore, 1964). Data are presented indicating that the presence of fibronectin inthe collagen substratum stimulates the migration of the melanoma cells, but inhibitsthe migration of fibroblasts into the collagen gel.

MATERIALS AND METHODS

Cell cultures

RPMI-3460 Syrian hamster melanoma cells were originally obtained from Dr M. Steinberg(Department of Pathology, New York University Medical Center) and human fibroblasts wereisolated 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'sMEM 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 tobe used in the experiments were brought into suspension from stock cultures by exposure to005 % 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 serumand 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 collagensolution with 1 ml of 10 x concentrate MEM and 0-5 ml of 4'4% sodium bicarbonate andpipetting 2-ml aliquots into the dishes. Gels set within 5 min and were incubated at 37 °C for24 h in a humidified COt incubator before use. These gels consist of a hydrated meshwork ofnative 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 aspreviously 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 determinedusing 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 SY2photographic graticule defining an area of 09 x 065 cm1. The number of cells both on the gelsurface and within the 3-dimensional collagen matrix was determined in approximately20 regions of the gel surface selected at random moving across the diameter of the gel. Datacollected from at least 3 such gels were used to calculate the mean ± S.D. of the percent of cellswithin the gel matrix for each result presented.

Isolation of fibronectin

Fibronectin was prepared from human serum by affinity chromatography on gelatin-Sepharosecolumns 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 andthe 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 removeammonium sulphate. The protein concentration was adjusted to 2-4 mg/ml (depending on theexperiment) and SDS electrophoresis showed a major band (doublet) at mol.wt 220000 andminor 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 ofpenicillin and streptomycin).

RESULTS

Fibronectin binds avidly to a specific region of the ai chain of type I collagenbetween 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 incubatinggels with 1 ml of serum-free growth medium containing either o, 1, 5 or 50 fig offibronectin for 1 h at 37 °C and then washing the gels 5 times with serum-free mediumto remove unbound material. The melanoma cells were detached from stock culturesby exposure to trypsin, resuspended in serum-free growth medium containing1 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 ofthis 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 andlactalbumin hydrolysate resulted in improved cell survival in serum-free mediumduring the course of the experiment. Cultures were then incubated at 37 °C and bothtotal cell number and the percentage of cells within the gel were measured daily aspreviously described (Schor, 1980). As can be seen in Fig. IA, there was no increasein total cell number in serum-free medium during the 3-day incubation period oneither control gels or gels preincubated with fibronectin. Cell viability was estimatedat the end of the experiment by trypan blue exclusion and judged to be greater than90% in all cases. The effects of fibronectin on cell migration into the gel are shown inFig. 1 B. Cell migration into the gels proceeded in an approximately linear fashion onall substrata and fibronectin was observed to have a dose-dependent stimulatoryeffect on cell migration, with 28% of the cells present within gels preincubated with

304 S. L. Schor, A. M. Schor and G. W. Bazill

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Fig. i. The effects of fibronectin on the proliferation (A) and migration (D) of melanomacells in serum-free medium. Collagen gels were incubated for i h at 37 °C with 1 mlof serum-free growth medium containing either no additions (i.e. control), or 1, 5 or50 fig fibronectin, they were washed 5 times with serum-free medium to removeunbound fibronectin and then overlaid with 1 ml of serum-free medium. Melanomacells were brought into suspension from stock cultures by exposure to trypsin, re-suspended at a concentration of 4 x io4 cells/ml in serum-free medium containinglactalbumin hydrolysate (10%, v/v) and bovine serum albumin (1 nag/ml) and 1 mlof this cell suspension plated on both control and fibronectin-preincubated gels. Allcultures were maintained for 3 days at 37 °C in an incubator gassed with air containing5 % CO,, A. Total cell number was determined at 24-h intervals. B. The percentagesof cells within the collagen gel were also determined at 24-h intervals by direct micro-scopic observation as previously described (Schor, 1980). Three replicate cultures wereused to determine the mean values shown in the figure and the s.D. (not shown) wasless than 12 % of the mean in all cases. O> control gels; # , gel preincubated with 1 figfibronectin; A, gel preincubated with 5 fig fibronectin, x , gel preincubated with 50 figfibronectin.

50 jig fibronectin after 3 days in culture compared to only 10% within control gelsat this time. These results also indicate that the migration of melanoma cells into thecollagen gels (either with or without fibronectin) may occur in serum-free mediumand is thus not dependent on factors supplied by serum.

The effects of fibronectin on cell proliferation and migration in the presence ofserum containing media are shown in Fig. 2. Gels preincubated with 50 /tg of fibro-nectin in serum-free medium were prepared as described above and then both controland fibronectin-preincubated gels were overlaid with 1 ml of growth medium con-taining 10% fibronectin-depleted foetal calf serum. Control gels overlaid with 1 ml ofgrowth medium containing 10% whole foetal calf serum (i.e. containing fibronectin)were prepared at the same time. Melanoma cells were suspended at 4X io4 cells/mlin growth medium containing 10% of either fibronectin-depleted or whole foetal calfserum and 1 ml of these cell suspensions plated onto gels overlaid with the correspond-ing medium. Total cell number and the percentage of cells within the gel weremeasured daily for the next three days. As shown in Fig. 2, cell proliferation occurredat the same exponential rate in the presence of both 10% fibronectin-depleted serum


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Fig. 2. The effects of fibronectin on the proliferation (A) and migration (B) of mela-noma cells in serum-containing media. Cells were cultured in growth medium con-taining 10 % fibronectin-depleted foetal calf serum on control gels (O) and gels pre-incubated with 50 fig fibronectin (#) or in medium containing 10 % whole foetal calfserum on control gels ( • ) ; see text for experimental details. Data are presented for(A) total cell number, and (B) the percentage of cells within the collagen gels. Threereplicate cultures were used to determine the mean values shown in the figure and theS.D.s were less than 12 % of the mean in all cases (not shown).

(on control and fibronectin-preincubated gels) and 10% whole serum (on control gels).In contrast, there was significantly more cell migration into those gels preincubatedwith fibronectin compared to control gels, with approximately 28 % of the cells foundwithin the fibronectin-preincubated gels after 3 days of growth compared to onlyI7 '5% within control gels. These data indicate that melanoma cells migrate to agreater extent into a collagen substratum containing fibronectin compared to a simplecollagen substratum under conditions which support cell proliferation (i.e. in thepresence of serum). The data presented in Fig. 2 also indicate that the amount offibronectin in medium containing 10% whole foetal calf serum is not sufficient toinfluence cell migration; this result was not due to possible differences in the bio-logical activity of human and bovine fibronectins, since the migratory behaviour ofmelanoma cells on gels preincubated with 50 /ig of bovine fibronectin was indis-tinguishable from that shown in Figs. 1 and 2 for gels preincubated with humanfibronectin (data not shown).

Results identical to those shown in Figs. 1 and 2 were obtained when fibronectinwas present continuously in the medium during the 3-day duration of the experimentrather than preincubated with the gels. Melanoma cells were suspended at a con-centration of 4X io4 cells/ml in either serum-free medium (containing bovine serumalbumin and lactalbumin hydrolysate) or medium containing 10% fibronectin-

306 5. L. Schor, A. M. Schor and G. W. Bazill

depleted foetal calf serum and i-ml aliquots were then plated onto collagen gelsoverlaid with i ml of the corresponding medium (control) or i ml of medium con-taining 50 /tg fibronectin. The percentage of cells attached two hours after plating wasdetermined as previously described (Schor & Court, 1979) and the remaining gelswere incubated for 3 days at 37 °C, at which time both total cell number and thepercentage of cells within the gel were measured. As can be seen in Table 1, melanoma

Table 1. The effects of fibronectin on the initial attachment, proliferation and migrationof melanoma cells in serum-free and serum-containing media

Initial cellattachment, Final cell Final cell migration

Medium Fibronectin % no. x io~* into gel, %

Serum-free Control 79-1 ±7-8 4-110-3 1Continuously 82-419-2 4-010-5 27-513-1present inmedium

15 % Fibronectin- Control 80-216-5 26-1 i i - 8 i6-il2-odepleted foetal Continuously 80-9 + 91 28-913-1 29-112-5 (P < 001)calf serum present in

mediumMelanoma cells were suspended at a concentration of 4 x io4 cells/ml in cither serum-free

medium or medium containing 10 % fibronectin-depleted foetal calf serum and 1 nil of eachsuspension was then plated onto collagen gels overlaid with 1 ml of the corresponding medium(control) or 1 ml of medium containing 50 fig fibronectin. The percentage of cells attached2 h after plating was determined as previously described (Schor & Court, 1979) and the re-maining gels were incubated for 3 days, at which time both total cell number and the percentageof cells within the gel were determined. Three replicate gels were used to obtain the mnan ands.D. for all determinations presented.

cells migrated into the collagen gels to a greater extent in the presence of mediumcontaining fibronectin compared to controls. These data also indicate that the initialattachment of melanoma cells to the collagen gel (2 h after plating) was not signific-antly higher in the presence of fibronectin, a finding consistent with our previousresults (Schor and Court, 1979; Schor, 1979).

All concentrations of fibronectin examined (5-400 /tg/ml) stimulated the migrationof melanoma cells into the collagen gel, although in certain experiments concentrationsof fibronectin greater than 100/ig/ml resulted in a lower stimulation than obtainedwith 50 fig/ml (data not shown).

The morphology of melanoma cells after 3 days of culture on collagen was notaffected by the presence of fibronectin (Fig. 3). Cells in the presence or absence offibronectin were spherical in appearance, both on the gel surface and within the3-dimensional collagen matrix.

The effects of fibronectin on the proliferation and migration of human foreskinfibroblasts in serum-containing medium are shown in Fig. 4. Cells were suspended ata concentration of 2 x io4 cells/ml in medium containing 15% fibronectin-depleted


Fig. 3. The effects of fibronectin on the morphology of melanoma cells. Cells wereincubated in serum-free medium either on control gels (A) or gels preincubated with50 fig fibronectin (B), as described in the legend of Fig. 1. No difference in cellmorphology is apparent after 1 day of growth, with the majority of cells on bothsubstrata spherical in appearance, x 300.

foetal calf serum and 1 ml of this suspension was then plated onto collagen gelsoverlaid with 1 ml of this medium (control) or medium containing 100/tg fibronectin.Cultures were incubated for 3 days at 37 °C. Fibronectin had no effect on cell pro-liferation during this period (Fig. 4). In contrast, fibronectin did have a significantinhibitory effect on fibroblast migration into the gel, with 5-9% of the cells within thegel after 3 days of growth in the presence of fibronectin compared to 15*5 % in theabsence of fibronectin. Fibronectin had no observable effect on cell morphology duringthis 3-day period (Fig. 5).

A similar inhibitory effect of fibronectin on fibroblast migration was obtained whenfibronectin-preincubated collagen gels were used. Collagen gels were incubated with1 ml of either serum-free medium (control) or serum-free medium containing 200 figfibronectin. After washing 5 times with serum-free medium, gels were overlaid with

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Fig. 4. The effects of fibronectin on the proliferation (A) and migration (B) of humanforeskin fibroblasts in serum-containing media. Cells were suspended at a concentra-tion of 2 x io4 cells/ml in medium containing 15% fibronectin-depleted foetal calfserum and 1 ml of this suspension was then plated onto collagen gels overlaid with1 ml of this medium (i.e. control) (O), or 1 ml of this medium containing 100 figfibronectin ( • ) . All cultures were incubated at 37 °C for 3 days. Data are presentedfor (A) total cell number and (B) the percentage of cells within the collagen gel. Threereplicate cultures were used to determine the mean values shown in the figure and theS.D.S were less than 12 % of the mean in all cases (not shown).

Table 2. The effects of fibronectin on the initial attachment, proliferation and migrationof human foreskin fibroblasts in serum-free and serum-containing media

MediumFibronectinattachment 0//o

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Final cell migrationinto gel, %

Serum-free

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ControlPreincubated

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Collagen gels were incubated with 1 ml of either serum- free medium (control) or serum-freemedium containing 200 fig fibronectin. After washing 5 times with serum-free medium, gelswere overlaid with 1 ml of either serum-free medium (containing lactalbumin hydrolysateand bovine serum albumin) or medium containing 15 % fibronectin-depleted foetal calf serum.Fibroblasts were suspended at a concentration of 2 x io4 cells/ml in either serum-free mediumor 15 % fibronectin-depleted foetal calf serum medium and 1 ml plated onto control or fibro-nectin-preincubated gels overlaid with the corresponding medium. The percentage of cellsattached 2 h after plating was measured and the remaining cultures were incubated for 3 days,at which time both the total cell number and percentage of cells within the collagen gels weremeasured. Three replicate gels were used to determine the mean and S.D. for each parametermeasured.

Effects of fibronectin on cell migration 3°9

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Fig. 5. The effect of fibronectin on the morphology of fibroblasts in serum-con-taining media. Phase-contrast micrographs of human foreskin fibroblasts from theexperiment shown in Fig. 4 after 3 days of growth in: (A) medium containing 15 %fibronectin-depleted foetal calf serum and (B) medium containing 50 fig fibro-nectin/ml. x 350.

1 ml of either serum-free medium (containing bovine serum albumin and lactalbuminhydrolysate) or medium containing 15% fibronectin-depleted foetal calf serum.Fibroblasts were suspended at a concentration of 2 x io4 cells/ml in either serum-freemedium or 15% fibronectin-depleted serum medium and 1 ml plated onto control orfibronectin-preincubated gels overlaid with the corresponding medium. The per-centage of cells attached 2 h after plating was measured and the remaining gels wereincubated for 3 days at 37 °C, at which time both the total cell number and percentageof cells within the collagen gel were measured. As can be seen in Table 2, the migra-tion of fibroblasts into fibronectin-preincubated gels was significantly lower comparedto controls, both in serum-free and serum-containing media. It should also be notedthat fibroblast migration into the gel occurred to approximately the same extent in


the presence of both serum-free and serum-containing media, indicating that fibro-blast migration into the collagen gel (as is the case with melanoma cells) does notdepend on factors supplied by serum. These results are in contrast with the reportedserum requirement for the migration of 3T3 cells on plastic tissue culture dishes(Lipton, Klinger, Paul & Holley, 1971).

As can also be seen in Table 2, there was an approximate doubling in total cellnumber during the 3-day period in serum-containing medium and no increase in cellnumber in serum-free medium, on both control and fibronectin-preincubation gels.Furthermore, the initial attachment of fibroblasts (within 2 h) to the native collagengel was greater than 90% under all conditions examined and thus not dependent onthe presence of exogeneous fibronectin.

We did not observe differences in the migratory behaviour of fibroblasts in mediumcontaining 15% fibronectin-depleted foetal calf serum compared to whole foetal calfserum (data not shown); again, this is presumably due to an insufficient quantity offibronectin in medium containing 15% whole foetal calf serum to affect cell migra-tion. Concentrations of fibronectin in the growth medium as low as 50 fig/ml resultedin a significant inhibition of fibroblast migration (data not shown) and a stimulationof fibroblast migration was never observed with any concentration of fibronectinexamined (5-400 /ig/ml).

Both the melanoma cells and fibroblasts used in this study were examined for thepresence of endogenous, surface-associated fibronectin using anti-fibronectin anti-body ; the fibroblasts were found to have considerable surface fibronectin organized ina similar fashion as previously described (Hynes et al. 1978), while the melanomacells did not have fibronectin which could be detected by this means (N. Tolson,personal communication).

DISCUSSION

The collagen gels used in this study consist of a hydrated, random meshwork ofnative type I collagen fibres (Elsdale & Bard, 1972; Schor, Allen & Harrison, 1980).Our previous studies have indicated that cells vary in their capacity to migrate fromthe gel surface into the 3-dimensional collagen matrix; all epithelial cells examined(both normal and tumour) do not migrate into the gel, while all fibroblasts (bothnormal and virally transformed) and certain tumour cells of non-epithelial origin (e.g.melanoma) do migrate into the collagen gel at rates which vary considerably accordingto cell type (Schor, 1980). The migration of the R3460 melanoma cells and humanskin fibroblasts into the collagen gel has previously been shown not to depend on theenzymatic digestion of the collagen substratum (Schor et al. 1980).

In this study, quantitative data regarding cell migration from the surface of thecollagen gel into the 3-dimensional gel matrix (previously referred to as 'infiltration';Schor, 1980) have been obtained by first plating cells on the gel surface and thenmeasuring the percentage of the total cell number within the collagen matrix atvarious times thereafter. Two distinct parameters of cell migratory behaviour aremeasured by this experimental approach; these are (1) the ability of cells to move


from an essentially 2-dimensional substratum (i.e. the gel surface), where cells makecontact with the collagen fibres only at their basal surface, into an isotropic, 3-dimensional environment (i.e. the gel matrix), where cells are potentially able toestablish adhesive interactions with collagen fibres over their entire surface, and (2)the ability of cells to migrate along the collagen fibres once they are within the3-dimensional collagen matrix. These 2 parameters appear to be capable of inde-pendent expression, since certain cell types (e.g. HeLa) are able to migrate on the gelsurface or within the 3-dimensional collagen matrix (when initially placed there as asingle cell suspension when the gel is formed), but are not able to migrate from thegel surface into the gel interior (Schor, 1980).

In this communication we present evidence that fibronectin isolated from serumstimulates the migration of R3460 Syrian hamster melanoma cells into 3-dimensionalgels of native collagen fibres, but inhibits the similar migration of normal human skinfibroblasts. This is the first study in which quantitative data have been presentedregarding the effects of fibronectin on cell migration into a 3-dimensional macro-molecular matrix. Fibronectin did not affect cell proliferation or cell morphologyunder the experimental conditions used in this study.

As discussed above, fibronectin may influence the migration of cells into the collagengel by altering the ability of cells to translocate from the gel surface into the collagenmatrix as well as by an effect on the rate of cell migration once within the 3-dimensionalgel environment. At this point we are unable to conclude with certainty by whichmeans fibronectin produces the observed effects on cell migration into the gel. Aspreviously described (Schor, 1980), a qualitative assessment of cell migration withinthe collagen gel matrix (as well as on the gel surface) may be obtained from therelative position of daughter cells within individual colonies. However, in this par-ticular case, both the hamster melanoma cells and human skin fibroblasts producerather diffuse colonies on and within the collagen gel (indicating that daughter cellsare capable of migration under these conditions) and the presence of fibronectin didnot result in apparent changes in colony morphology (data not shown). Quantitativedata regarding cell migration on and within the gel may be obtained using time-lapsecinematography and this information will be presented in a subsequent communication.

Previous studies have indicated that fibronectin stimulates the migration of severaltransformed cell lines on plastic tissue culture dishes, as assessed by the proximity ofdaughter cells in individual colonies (Ali & Hynes, 1978), the distance of cell migra-tion from cell aggregates (Yamada, Olden & Pastan, 1978) and the length of individual celltracks recorded by time-lapse cinematography (Poussegur, Willingham & Paston, 1977).An inhibitory effect of fibronectin on cell migration was suggested by Couchman &Rees (1979) who observed that the cessation of fibroblast migration from chick heartexplants was temporally correlated with the appearance of fibronectin at the cell surface.

The mechanism by which fibronectin influences cell migration is not known. Cellsurface fibronectin has been observed to be in close anatomical proximity to thefilamentous elements of the cytoskeleton, possibly in continuity with these elementsvia transmembrane linkages (Hynes & Destree, 1978; Singer, 1979), and fibronectinmay therefore be expected to influence cell migratory behaviour by virtue of this

5. L. Schor, A. M. Schor and G. W. Bazill

association. Albrecht-Buehler (1977) has reported that the rate of centripetal mem-brane flow at the apical surface of migrating 3T3 cells is reduced in regions of themembrane containing associated fibronectin; since centripedal membrane flow hasbeen postulated by Harris (1973) to result from tension applied to the surface mem-brane due to interaction with underlying elements of the cytoskeletal system, theresults reported by Albrecht-Buehler provide further indirect evidence that fibronectinmay influence cell migration by modulating the functional relationship betweensurface membrane and cytoskeleton.

Fibronectin binds to native type I collagen fibres (Kleinman et al. 1976). Our dataobtained using gels preincubated with fibronectin indicate that fibronectin bound tocollagen influences cell migration. Previous studies have suggested that fibronectinmediates the attachment of a number of cell types to collagen-coated dishes and thatthis is a 2-step process in which fibronectin first binds to the collagen substratum,followed by cell attachment to the fibronectin-collagen complex (Klebe, 1974; Pearl-stein, 1978). The molecular organization of collagen on these coated dishes is notknown, but since they were treated with 8 M urea during the course of their prepara-tion, it is likely that a majority of the collagen molecules are denatured (Schor &Court, 1979). Other studies have indicated that exogenous fibronectin is not requiredfor the attachment of a variety of cell types to native type I collagen fibres (Grinnell &Minter, 1978; Schor & Court, 1979; Schor, 1979), a conclusion consistent with theresults presented here in Tables 1 and 2. It is, however, possible that although fibro-nectin is not required for the initial attachment of certain cell types to native type Icollagen fibres (as measured by the kinetics of cell attachment during the first fewhours after plating), it may nevertheless increase the strength of cell adhesion tocollagen; this possibility has not been experimentally verified since the techniquesavailable for measuring the strength of cell adhesion to a substratum (Gail & Boone,1972) cannot easily be applied to a fragile structure such as the collagen gel. If fibro-nectin does indeed increase the strength of cell adhesion to the collagen substratum,then the effects of fibronectin on cell migration reported here may be viewed as anexample of haptotaxis, as defined by Carter (1965). According to this view, the stimu-latory effect of fibronectin on the migration of melanoma cells (which lack endogenouscell surface fibronectin) may be due to an increase in the strength of cell adhesiontowards a value which is optimal for cell migration, while the inhibitory effect offibronectin on the migration of fibroblasts (which possess abundant cell surfacefibronectin) may result from an increase in cell adhesion beyond this optimal value.Indeed, Hynes et al. (1978) have suggested that fibronectin at sufficiently high con-centration might be expected to inhibit cell migration by virtue of its enhancement ofcell adhesion, although experimental evidence indicating such an inhibitory effect wasnot obtained, possibly because plastic tissue culture dishes were used as a substratumfor cell migration. This conclusion is consistent with the results reported by Yamadaet al. (1978) who observed that the effect of exogenous fibronectin on the alignment oftransformed cells is more pronounced when the cells are cultured on a collagen sub-stratum compared to plastic, which again serves to emphasize the important roleplayed by the substratum in the control of various aspects of cell behaviour.


Whatever the mechanism by which fibronectin acts, our data suggest that themigration of different cell types in tissues containing type I collagen fibres (e.g. dermis)may be affected in diverse ways by the presence of fibronectin in the surroundingextracellular matrix. This differential response to fibronectin may play an importantrole in maintaining normal tissue structure as well as contribute to the pattern oftumour cell invasion.

We wish to thank Mr G. Rushton and Mr B. Winn for excellent technical assistance duringthe course of this work.

REFERENCES

ALBRECHT-BUEHLER, G. (1977). Local inhibition of centripedal particle transport where LETSprotein patterns appear on 3T3 cells. Nature, Land. a66, 454-456.

ALI, I. U. & HYNES, R. O. (1978). Effects of LETS glycoprotein on cell motility. Cell 14, 439-446.

BORNSTEIN, P. & ASH, J. F. (1977). Cell surface-associated structural proteins in connectivetissue cells. Proc. natn. Acad. Sci. U.S.A. 74, 2480-2484.

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(Received 30 June 1980)

the effects of fibronectin on the migration...

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