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. 2001 Jun;82(3):193–200. doi: 10.1046/j.1365-2613.2001.iep0082-0193-x

Effects of hyaluronan on the invasive properties of human breast cancer cells in vitro

Andrea Herrera-Gayol *, Serge Jothy **
PMCID: PMC2517708  PMID: 11488992

Abstract

Hyaluronan (HA) is a high molecular weight glycosaminoglycan present mostly in the extracellular matrix (ECM). HA binds to specific receptors such as CD44. Its production is increased at the tumour–stroma interface, including those in breast cancer tumours. It has been suggested that it facilitates invasion of tumour cells into the ECM by a hydrodynamic effect, or by altering tumour cell behaviour. Using in vitro tests we studied the effect of immobilized (iHA) and soluble (sHA) HA on the invasive properties of four human breast cancer cell lines with different levels of CD44 expression. Our results show that iHA acts as an adhesive, haptotactic, and motility stimulating factor for the CD44 positive Hs578T cells and induces the expression of membrane CD44. sHA also changes the motility properties of the Hs578T and MDA-231 cells and increases their CD44 expression. sHA or iHA have no measurable effect on the adhesion, motility or CD44 expression of the ZR-75–1 and MCF-7 breast cancer cells. Our results establish that in high CD44 expressing breast cancer cells HA modulates tumour cell adhesion and motility and also increases the expression of its own receptor, CD44.

Keywords: hyaluronan, CD44, breast cancer

Introduction

Tumour cell invasion is a three-step process characterized by altered cellular adhesion, cell motility and degradation of the extracellular matrix (ECM). The extracellular environment interacts with tumour cells facilitating, or inhibiting invasion. Hyaluronan (HA) is a glycosaminoglycan present mostly in the extracellular matrix of connective tissues. HA binds to different cellular receptors, such as CD44, generating signals acting on the cytoskeleton (reviewed in Entwistle et al. 1996). CD44 is involved in binding to its HA ligand, followed by its degradation (Culty et al. 1994). Degradation of HA is an important phenomenon, as the intact polymer has different effects than its fragments. Experiments performed both in vitro and in vivo indicate that intact polymer is an important determinant of cell motility (Goebeler et al. 1996; Thomas et al. 1992; Turley 1992; Haddon and Lewis 1991). HA fragments can induce chemokine gene expression (McKee et al. 1996; Horton et al. 1998); production of cytokines (Boyce et al. 1997); up-regulation of immediate early genes such as c-fos and c-jun (Deed et al. 1997); tyrosine kinase activity (Slevin et al. 1998) and activation of the transcriptional regulator, NF kappaB (Noble et al. 1996; McKee et al. 1997). Interestingly, antibodies against CD44, but not against RHAMM or ICAM-1, inhibit HA binding and gene induction in macrophages (McKee et al. 1996). HA fragments of specific length induce angiogenesis (West et al. 1985; West & Kumar 1989a; and West et al. 1989b; Deed et al. 1997).

Tumour cells in breast cancer tumours often express CD44 and the tumour–stroma interface is enriched in HA (Bertrand et al. 1992; de la Torre et al. 1993), but how this might alter tumour cell functions is not well understood. Considering the involvement of CD44 in cell adhesion to HA (Aruffo et al.1990), we hypothesized that HA is an important determinant of breast tumour cells migration. We have previously demonstrated that the Hs578T breast cancer cell line is motile and invasive in in vitro assays and that CD44 is involved in those properties (Herrera-Gayol & Jothy 1999). Therefore, the effect of immobilized (iHA) and soluble HA (sHA) on the invasive properties of four human breast cancer cell lines in vitro was studied, including: adhesion, haptotaxis, motility, CD44 expression and proliferation.

Materials and methods

Cell culture

Four human breast cancer cell lines were studied: Hs578T, a high CD44 expressor cell line; MBA-MB-231, a medium-high CD44 expressor cell line; MCF-7, a low CD44 expressor cell line and ZR-75–1, a CD44 negative cell line (Culty et al. 1994). All cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The culture medium for the cell lines Hs578T, MDA-MB-231, MCF-7 was composed of: DMEM (Gibco, Burlington, Ontario, Canada) supplemented with 5% fetal bovine serum (FBS, Gibco), 2 mM glutamine (Gibco) and gentamycin (Gibco). The cell culture medium for the cell line ZR-75–1 was composed of RPMI (Gibco) and 10% FBS. Cells were exposed to: (1), HA (Sigma, St Louis, MO) in solution (sHA) at 1 mg/mL diluted in regular culture medium (RCM) for 1–6 days; (2) immobilized HA (iHA) where different solid surfaces were coated with HA; (3) hyaluronidase Type I (Sigma), 500 U or 1000 U per ml of RCM.

Antibodies

An antibody against all CD44 isoforms, also called anti-CD44s (CD44s, clone F.10.44.2) was obtained from Novocastra Laboratories (Newcastle, UK) and used at a 1/50 concentration in flow cytometric experiments. A FITC labelled goat antimouse IgG (Cedarlane, Hornby, Ontario, Canada) was used as secondary antibody.

Flow cytometry

At least 3 × 105 unfixed cells were resuspended in 0.05% NaN3 in PBS. Primary and secondary antibodies were diluted in NaN3/PBS at a 1/50 and 1/250 concentration, respectively. Background fluorescence was evaluated by omitting the primary antibody. The analyses were performed on an EPICS® scan (Coulter Corporation, Hialeah, Fl) flow cytometer.

Adhesion assays

The adhesion assays were performed as described previously (Herrera-Gayol & Jothy 1999). Briefly, cells were cultured for different time points in regular culture medium (RCM) or in RCM plus HA (sHA) 1 mg/mL, trypsinized and plated on 96-well plates which have been previously coated with HA at 500 µg/mL, 1 mg/mL or 5 mg/mL or bovine serum albumin (BSA, Sigma). After 1–2 or 24 h of incubation at 37 °C, wells were washed and the adherent cells were fixed with cold methanol. Cells were stained with Gill's haematoxylin and counted under an inverted microscope.

Haptotaxis and chemotaxis

Haptotaxis was studied using a modified Boyden chamber system as described previously (Thomas et al. 1993; Herrera-Gayol & Jothy 1999). To test the haptotactic effect of HA, the underside of inserts' membranes were coated with HA at 1 mg/mL or 5 mg/mL. Uncoated inserts were used as controls. After 4 h, cells that had not responded to the haptotatic signal were removed by wiping the upper surface of the membrane with a cotton swab. Inserts were washed and fixed in cold methanol. The cells were stained with Gill's haematoxylin. Cells were counted in 5 different 200x areas.

Chemotaxis was studied using a modified Boyden chamber system. Cells were cultured on uncoated inserts in 0.5% BSA in DMEM plus antibiotics. Wells were filled with 600 µL of HA 100 µg/mL or HA 1000 µg/mL in 0.5% BSA in DMEM or 0.5% BSA in DMEM (negative control). Cells were allowed to migrate for 4 h. Inserts were analysed as described before.

Motility assays in a 2 dimension system: ‘wound assay’

The wound assay used to study the effect of iHA on cell migration and random motility was modified from Thomas et al. (1992). Round glass coverslips (22 mm diameter) were placed in 6-well plates. HA at 5 mg/mL was applied to the coverslips, left at 4°C overnight, dried at room temperature for one hour, washed in PBS, and incubated with 0.2% BSA (Sigma) for 2 h at 37°C. Control coverslips were incubated with BSA alone. The coverslips were washed again in PBS before plating 1 × 105 cells in 100 µL of RCM per coverslip. After reaching confluence, cells were wounded with the tip of a rigid 1 mm tissue culture scraper along the entire diameter of the coverslip, creating a cell-free area. The medium was removed and was replaced by DMEM with or without serum. Cells were allowed to migrate into the wound area for 24 h. Coverslips were washed with PBS, fixed with cold methanol and stained with Gill's haematoxilin. Two photographs were taken from each coverslip and projected onto paper sheets, and cells inside the margins of the wound area were counted. One coverslip per assay was fixed, stained and photographed after wounding to establish the wound area before cell migration.

The effect of sHA on random motility was also studied by the wound assay technique. Cells were plated on HA-uncoated coverslips and incubated for 2 or 3 days in RCM or in RCM plus sHA. Motility was assessed as described before.

Cell proliferation assay

Cells were plated on 6-well plates containing RCM, sHA 1 mg/mL in RCM or on HA-coated wells. Cells were allowed to grow for 1, 2, 3, or 6 days, trypsinized, and counted with a haemacytometer after trypan blue staining.

Statistics

Comparisons were performed using one way analysis of variance (ANOVA) followed by post tests or paired t-tests depending on the assays.

Results

Effect of immobilized hyaluronan (iHA) on cell adhesion and motility

When the Hs578T cells were plated on wells coated with HA and allowed to adhere for 1 or 24 h, the presence of iHA increased tumour cell adhesion to the wells up to sevenfold (Figure 1). Although important, this effect was less striking at 24 h (P < 0.05; data not shown). No differences in adhesion were observed using the two HA concentrations therefore a concentration of 1 mg/mL of HA was used thereafter for coating. This concentration is in the range used in studies where cultured tumour cells were exposed to HA (Thomas et al. 1992; Goebeler et al). iHA showed a trend to increase the adhesion of the MDA-MB-231 cells by 20% after 24 h and had no effect on the adhesive properties of the MCF-7 or ZR-75–1 cells (data not shown).

Figure 1.

Figure 1

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Adhesion of Hs578T breast cancer cells to different concentrations of immobilized hyaluronan (iHA).The adhesion of cells to BSA and two different concentrations of HA (500 (μg/mL and 5 mg/mL) was evaluated at 1 h. Up to a sevenfold increase in cell adhesion was observed when cells were plated on HA 500 (g/mL and 5 mg/mL, respectively, compared to controls. Bars represent mean values of percentage of adherent cells ± SD.

The haptotactic migratory capacity of the Hs578T cell line towards different concentration of iHA was investigated. After 4 h of incubation, 100% to 170% more cells (P < 0.01) could be observed in the underside of the membranes coated with 1 mg/mL or 5 mg/mL hyaluronan, respectively (Figure 2). No significant differences were observed between the two HA concentrations. iHA showed a trend to increase the haptotatic migration of the MDA-MB-231 cells by 30% and had no effect on the MCF-7 or ZR-75–1 cells (data not shown).

Figure 2.

Figure 2

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Immobilized hyaluronan (iHA) as an haptotactic factor. The haptotactic migratory capacity of the Hs578T cell line was evaluated towards two different concentrations of iHA (1 mg/mL or 5 mg/mL) as described in Materials and methods. Hyaluronan acts as an haptotactic factor compared to ‘random motility’ (uncoated membranes). No significant difference was observed between the two iHA concentrations. Bars represent mean values of percentage of cells ± SD.

To evaluate the effect of iHA on cell motility in a two-dimensional system, cells were cultured on glass coverslips which had been coated with HA at 5 mg/mL and incubated with RCM or serum free medium. As shown in Figure 3, iHA increased motility 160% (P < 0.01) when the Hs578T cells were incubated in the absence of serum. When both products were used together (serum and iHA) motility was increased by 2.4 fold (P < 0.01). Similar effects were observed when the MDA231-cells were used. In the absence of serum, iHA stimulated motility slightly as shown by an 11% increase in migrated cells. The presence of serum in the medium without exogenous HA increased by 25% the number cells in the wound area. Plating the cells on a HA-coated culture surface (iHA) and adding serum to the culture medium increased the number of migrated cells by 37%. iHA had no effect on the migration of the MCF-7 and ZR-75–1 cells.

Figure 3.

Figure 3

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Effect of immobilized hyaluronan (iHA) on the motility of Hs578T cells. Hs578T cells were plated on HA-coated or HA-uncoated coverslips, wounded, and allowed to migrate in the absence of serum in the culture medium for 24 h as described in Materials and methods. Cells show a 160% increase in motility, error bars refer to SD.

Effect of immobilized hyaluronan (iHA) on CD44 expression

To evaluate the effect of iHA on the expression of CD44, cells were plated on HA-coated wells for up to 6 days. Flow cytometric measurements showed that iHA increased CD44 expression of Hs578T cells by 65% (P = 0.05) and 150% (P = 0.01) at 1 day and 3 days, respectively (Figure 4 and Figure 6b) as compared to controls. iHA showed a trend to increase CD44 expression of MDA-MB-231 by 40% at 2 and 3 days in culture (data not shown). No effect was observed on CD44 expression of the MCF-7 and ZR-75–1 cells (data not shown).

Figure 4.

Figure 4

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Effect of immobilized hyaluronan (iHA) on CD44 expression by Hs578T cells, at different time points. Cells were grown on uncoated or HA-coated wells for different periods of time. CD44 expression was measured by flow cytometry. Cells grown on iHA show increases of CD44 expression of 60%, 150% and 8% at 1, 3 and 6 days in culture, respectively, as compared to control. Values are expressed in percentage of control, error bars refer to SD.

Figure 6.

Figure 6

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Effect of soluble and immobilized hyaluronan on CD44 expression by Hs578T cells. (a): Hs578T cells grown with sHA ((c): white profile) for 3 days express more CD44 molecules on their membrane than cells that were cultured in RCM ((b): grey profile). (a): black profile represents background fluorescence, omitting primary antibody. LFL1: fluorescence, COUNT: number of cells. (b): Hs578T cells grown on iHA ((c): white profile) for 3 days express more CD44 molecules on their membrane than cells that were cultured in RCM ((b): grey profile). a: black profile represents background fluorescence, omitting primary antibody.

Effect of soluble hyaluronan (sHA) on chemotaxis

Taking into account that iHA has an haptotactic effect on the cell line Hs578T, it became relevant to test if its soluble form has a chemotactic effect. Chemotaxis was evaluated as described in Material and Methods. sHA did not act as a chemotactic stimulus for the various breast cancer cell lines cells (data not shown).

Effect of soluble hyaluronan (sHA) on cell adhesion, motility, CD44 expression and proliferation

Under in vivo conditions such as malignant effusions, in areas of active degradation of the extracellular environment, and in the lymphatic circulation, tumour cells are in contact with soluble HA (sHA). Therefore, we used in vitro experiments reproducing these conditions where tumour cells were grown in medium containing this form of the product. As CD44 expression increased significantly at 3 days on iHA, cells were first cultured for the same period of time with sHA 1 mg/mL in RCM. The Hs578T and the MDA-MB-231 cells were shown to be more adhesive to HA-coated wells in adhesion assays for 24 h and less motile towards iHA in haptotactic assays for 4 h, than those cultured in RCM (Figure 5). No differences in random motility at 24 h were observed (data not shown). A significant increase of membrane CD44 expression was observed by flow cytometry on both cell lines (Figure 5 and Figure 6a). Breast cancer cells expressing lower (MCF-7) or no detectable CD44 expression (ZR-75–1) were not affected by soluble HA in these functional assays.

Figure 5.

Figure 5

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Effect of soluble hyaluronan (sHA) on adhesion, haptotaxis, CD44 expression and proliferation of Hs578T and MDA-MB-231cells. Cells were grown in regular culture medium (RCM) or in RCM containing soluble hyaluronan (sHA) for 3 days. (a): Hs578T cells that were grown with sHA are more adhesive to HA, less motile towards HA and express more CD44 molecules on their membrane. No differences in proliferation are observed. (b): MDA-MB-231 cells that were grown with sHA express more CD44 molecules on the cell membrane and are less motile towards hyaluronan. Proliferation rate is the same.

Effect of hyaluronidase on CD44 expression

As tumour cells have the ability to degrade HA in oligomeric fragments, it is relevant to test whether hyaluronidase has an effect on CD44 expression. All cell lines were cultured with 1000 U/mL or 500 U/mL of hyaluronidase type I for up to 3 days. CD44 expression was upregulated 90% by the Hs578T cells at 3 days in culture (P < 0.05) and 50% by the MDA-MB-231 cells at 2 days in culture (data not shown). No effect was observed on the MCF-7 and ZR-75–1 cells.

Discussion

This study was designed to explore whether HA has a passive role in tumour cell invasion, or alternatively, if it modifies tumour cell behaviour, promoting the invasive cascade. Specifically we investigated the functional consequences of HA on the adhesive and migratory capacity of tumour cells.

The human breast cancer cell lines Hs578T and MDA-231 were chosen because of their aggressive phenotype as indicated by their expression of vimentin, loss of oestrogen receptor expression, ‘fibroblastic’-type morphology and in vivo and in vitro invasive capacities (Thompson et al. 1992; Bae et al. 1993; Sommers et al. 1994). A notable difference, however, is that the Hs578T cells express more CD44 and are able to bind more HA than the MDA-MB-231 cells (Culty et al. 1994). Also, the Hs578T cells can degrade and produce more HA than the MDA-MB-231 cells (Culty et al. 1994; Heldin et al. 1996). The ZR-75–1 and MCF-7 cell lines are well differentiated cancer cell lines, oestrogen positive and vimentin negative, poorly invasive in vitro and non invasive in vivo (Thompson et al. 1992). While the MCF-7 express low levels of CD44 (Culty et al. 1994 and our own data) the ZR-75–1 cells are CD44 negative (Culty et al. 1994 and our own data). Both cell lines have very low binding to HA and HA degradation capacities (Culty et al. 1994) and do not synthesize HA (Heldin et al. 1996).

The effect of immobilized and soluble HA was tested because HA can be found attached to cells and to other ECM components, as well as being exposed to soluble forms of HA in blood and in the lymphatic circulations in vivo. In areas of tumour invasion, it has been documented that HA is present at higher concentrations where active degradation of the ECM takes place (Bertrand et al. 1992; de La Torre et al. 1993). Therefore, polymeric HA, whether it is immobilized or in a soluble form, has the potential to alter cell behaviour.

The results of this study show that iHA acts as a promoter of adhesion for the breast cancer cell line Hs578T. A sevenfold increase in cellular adhesion was observed at 1h whereas at 24 h a less striking effect was observed. This can be explained by the ability of tumour cells to produce enough HA themselves at 24 h, to mask the effect of exogenously added HA.

Similarly, it is possible to explain the haptotactic stimulating capacity of HA on the Hs578T cells. The Hs578T cells can sense the HA coat underneath the modified Boyden chamber membranes and move throughout the pores towards it. CD44 molecules normally expressed by the Hs578T cells would be involved in this phenomenon, as we previously reported (Herrera-Gayol & Jothy 1999).

We found that iHA acts as a motogenic factor for the Hs578T cell line, where cell motility increased between 160% to 340% depending on the presence of serum. This might be due to additional soluble factors with motogenic stimulatory properties present in the serum. It is also possible that the presence of serum increases HA secretion by the cells during their migration, therefore increasing their motility.

CD44 expression by Hs578T cells increased significantly after 3 days in culture on HA-coated surfaces. This represents another example showing that an ECM ligand can induce the synthesis of its own receptor. In vivo this finding would indicate that breast cancer cells expressing a high level of CD44 acquire a selective motility advantage mediated by their interaction with HA immobilized on the ECM. These data are consistent with in vitro studies using melanoma cell lines where iHA increased both CD44 expression and motility (Yoshinari et al. 1999). A probable mechanism relates to the capacity of the cells to degrade the intact HA into fragments. HA fragments would in turn stimulate the synthesis of new CD44 molecules. Our hypothesis that HA fragments are responsible for the cellular behavioural changes is supported by the data of this study showing that exogenous hyaluronidase increases CD44 expression on cells without the need to add exogenous HA.

The Hs578T and MDA-MB-231 cells cultured with sHA changed their migratory patterns according to time in culture. It is important to notice that an intermediate level of adhesion and critical concentration of ECM components are necessary to achieve a maximum migratory capacity (Huttenlocher et al. 1996; Horwitz et al. 1998). The cell membrane at the leading edge of migrating cells generates traction forces for cell movement. At the trailing-end of migrating cells, antiadhesive mechanisms take place allowing the cell to be released from its substrate. In our model, the adhesion of cells to ECM involving CD44 and other adhesion molecules vary dynamically in both intensity and cellular site of expression modifying cell migration.

In conclusion, the presence of immobilized and soluble HA modifies cell behaviour, by increasing membrane CD44 expression and changing cell adhesion and motility, depending on cell type. HA modulates the adhesive and migratory characteristics of tumour cells according to the level of CD44 expression: Hs578T (high CD44 expressor) and MDA-MB-231 cells (medium-high CD44 expressor) respond to HA; but HA had no effect on the MCF-7 (low CD44 expressor) and ZR-75–1 cells (CD44 negative).

In the general context of the invasion cascade the results of this study are consistent with the following model: HA present in areas of active invasion would induce a high level of one of its receptors, CD44, thereby modifying adhesion and motility. In vivo, it would be the concentration of HA, its location within the tissue, the proportion of its soluble form, the relationship between polymeric and oligomeric HA, and the upregualtion of CD44 that would determine the adhesive and migratory behaviour of tumour cells during the invasion process.

Acknowledgments

We would like to thank Dr F. Halwani for his technical advice; Drs F. Babaï, T.C. Laurent, W. Knudson, and Mr L. LeDuy for their critical comments and Mrs. S. Schiller for her technical assistance with the flow cytometric analysis.

This study was supported by operating grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada to SJ. AH-G is supported by a Fellowship from the FRSQ-FCAR Santé, Quebec, Canada.

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