J. Cell Set. 49, 299-310 (1981) 299Printed in Great Britain © Company of Biologists Limited 1981

THE EFFECTS OF FIBRONECTIN ON THE

ADHESION AND MIGRATION OF CHINESE

HAMSTER OVARY CELLS ON

COLLAGEN SUBSTRATA

SETH L. SCHOR, ANA M. SCHORCancer Research Campaign Department of Medical Oncology, Christie Hospital andHolt Radium Institute, Wilmstoio Road, Manchester M20 gBX, England

AND GEORGE W. BAZILLPaterson Laboratories, Christie Hospital and Holt Radium Institute, Wilmslcw Road,Manchester M20 gBX, England

SUMMARY

Data are presented indicating that the adhesion of Chinese hamster ovary cells (CHO) tofilms of denatured type I collagen occurs by a fibronectin-dependent mechanism, whereasthe adhesion of these cells to 3-dimensional gels of native type I collagen fibres may occurby either a rapid, fibronectin-dependent mechanism or by a slower, fibronectin-independentmechanism. Data are also presented indicating that fibronectin promotes the migration ofCHO cells on native type I collagen fibres.

INTRODUCTION

Extensive cell migration occurs during normal embryological development, but isgenerally restricted in the adult organism to pathological conditions, such as woundhealing and tumour-cell invasion (Martinez, 1972; Willis, 1973; Strauli & Weiss,1977). Various factors are believed to contribute to the control of cell migration,including the ability of the cells to adhere to the elements of the extracellular matrixwhich form the substratum upon which they move (Carter, 1965; Gail & Boone,1972). In view of the important role played by the extracellular matrix in the controlof cell movement, it is important that cell migratory behaviour in vitro be examinedon an appropriate macromolecular substratum. Collagen is a major constituent ofthe extracellular matrix (Miller, 1977) and we have previously described techniques forobtaining quantitative data concerning cell adhesion and migration on 3-dimensionalgels of native collagen fibres (Schor & Court, 1979; Schor, 1980). We now intendto examine the role played by other matrix components in the control of cell adhesionand migration on collagen and have chosen to begin with fibronectin because of itstypical association with collagen in the extracellular matrix (Linder, Stenman, Lehto& Vaheri, 1978).

Information concerning the biochemistry of fibronectin, its distribution and itseffects on cell behaviour may be found in a number of reviews (Vaheri & Mosher,

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

1978; Yamada & Olden, 1978; Hynes, Destree, Perkins & Wagner, 1979). Previousstudies concerned with the effects of fibronectin on cell adhesion have indicatedthat various permanent cell lines (e.g. HeLa, BHK and PyBHK) attach to 3-dimen-sional gels of native collagen fibres by a fibronectin-independent mechanism andthat these same cells use a fibronectin-dependent mechanism when attaching tofilms of denatured collagen (Grinnell & Minter, 1978; Schor & Court, 1979; Schor,1979). These results are consistent with earlier observations indicating that fibro-nectin is required for the attachment of various cell lines to films of collagen exposedto 8 M-urea (Klebe, 1974; Pearlstein, 1976).

In this communication we are concerned with the effects of fibronectin on theadhesion and migration of Chinese hamster ovary (CHO) cells on both native anddenatured collagen substrata. Data are presented indicating that CHO cells mayattach to native collagen fibres either by a slow, fibronectin-independent mechanismor by a more rapid, fibronectin-dependent mechanism. CHO cells therefore differfrom the cell lines previously examined in that a fibronectin-dependent mechanismof cell attachment to native collagen fibres has been demonstrated. Fibronectin hasalso been observed to increase the migration of CHO cells on native collagen fibres.

MATERIALS AND METHODS

Cell cultures

Chinese hamster ovary cells (CHO) were originally obtained from Dr Charles Ockey ofthe Paterson Laboratories, Manchester. Stock cultures were grown in plastic tissue-culturedishes in Eagle's MEM supplemented with 10% foetal calf serum, 2 mM-glutamine, 1 mM-sodium pyruvate, non-essential amino acids (Gibco-Biocult) and 100 units/ml of penicillinand streptomycin. Stock cultures were subcultured once a week and the medium changed3 times a week. Cells to be used in the experiments were brought into suspension from stockcultures by exposure to 0-05 % trypsin (Sigma Ltd, cat. no. T-8253) >n phosphate-bufferedsaline for 5 min at 37 °C, an equal volume of growth medium containing 10% foetal calfserum was then added and the cells collected 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-culturedishes (Gibco-Biocult Ltd, Uxbridge, cat. no. 53066) by rapidly mixing 8-5 ml of the collagensolution with 1 ml of 10 x concentrated 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 °Cfor 24 h in a humidified CO2 incubator before use. These gels consist of a hydrated meshworkof native collagen fibres (Elsdale & Bard, 1972).

Heat-denatured collagen was prepared by incubating aliquots of the aqueous collagensolution at 60 °C for 30 min and films of heat-denatured collagen were prepared on 35-mmplastic tissue culture dishes as previously described (Schor & Court, 1979).

Cell adhesion

The percentage of cells attached to the different substrata was determined as previouslydescribed (Schor & Court, 1979). Accordingly, 1 ml of the appropriate cell suspension wasplated onto 35-mm dishes containing the different substrata previously overlaid with 1 ml ofmedium. The dishes were incubated at 37 °C and the number of adherent cells determined

Cell adhesion and migration on collagen 301

at various times thereafter by counting the number of unattached cells with a Coulter electronicparticle counter and subtracting this number from the number of cells initially plated on thedish.

Determination of total cell number and migration of cells into collagen gel matrix

The total number of cells present 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, cultureswere examined with phase-contrast optics using a Leitz Diavert microscope fitted with anSY2 photographic graticule defining an area of 0'9 cm x 0-65 cm. The number of cells bothon the gel surface and within the 3-dimensional collagen matrix was determined in approxi-mately 20 regions of the gel surface selected at random moving across the diameter of thegel. Data collected from at least 3 such gels were used to calculate the mean + s.D. of thepercentage 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 & Ruoslahti (1977). After application of theserum, the column (12 cmx 1-5 cm) was washed with 015 M-NaCl in 001 M-potassium phos-phate buffer, pH 74 (PBS), then with PBS containing i-6 M-urea. Fibronectin was elutedwith 6 M-urea in PBS, and the solution was dialysed overnight against 50 vol. of buffercontaining 10 mM-cyclohexylaminopropane sulphate, 015 M-NaCl and 1 mM-CaCl.. (CAPSbuffer) (Yamada & Kennedy, 1979). Fibronectin was concentrated by precipitation withammonium sulphate at 40 % saturation. The precipitate was dissolved in CAPS buffer anddialysed against this buffer to remove ammonium sulphate. The protein concentration wasadjusted to 2—4 mg/ml (depending on the experiment) and SDS/polyacrylamide electro-phoresis showed a major band (doublet) at a molecular weight of 220000 and minor bands(< 10%) of low molecular weight components. Growth medium containing fibronectin-depleted foetal calf serum was prepared by applying foetal calf serum to the gelatin-Sepharosecolumn and washing with growth medium (Eagle's MEM containing 2 mM-glutamine,i mM-sodium pyruvate, non-essential amino acids (Gibco-Biocult) and 100 units/ml of penicillin andstreptomycin).

RESULTS

Cell adhesion

Data are presented in Fig. 1 concerning the effects of different concentrations offoetal calf serum (A), fibronectin-depleted foetal calf serum (B) and fibronectindissolved in serum-free medium (c) on the initial attachment of CHO cells to plastictissue-culture dishes, heat-denatured collagen films and 3-dimensional gels of nativecollagen fibres. In this experiment, trypsinized cells were resuspended in serum-freegrowth medium at a concentration of io5 cells/ml and 1 ml of this cell suspensionwas plated onto each of the different substrata, these having been previously overlaidwith 1 ml of growth medium containing the various additives to give the finalconcentrations shown in Fig. 1. All cultures were incubated for 2 h at 37 °C andthen the percentage of cell adhesion in each culture was measured as previouslydescribed (Schor & Court, 1979). In this experiment, approximately 50% of theCHO cells were attached both to the heat-denatured collagen films and to the3-dimensional gels of native collagen fibres in the presence of serum-free medium.

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

As can be seen in Fig. i A, cell attachment (relative to that observed in serum-freemedium) to both types of collagen substrata was inhibited by the presence of lowconcentrations of foetal calf serum and enhanced in the presence of high concentrationsof serum; cell attachment to the denatured collagen films reached a plateau value ofapproximately 90% in the presence of 5% foetal calf serum, whereas the percentageof cells attached to the gels of native fibres continued to rise as the concentration

% Foetal calf serum % Fibronectin-free foetal calf serum Fibronectin (/jg/ml)

Fig. 1. The effects of different concentrations of foetal calf serum, fibronectin-depletedfoetal calf serum and fibronectin on the adhesion of CHO cells to collagen substrata.Trypsinized cells were resuspended in serum-free growth medium at a concentrationof io6 cells/ml and 1 ml of this cell suspension plated onto plastic tissue-culturedishes ( • ) , denatured collagen films (x) and 3-dimensional collagen gels (O);these substrata having been previously overlaid with 1 ml of growth medium containingthe appropriate amount of foetal calf serum (A), fibronectin-depleted foetal calfserum (B) and fibronectin (c) to give the concentrations shown above. All cultureswere incubated for 2 h at 37 °C and then the percentage of cell adhesion in eachculture was measured as previously described (Schor & Court, 1979).

of serum was increased, with 60% of the cells attached in the presence of 25%serum. Concentrations of bovine serum albumin in serum-free medium within therange of 0-05-10-0 mg/ml did not affect the percentage of cell attachment to eitherthe denatured or native collagen substrata compared to the values obtained in serum-free medium alone. The serum factor(s) responsible for the inhibition of CHO cellattachment to the collagen substrata observed at low concentrations of serum havenot been identified.

The effects of different concentrations of fibronectin-depleted foetal calf serumon CHO cell attachment to the collagen substrata are shown in Fig. 1B. These dataindicate that cell attachment to the denatured and native collagen substrata isinhibited (relative to the results obtained in serum-free medium) at all concentrationsof fibronectin-depleted foetal calf serum examined (1-10%), with a plateau ofapproximately 12% adhesion obtained on both substrata. In contrast, the datapresented in Fig. 1 c indicate that all concentrations of fibronectin in serum-freemedium examined (1-50/tg/ml) enhance CHO cell attachment to both denaturedand native collagen substrata with plateau values of 92% adhesion to denatured

Cell adhesion and migration on collagen 303

collagen films and 80% adhesion to native collagen gels. Identical results to thoseshown in Fig. i c were obtained when the fibronectin was dissolved in 10% fibro-nectin-depleted foetal calf serum medium (data not shown). Finally it should benoted that cell attachment to the plastic tissue-culture dishes was over 90% underall conditions examined.

Further data are presented in Table 1 comparing cell attachment to denaturedand native collagen substrata after 2 h and 24 h of incubation in growth medium

Table 1. Adhesion of CHO cells to native and denatured collagen substratain the presence of different media

Substratum

Denatured collagen film

Native collagen gel

Time(h)

2

2 42

24

Percentage of cell attachment

Serum-free

3 1 23 8 0

35-3956

in different mediaA . „ ,

Wholefoetal calf

serum(10%)

8 2 1

91-742-09 2 5

Fibronectin-depleted

foetal calfserum

(10%)

I2-I

I S !15-6898

Trypsinized CHO cells were resuspended in serum-free growth medium at a concentrationof io6 cells/ml, and 1 ml of this cell suspension plated onto denatured collagen films andnative collagen gels overlaid with 1 ml of serum-free growth medium or growth mediumcontaining 20 % foetal calf serum or fibronectin-depleted foetal calf serum (to give a finalconcn. of 10% serum). Cultures were incubated at 37 °C and the percentage of cell adhesionwas determined as previously described (Schor & Court, 1979) after 2 and 24 h. The adhesiondata shown above represent the mean of 3 replicate cultures and the S.D. was less than 10 %of the mean in all cases.

with the different sera. These data indicate that cell attachment to the denaturedcollagen films at both times is significantly greater in 10% whole foetal calf serummedium compared to either serum-free medium or 10% fibronectin-depleted foetalcalf serum; these results are consistent with those presented in Fig. 1 and indicatethat the percentages of cell attachment to the denatured collagen films measured at2 h remain unchanged after 24 h of incubation. Different cell behaviour was observedon the native collagen fibres; cell attachment to this substratum increased significantlybetween 2 and 24 h of incubation in the presence of all these media, giving finalvalues of greater than 90% adhesion in all cases. These data indicate that althoughfibronectin increases the initial rate of CHO cell attachment to native collagen fibres(see Fig. 1), these cells will eventually adhere well to the native collagen gels in thecomplete absence of serum or fibronectin.

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

Cell morphology

The different morphologies of the CHO cells 2 h after plating on the 3-dimensionalgels of native collagen fibres in the presence and absence of fibronectin are shownin Fig. 2. Cells cultured either in serum-free medium (Fig. 2A) or 10% fibronectin-

Fig. 2. CHO cells were trypsinized and then resuspended at a concentration of10s cells/ml in either serum-free growth medium or growth medium containing10% fibronectin-depleted foetal calf serum and then 1 ml of cell suspension platedonto gels of native collagen fibres overlaid with 1 ml of the same medium, eitherwith or without added fibronectin at 50 /tg/ml. Cultures were incubated for 2 h at37 °C and then photographed. Cells in serum-free medium without fibronectin (A),with fibronectin (B); cells in. medium containing 10% fibronectin-depleted foetalcalf serum without added fibronectin (c), with added fibronectin (D). Bar, 50 /tm.

depleted foetal calf serum (Fig. 2 c), were generally round in appearance and poorlyspread on the collagen substratum. The presence of 50 /tg/ml fibronectin in bothserum-free medium (Fig. 2B) and 10% fibronectin-depleted foetal calf serum(Fig. 2D) resulted in a significant alteration in cell morphology, with the majorityof cells now being polygonal in shape and relatively well spread on the collagensubstratum. The morphology of the CHO cells on the collagen gels 24 h after platingis shown in Fig. 3 for cells cultured in serum-free medium (A), serum-free mediumplus 50/tg/ml fibronectin (B), 10% fibronectin-depleted foetal calf serum (c) and

Cell adhesion and migration on collagen

Fig. 3. The same experiment as described in the legend to Fig. 2, except cultures wereincubated for 24 h before being photographed. Cells in serum-free medium withoutfibronectin (A), with fibronectin (B); cells in medium containing 10% fibronectin-depleted foetal calf serum without added fibronectin (c), with added fibronectin (D).Bar, 50 /im.

this latter medium containing 50 /<g/ml fibronectin (D). AS these micrographs show,there was no difference in the morphology of CHO cells 24 h after plating on thenative collagen gels; note that all cells had spread and assumed an elongated, spindle-shaped morphology, even in the complete absence of serum or fibronectin.

Cells plated onto denatured collagen films under the same conditions as describedfor Figs. 2 and 3 were observed to assume an elongated, spindle-shaped morphologyby 2 h in the presence of fibronectin, while the small number of cells that did attachin the absence of fibronectin remained round and poorly spread, even after 24 h(data not shown). These observations suggest that the attachment and spreading of

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

CHO cells on the native collagen fibres does not result from the synthesis of endo-genous fibronectin, a conclusion verified by immunofluorescent studies using anti-fibronectin antibodies (data not shown).

Cell migration and proliferation

The effects of fibronectin on the migration of CHO cells into the 3-dimensionalgels of native collagen fibres are shown in Fig. 4. Trypsinized cells were resuspended

15 1-

10

8h

10

.0

Time (days)

Fig. 4. The effects of fibronectin on the proliferation and migration of CHO cells into3-dimensional gels of native collagen fibres. Trypsinized cells were resuspended inserum-free medium at a concentration of 2-5 x io4 cells/ml and 1 ml plated ontocollagen gels overlaid with either 1 ml of serum-free medium (O) or serum-freemedium containing 50 /tg/ml fibronectin (#) . Cultures were incubated at 37 °C, andboth total cell number and the percentage of cells that had migrated into the gel weredetermined at 24-h intervals as previously described (Schor, 1980).

in serum-free medium at a concentration of 2-5 x io4 cells/ml and 1 ml plated ontocollagen gels containing 1 ml of serum-free medium either with or without 50 //g/mlfibronectin. Cultures were incubated for 3 days and both cell number and thepercentage of cells that had migrated into the gel were determined at 24-h intervalsas previously described (Schor, 1980). As can be seen in Fig. 4, fibronectin enhancedthe migration of the CHO cells into the gel, with 12-5% of the cells within thecollagen matrix after 3 days of culture in the presence of fibronectin compared toonly 2-2% of cells within the gel in the absence of fibronectin. The CHO cells didnot proliferate in serum-free medium during the 3-day incubation period, either inthe presence or absence of fibronectin.

The effects of fibronectin on the migration of CHO cells in the presence of serum-containing media are shown in Table 2. Fibronectin-preincubated gels were preparedby incubating gels with 1 ml of serum-free medium containing 50 /tg/ml fibronectinfor 1 h at 37 °C, and then washing the gels 5 times with serum-free medium to removeany unbound fibronectin. Trypsinized cells were then resuspended in mediumcontaining 10% fibronectin-depleted foetal calf serum at a concentration of 25 x io4

Cell adhesion and migration on collagen 307

Table 2. The effects of fibronectin on the proliferation and migration of CHO cellsinto three-dimensional gels of native collagen fibres

Substratum

Control gelControl gel

Fibronectin-preincubatedgel

Medium

10 % Whole foetal calf serum10% Fibronectin-depletedfoetal calf serum

10 % Fibronectin-depletedfoetal calf serum

Totalcell number

(XIO»)

5-5±°-65-0 + 0-8

63+0-9

% of cellswithin collagen

gel matrix

i2-4±i-86-i ±0-9

12-2 ±2-0

Fibronectin-preincubated gels were prepared by incubating collagen gels with 1 ml ofserum-free growth medium containing 50 /ig/ml fibronectin for 1 h at 37 °C and then washingthe gels s times with serum-free medium to remove any unbound fibronectin. Trypsinizedcells were then resuspended in medium containing 10% fibronectin-depleted foetal calf serumat a concentration of 2-5 x io4 cells/ml and 1 ml of this suspension plated onto both controland fibronectin-preincubated gels overlaid with 1 ml of 10 % fibronectin-depleted foetal calfserum medium. Another aliquot of trypsinized cells was resuspended in growth mediumcontaining 10 % whole foetal calf serum to give a concentration of 2-5 x io4 cells/ml, and 1 mlplated onto control gels overlaid with 1 ml of the same medium. Cultures were incubatedfor 3 days at 37 °C, at which time both total cell number and the percentage of cells withinthe gel matrix were determined as previously described (Schor, 1980). Three replicate cultureswere used for each measurement.

cells/ml and 1 ml of this suspension plated onto both control and fibronectin-preincubated gels overlaid with 1 ml of 10% fibronectin-depleted foetal calf serummedium. Another aliquot of trypsinized cells was resuspended in growth mediumcontaining 10% whole foetal calf serum to give a concentration of 2-5 x io* cells/mland 1 ml of this suspension plated onto control collagen gels containing 1 ml of thesame medium. Cultures were incubated for 3 days at 37 °C, at which time bothtotal cell number and the percentage of cells within the gel were measured. Fibro-nectin had a significant effect on cell migration with approximately 6% of the cellswithin the gel after 3 days of growth in the absence of fibronectin compared toapproximately 13% in the presence of fibronectin. Fibronectin did not have aneffect on the 20-fold increase in cell number that occurred under all conditions.

DISCUSSION

This communication is primarily concerned with the effects of fibronectin on theadhesion and migration of CHO cells on 3-dimensional gels of native collagenfibres. Data are presented indicating that CHO cells may attach to native collagenfibres either by a slow, fibronectin-independent mechanism or by a more rapid,fibronectin-dependent mechanism; in both cases cells become well spread on thesubstratum and adopt an elongated, spindle-shaped morphology. Previous studiesusing other cell lines (e.g. HeLa, BHK and PyBHK) indicated that these cellsattach to native collagen fibres by a rapid, fibronectin-independent mechanism

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

(Grinnell & Minter, 1978; Schor & Court, 1979); however, because of the efficientmanner in which these cells attach to native collagen in the absence of fibronectin, itmay not be possible to demonstrate whether they also employ a fibronectin-dependentmechanism of adhesion to native collagen. It should be noted that like the othercell lines examined, CHO cells attach to denatured collagen films by a fibronectin-dependent mechanism. Finally, our conclusion that the slow attachment of the CHOcells to native collagen fibres in serum-free medium is mediated by a fibronectin-independent mechanism (and not by the synthesis of endogenous fibronectin by thecells) is supported by the observations that no cell-surface fibronectin was detectedby immunofluorescence techniques and that cells did not attach to denatured collagenfilms after 24 h in the presence of serum-free medium (Table 1).

Recently, Kleinman, McGoodwin, Rennard & Martin (1979) have observed thatCHO cells bind to native collagen fibres in serum-free medium containing unphysio-logically high levels of phosphate ion (greater than IOHIM); the attached cells didnot spread on the substratum and it was therefore suggested that cell adhesion tonative collagen in the presence of serum-free medium was actually mediated by anon-physiological mechanism requiring high levels of phosphate. Our data are atvariance with this conclusion since we find that the CHO cells do spread on thenative collagen fibres (after 24 h) although the concentration of phosphate in ourmedium is only 1 mM (i.e. equal to the concentration of phosphate ion in plasma).The fact that the native collagen substrata used by Kleinman et al. (19796) wereexposed to ammonia vapour may account for this discrepancy with our results.

We observed an inhibition of CHO cell attachment to the collagen substrata inthe presence of fibronectin-depleted foetal calf serum or low concentrations of wholefoetal calf serum. The simplest explanation for this observation may be that inaddition to fibronectin (which promotes cell attachment), foetal calf serum containsa factor or factors that inhibit attachment. The chemical nature of this presumedinhibitor and its relationship to other agents known to inhibit cell attachment tocollagen (Kleinman, Martin & Fishman, 1979) is not known.

Data are also presented here concerning the effects of fibronectin on the migrationof CHO cells into 3-dimensional gels of native collagen fibres (Table 2). We findthat significantly more cells migrate from the surface into the gel interior in thepresence of fibronectin, both in serum-free and serum-containing media. Previousstudies have indicated that fibronectin enhances the migration of a number of trans-formed cell lines on (2-dimensional) plastic tissue-culture dishes, as assessed by theproximity of daughter cells in individual colonies (AH & Hynes, 1978), the distanceof cell migration from adherent aggregates (Yamada, Olden & Pastan, 1978) andthe length of individual cell tracks recorded by time-lapse cinematography (Pouys-segur, Willingham & Pastan, 1977).

Our experiments using gels preincubated with fibronectin indicate that fibronectinbound to collagen is responsible for the observed effects on CHO cell migration.This is consistent with previous observations indicating that fibronectin promotescell adhesion to films of type I collagen by a 2-step mechanism involving (1) theinteraction of fibronectin with a specific region of the ai chain of type 1 collagen

Cell adhesion and migration on collagen 309

between amino acid residues 757 and 791 (Kleinman et al. 1979 a), and (2) celladhesion to this collagen-fibronectin complex (Klebe, 1974; Pearlstein, 1976).

On a priori grounds it would seem reasonable to suggest that cell adhesion andmigration are related phenomena since migration would be expected to require thecontinuous making and breaking of cell contacts with the substratum. IndeedHynes et al. (1979) have suggested that the varied effects of fibronectin on cellbehaviour on plastic tissue-culture dishes (including increased mobility) could resultfrom the primary effect of fibronectin in increasing cell adhesion to the substratum.Our results indicating a fibronectin-dependent mechanism of CHO cell attachmentto native collagen fibres are consistent with this hypothesis. In a previous communi-cation (Schor, Schor & Bazill, 1981) we reported that fibronectin increases themigration of a Syrian hamster melanoma cell line into the 3-dimensional collagengel, but inhibits the migration of normal human skin fibroblasts. We suggested thatthe different response of the 2 cell types might be due to fibronectin increasing thestrength of melanoma cell adhesion towards a presumed optimal value for migration,while increasing the adhesion of the fibroblasts (which already contain endogenouscell surface fibronectin) beyond this optimal value.

The manner in which fibronectin influences cell migration may, however, be morecomplex. An observation arguing against a simple relationship between cell adhesionand migration is that CHO cells attached ta native collagen fibres after 24 h in thepresence of serum-free medium (low migration) are well-spread and morphologicallyindistinguishable from cells attached in the presence of fibronectin (high migration).These results suggest that fibronectin may be affecting cell migratory behaviour bymore subtle means than simply facilitating adhesion, e.g. fibronectin might affectthe functional interaction between elements of the cytoskeletal system and plasmamembrane to alter the balance between membrane flow and the creation of stableadhesion plaques (Harris, 1973; Albrecht-Buehler, 1977; Singer, 1979).

We wish to thank Mr Brian Winn and Mr Graham Rushton for excellent technical assistanceand Miss Elaine Mercer for preparing the manuscript.

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(Received 30 September 1980)