Chemotaxis and Haptotaxis of Human Malignant Mesothelioma Cells: Effects of Fibronectin, Laminin, Type IV Collagen, and an Autocrine Motility Factor-Like Substance1

[CANCER RESEARCH 53. 4376-4.1K2. September 15. 199.1] Chemotaxis and Haptotaxis of Human Malignant Mesothelioma Cells: Effects of Fibronectin, Laminin, Type IV Collagen, and an Autocrine Motility Factor-like Substance1

Julius Klominek,2 Karl-Henrik Robert, and Karl-Costa Sundqvist

De/hirlment of Lung Medium1 /./. K.Â¡,Medicine [K-H. R.j. unii Clinical lininiinolog\\ [J. K,, K-(!. S.[. Karolinska Institute til Huililin^e University Hospital, S-ÃŒ41Kt>Huddinge. Sweden

ABSTRACT the highly invasive behavior clinically relevant mÃ©tastasesare not common in MM. Mesotheliomas are often accompanied by pleural A human malignant pleural mesothelioma cell line (STAY) was studied effusion which may contribute to the local tumor spread. However, in with respect to production of the extracellular matrix components lumi patients without effusion and when the tumor invades adjacent extra- nili, type IV collagen, and fibronectin, and interactions with these proteins in vitro. We also analyzed S'l'AV cell serum-free conditioned medium with thoracal locations, the dissemination probably requires an active lo respect to the possible presence of "autocrine motility factor-like" sub comotion of mesothelioma cells rather than reimplantation of floating stance. Sodium dodecylsulfale-polyacrylamide gel electrophoresis of bio- cells. synthetically labeled STAY serum-free conditioned medium showed that Tumor cell invasion is a complex multistep process that requires STAY cells released several proteins into the medium, including compo successful infiltration of tumor cells from the primary site through nents with molecular weights of 850,000, 540,000 and 440.000. Using tissue barriers. Invading MM cells most likely interact both with Western blotting we identified these proteins as luminili, type IY collagen, soluble ECM molecules present in pleural fluid (5, 6) and with in and fihronectin, respectively. By immunocytochemistry laminin, type IY soluble forms of such components in pleural and subpleural soft collagen, and fibronectin were detected as a matrix surrounding the cells. tissues. Plastic culture dishes coated with /ug quantities of luminili, type IY colla gen, and fibronectin induced attachment and spreading of STAY cells. Several substances that stimulate tumor cell locomotion HI vitro Laminin, type IY collagen, and fibronectin stimulated directional (chemo- have been described. These include factors derived from rcsorbing tactic) migration of STAY cells in Boyden chambers Pitted with 8 Â¿im bone (7), liver, lung (8). and smooth muscle (9), as well as growth filters. The same cells also migrated to insoluble step gradients of filter- factors such as insulin, insulin-related peptides (10), and bombesin bound extracellular matrix components (haptotaxis). When STAY serum- (11). Tumor cells can also respond to endogenous substances that in an free conditioned medium was separated by using fast protein liquid chro- autocrine fashion stimulate locomotion (12-14). matography SuperÃ³se 6 gel filtration, two motility-inducing protein peaks Normal mesothelial cells are known to produce ECM components were detected. The first peak contained proteins with molecular weight > such as collagen type I, III, and IV, as well as elastin, laminin, and 220,000 that had both chemotactic and haptotactic properties, while the fibronectin (15). It has been shown that MM cells in vitro produce second peak contained material with apparent molecular weights of ap collagens type I (16, 17) and IV (17) and the glycosaminoglycan proximately 67,000 that had chemotactic and chemokinetic (random mo tility) but not haptotactic properties. Analysis of the M, 67,000 material hyaluronic acid ( 18). Pleural effusions of patients with MM have been indicated that it was a heat-sensitive and trypsin-digestible protein. The found to contain high concentrations of soluble fibronectin (5). Fur production of both soluble and insoluble extracellular matrix components thermore, the amount of fibronectin was higher than in plasma, indi by human mesothelioma cells and the motile response to these molecules cating local synthesis (5). Another substance often present in pleural as well as the production of a M, 67,000 autocrine motility factor-like effusions of patients with MM is hyaluronic acid (6, 18-20). substance may be important for the highly invasive motile behavior of this Fibronectin, laminin, type IV collagen, and thrombospondin have tumor. been shown to stimulate locomotion of certain tumors, including Schwann cell tumor (21), B16 mouse melanoma (22), and A2058 INTRODUCTION human melanoma (23, 24). Hyaluronic acid, which is present in MM MM' are rare malignancies arising from mesothelial-lined surfaces. both in vitro (18) and in vivo (20), has been proposed to enhance tumor spread by increasing the fluid content of tissues and physically The most common location is the pleural cavity but peritoneum, "open up" matrices (25). pericardium, and paratesticular soft tissues may also be involved (1. Substances that stimulate cell locomotion can be presented to tumor 2). Mesotheliomas spread rapidly along serosal surfaces to involve cells either in soluble form or as an insoluble matrix. Chemotaxis, pericardium, contralateral hcmithorax, and peritoneal cavity by inva directional cell movement, describes cell migration toward concen sion through the diaphragm. The resulting tumor will often form tration gradients of solubilized attractants, while haptotaxis describes diffuse thickening of involved surfaces (3, 4) rather than solitary cell movement in response to adhesive gradients on the substratum rounded lesions as seen in other neoplasms. MMs often invade (26, 27). The important distinction between chemotaxis and haptotaxis through needle biopsy tracts or incisions in the chest wall (4). Despite has recently been underlined by evidence that the signals for chemo taxis and haptotaxis to type IV collagen are mediated by distinct Received 3/2/93; accepted 7/13/93. The costs of publication of this article were defrayed in part by the payment of page transduction mechanisms (24). charges. This article must therefore be hereby marked adtvrtisement in accordance with The highly invasive behavior of mesothelioma cells taken together 18 U.S.C. Section 1734 solely to indicate this fact. 1This work has been supported by grants from The Swedish Cancer Foundation. The with local abundance of both soluble and insoluble ECM components Swedish Medical Research Council. The Swedish Society of Medicine, the Swedish Heart indicate local synthesis of ECM by MM and suggest that those inter and Lung Foundation, and the Alex and Eva WallstrÃ¶mFoundation. actions between MM and ECM can cause local tumor spread. Several 2 To whom requests for reprints should be addressed, at Department of Lung Medicine. M 53. Huddinge University Hospital. S-14I 86 Huddingc. Sweden. investigators have previously described synthesis of ECM by tumor 'The abbreviations used are: MM. malignant mesothelioma; BSA, bovine serum cells (28-31). However, most of these studies concern melanomas or albumin; DTT. dithiotreitol; ECM, extracellular matrix; FPLC. fast protein liquid chro- other solitary tumors and to our knowledge there are no in vitro studies matography; PBS, phosphate-buffered saline; SFCM. serum-free conditioned medium; SS. serum-supplemented medium; SDS-PAGE. sodium dodecyl sulfatc-polyacrylamide of mechanisms involved in the invasion of MMs. We have previously gel electrophoresis; AMF, autocrine motility factor; FCS. fetal calf serum. described establishment of MM cell lines (18). In the present paper we 4376

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1993 American Association for Cancer Research. MOTIL.[TY OÃ-'MALIGNANT MliSOTIIlil.lOMA CÃ¯LLS examine one of these cell lines with respect to production of different uncoated plastic dishes (or coated with 10 ng/ml BSA) and less than 0.1% ECM components and "motility factors." We further investigate in adhered to plastic dishes coated with 10 mg/ml BSA. teractions with ECM components in order to elucidate whether these Chemotaxis was assayed by using 48-well chemotaxis chamber (Neuro could stimulate the locomotion of mesothelioma cells and thus explain Probe, Cabin John. MA) and 8 Â¡imNucleopore filters (Pleasanton. CA) coated with 100 fig/ml gelatin on both sides. Different concentrations of fibronectin, their distinctive invasive features. type IV collagen, and laminin were diluted into serum-free RPMI and added in triplicate to the lower wells, while upper wells were filled with STAV cells, 2 X 10'Vml in serum-free RPMI containing 1 mg/ml BSA. The chambers were MATERIALS AND METHODS incubated for 15 h in a well humidified incubator at 37Â°C.Control wells

Cells. Human malignant mesothelioma cell line STAY PCS (18) was cul received 1 mg/ml BSA in the lower wells. At the end of the assay, filters were tured in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum. removed, fixed in methanol, and stained with Giemsa. The filters were then This cell line grows as a substrate adherent monolayer and displays a sarco- placed onto glass slides and the cell pellets corresponding to the upper wells matuus growth pattern. After reaching conflucncy the cells in some areas form were wiped with cotton swabs. Cells that had migrated through pores to the a multilayer. Normal mesothclial cells were generously provided hy Dr. P. lower side of the filter were counted by light microscopy under high power Heldin (BiomÃ©dical Center, Uppsala, Sweden) and cultured as previously field (400 X). For each triplicate, the number of cells in three high power fields described (32). was determined and the counts were averaged. Preparation of Serum-free Conditioned Media. STAY PCS cells were Haptotaxis was assayed in triplicate by using 48-well migration chambers grown in SS medium until they were nearly confluent. SS medium was re and the same cell concentration as in chemotaxis assays. Before the motility moved and the cell monolayer was extensively washed with Dulbecco's PBS assays the uncoated 8 /im Nucleopore filters were placed in the chamber, the in order to remove remaining serum proteins. The monolayer was then incu dull side of the filter facing the upper wells. The upper wells were filled with different concentrations of ECM components in serum-free RPMI, control bated for 48 h in fresh RPMI without serum or any other protein. MeUibolically labeled SFCM for gel electrophoresis was prepared in the same way, but wells were filled with 1 mg/ml BSA. The lower wells remained empty. The methionine-free RPMI was used, and supplemented with 10 /xCi/ml of [15SJ- chambers were left overnight at 4Â°C,the filters were then removed and ex tensively washed on both sides, first in Dulbecco's PBS, then in distilled water, methionine (50 mCi/ml; Amersham Laboratory, Buckinghamshire, England). air dried, and finally placed in 48-well chemotaxis chambers with the coated Separation of Serum-free Conditioned Medium. One liter of SFCM was spots of the filler facing exactly toward the lower wells that were filled with prepared, eentrifuged at 300 X g for 15 min, filtered through a 45 /Â¿mfilter to remove cellular debris, concentrated 50-fold by using Amicon membranes YM RPMI containing 1 mg/ml BSA. This procedure was not difficult as there were clear pressure made marks on the filters indicating coated spots. We have also 30 (Amicon, Danvcrs, MA) that retain molecules a M, 30,000. dialyzed tested previously (24) described coating technique by floating 13-mm diameter against distilled water, and lyophilized. The lyophilized material was sus filters on different concentrations of ECM components in serum-free RPMI, pended in serum-free medium and used in chemotaxis and haptotaxis assays, washed them as described above, and used in haptotaxis assays. We found that or it was suspended in PBS, pH 7.4. and applied on FPLC SuperÃ³se 6 column our coating technique performed in 48-well chambers did not affect the results (Pharmacia, Stockholm, Sweden). The column was run Â¡it0.3ml/min and 1-ml (not shown) as compared to coating achieved by floating 13-mm filters on fractions were collected, dialyzed against serum-free medium, and used in ECM components. Thus we used our coating method because it was easier, ehemotaxis and haptotaxis assays (see below). Antibodies. Monoclonal anti-fibronectin antibody IST-1 was obtained faster, and required smaller amounts of ECM. FPLC fractions were either placed in the lower compartment of the Boyden from Sera Laboratory (Crowley Down, England), working dilution 1:100. A monoclonal anti-laminin antibody was purchased from Boehringer Mannheim chamber or were coated on Nucleopore filters as described above. Protein (Mannheim. Germany), working dilution 1:20; anti-laminin polyelonal anti content was determined as described (35). In all adhesion and motility assays the subconfluent cells were detached by brief incubation in 0.25% trypsin/0. body was purchased from Collaborative Research (Bedford. MA), working dilution 1:300(1. A monoclonal anti-type IV collagen antibody was purchased 02% EDTA and then allowed to regenerate for at least l h in SS medium at room temperature. In experiments involving synthetic peptides or pertussis from Dakopatts a/s (Glostrup. Denmark), working dilution 1:30; anti-type IV toxin the cells were preincubated for I h with indicated concentrations of collagen polyelonal antibody was from Collaborative Research, working dilu GRGDS, GRGES. or pertussis toxin (List Biological Laboratory, Campbell, tion 1:500. A monoclonal anti-CD-3 antibody was from Becton Dickinson CA) in scrum-free RPMI. The motility assays were then performed in contin Immunocytochemistry Systems (Mountain View, CA). working dilution 1:100. ued presence of the peptides or pertussis toxin. The peptides were made hy Gel Electrophoresis and Western Blotting. Metabolieally labeled SFCM using synthesizer (Model %00: Bioresearch. San Rafael, CA) and generously (50 ml), was prepared as described above. SFCM was then separated on provided by Dr. E. C. Kohn, (Bethesda, MD). 3.5-8% gradient SDS-PAGE gels under reducing (+50 mM DTT) or nonreducing (-DTT) conditions. The gels were prepared hy using the buffer system of Laemmli (33). In order to characterize different bands, the gels were elec- RESULTS troblotted to nitrocellulose as previously described (34) and stained with the Column Chromatography of Motility-stimulating Factors in use of antibodies to ECM components. The reactions were detected by using STAV SFCM. Unseparated mesothelioma cell SFCM was both che- the biotin-avidin-peroxidase system (Vectastain PK 4002 ABC Kit, Vector motactic and haptotactic for STAV cells in a dose-dependent fashion Laboratories, Burlmgame. CA). Enzyme label was demonstrated by using 3-amino-y-ethyl-earbazole (Sigma, St. Louis, MO). (Fig. 1). The strongest motility response was found when SFCM was Immunocytochemistry. Mesothelioma cells were cultured on glass slides concentrated 100 times, but even unconccntratcd SFCM augmented inserted into the 6-well culture plates until confluence was reached. The cells the motility of STAV cells. In order to investigate the possibility that growing on glass slides were fixed in ice cold methanol and stained by using the motility-inducing activity in SFCM was a scrum contaminant we anti-fibronectin. anti-laminin. and anti-type IV collagen monoclonal antibod used different dilutions of SS medium (20% FCS-0.01% PCS) as ies. Monoclonal anti-CD-3 antibody Leu-4 was used as a negative control. The attractants in motility assays. None of these concentrations of SS reactions were detected by using the biotin-avidin-peroxidase system (see medium induced motility of STAV cells which excludes that serum above). factors were responsible for the motile response of STAV cells (not Cell Adhesion and Motility Assays. Adhesion assay was carried out in shown). In order to characterize the factors responsible for cell trans- triplicate in Petri dishes coaled overnight at 4Â°Cwith 10 fig/ml of fibronectin location we separated mesothelioma SFCM proteins by using molecu (Boehringer Mannheim), laminin (Sigma), or type IV collagen (Sigma). Con lar sieve Chromatography on a FPLC SuperÃ³se 6 column. All fractions trol dishes were coated with 10 mg/ml BSA (negative control) or 10 /xg/ml were assayed for motility-inducing activity in chemo- and haptotactic BSA (positive control). One million STAV cells in RPMI containing 1 mg/ml BSA were added to each dish and incubated. After 40 min the nonadherent cells assays. Two peaks that induced significant motile responses were were removed and counted by using trypan blue exclusion, and the percentage found (Fig. 2). The first peak corresponded to proteins with molecular of adherent cells was calculated. In all experiments 100% cells adhered to weights >200,000. These fractions caused both chemotactic (a) and 4377

80- The substance used to coat the filters was important for the detec tion of chemotactic activity. Thus, the first peak of motility-inducing 70- activity could be detected on both gelatin- and type IV collagen- 60- coated filters. However, in order to identify the second peak of mo tility-inducing activity it was necessary to use filters coated on both 50- sides with 40 /ng/ml of type IV collagen. Furthermore, if gelatin-

40- coated filters were used the motile responses recorded were weak or absent (not shown in Fig. 2), indicating that the presence of type IV 30- collagen on the filters was necessary for motility of MM cells to the

20- M, 67,(HH)material. In control experiments we found that coating of filters on both sides with type IV collagen did not induce motility of 10- mesothelioma cells as compared to haptotactic migration to type IV

"I collagen (see below). 0 The motility-inducing activity in M, 67,000 fractions was inacti I 10 100 1000 vated by heat and protease. We further observed that if STAV cells Concentration of SFCM (x) were pretreated with ng concentrations of pertussis toxin the motile Fig. I. Migration of STAV cells to their own serum-free conditioned medium. Condi response to fractions containing high molecular weight material (first tioned media were prepared as described in "Materials and Methods." Lyophilized SFCM was dissolved in fresh serum-free medium (2(K) x concentrated SFC'M) and then diluted. peak) remained virtually unaffected (not shown), whereas the motile For determination of haptotaxis (D), different dilutions of SFC'M were used to coal the response to fractions within the second peak was completely abol filters, while for determination of chemotaxis ( 0 ). different dilutions of SFCM were ished. This observation indicates that the motility-inducing signals placed in the lower compartment of Boyden chambers. Migration to control proteins (BSA, 1-1000 ngjml, or different concentrations of RPMI + PCS) was 0-1 cells/high delivered by molecules in the first and the second peak are mediated power field (HPF). by different transduction mechanisms. The total inhibition of chemo taxis by pertussis toxin to fractions of molecular weight Â«=67,000 indicates that G-proteins are involved in signal transduction. M r 200 116 90 67 43 13 Detection of ECM Components in STAV SFCM by SDS-PAGE. In order to characterize the high molecular weight substances respon sible for the chemotactic and haptotactic migration of mesothelioma cells we separated STAV [35S]methionine-labeled SFCM on 3.5-8% SDS-PAGE gels under reducing and nonreducing conditions (Fig. 4).

SF 10ug 20 ng 40 ug

v 5^15 12vj34

Fractions ^3763\ Fig. 2. Determination of motility-inducing activity in STAV serum-free conditioned medium separated by using FPLC SuperÃ³se t>column chromatography. The dark areas denote fractions containing motility-inducing activity. Numbers within the graph, number so i of chemotactically and haptotactically migrating cells. For determination of chemotaxis ^6027Vv\702624V\7^ (a), the fractions were pooled and placed in the lower compartment of the Boyden 8 chamber. In determinations of haptotaxis (b). STAV cells migrated to pooled fractions coated onto the distal surface of filters. Top abscissa, molecular weight in thousands. haptotactic (b) responses in a concentration-dependent fashion (not shown). The second peak had an apparent molecular weight of 67,000.

To assess whether the motility induced by the M, 67,000 peak was Fig. 3. Checkerboard analysis of migration induced by pooled FPLC SuperÃ³se 6 directional or random we performed a checkerboard analysis. In this fractions with M, 67,(KH).Varying concentrations of pooled FPLC fractions were added to the upper chamber with the cells or to the lower chambers, as indicated. The M, 67,tKM( assay cells are exposed to different gradient conditions obtained by protein acts in a dose-dependent fashion and is both chemotactic (values below the varying the amounts of attractant in the upper and lower compart diagonal) and chemokinetic (values on the diagonal). SF, serum-free medium. ments of the Boyden chamber. Pooled fractions within this peak induced both chemotactic (to a gradient) and chemokinetic (in the Table I Propertiesâ€¢ofpooled M, 67,000 FPLC fractions absence of the gradient) motility (Fig. 3). The M, 67,000 peak was further characterized as shown in Table 1. Treatment No. of migrated cells We also examined if mesothelioma-derived material corresponding None (control) 57 Â±7Â° 1WC 5 min* 5Â±2 to M, 2(X),()00and M, 67,000 motility peaks affected the migration of Trypsin, KM)u,g/ml' 5Â±2 normal mesothelial cells. In this experiment normal mesothelial cells PMSF, 5 mm 55 Â±6 Pertussis toxin, (1.5 u.g/ml'' were placed in the upper compartment of Boyden chambers and 6Â±2 " Mean Â±SD. exposed to pooled FPLC fractions containing Mr 200,000 and Mr h Pooled fractions were boiled for 5 min. 67,000 material, respectively. Normal mesothelial cells showed a ' Pooled fractions were incubated with trypsin for l h at 37Â°Cand then 5 Ã•TIM weak motile response to the high molecular weight ECM-containing phenylmethylsulfonyl fluoride (PMSF) was added to inactivate the enzyme. PMSF alone was used as a control. peak, whereas no migration was observed to M, 67,000 material (data '' STAV cells were preincubated with pertussis toxin for I h and placed in the upper not shown). compartment of a Boyden chamber; pooled FPLC fractions were used as chemoattraclant. 4378

which was used as a control. Other bands detected on top of the gel 850 had molecular weights in the range 500,()0()-2()(),(K)0. SDS-PAGE-separated STAY [35S]methionine SFCM was electrob- lotted onto nitrocellulose and reacted with antibodies to laminin, fibronectin, and type IV collagen. This identification, using antibodies 400 >â€¢ against each ECM component, demonstrated that the mesothelioma cells secreted laminin, fibronectin, and type IV collagen into the culture medium. Under nonreducing conditions, anti-laminin poly 200 clonal antibody recognized a M, 850,000 band. Two different type IV collagen antibodies recognized a M, 540,000 band, while fibronectin antibody recognized a M, 440,000 band. In all cases the antibody reactive bands corresponded to fluorogram bands, indicating that they represented endogenous ECM components. Under reducing condi 116 tions laminin was detected as a M, 200.000 band corresponding to the 1 8 ÃŸchain of the molecule. The Mr 400,000 a chain of laminin stained Fig. 4. Separation of proteins released by STAY cells using 3.5-8% SDS-PAGE gels. weakly. Fibronectin was detected as a M, 220,000 band. The mono Fluorogram of STAY [15S]methionine scrum-free conditioned medium. Lane 1 (-DTT) clonal anti-type IV collagen antibody did not detect reduced forms of and Lane 6 ( + 50 HÃŒMDTT).Immunoblol stained with anti-CD-3 monoclonal antibody (Lane 2). Immunoblot stained with anti-laminin polyclonal antibody. Lane 3 (-DTT) and this molecule (not shown), while the polyclonal anti-type IV collagen Lane 7 (+ DTT). Immunoblot stained with anti-fibronectin monoclonal antibody. Lane 4 antibody recognized Mr 180,000 and Mr 230,000 bands. A third anti (-DTT) and Lane 8 ( + DTT). Immunoblot stained with anli-lypo IV collagen polyclonal antibody. Lane 5 (-DTT) and Lane 9 ( + DTT). Ordinate, molecular weight in thousands. type IV collagen polyclonal antibody (generously provided by Dr. D. Shuppan, Berlin, Germany) also detected M, 180,000 and M, 230,000 bands (not shown). Several high molecular weight bands were detected by using fluorog- Immunocytochemistry. STAV cells cultured on glass slides were raphy, showing that these components were indeed produced by the reacted with antibodies against fibronectin, type IV collagen, and mesothelioma cells. The component with highest molecular weight laminin, (Fig. 5). The strongest immunoreactivity was observed with (>850,000) comigrated with commercially available laminin (Sigma) fibronectin and laminin antibodies. Fibronectin was detected as an

m^m:"m*' Â¿V-^Ã&^-'t^V â€¢¿-*-. V ;"7H S

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Fig. 5. Immunoslaining nf STAV cells with anli-fibronectin (a), anti-type IV collagen (h), anli-laminin (c) and anti-CD-3 monoclonal antibodies (d) (X243). 4379

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1993 American Association for Cancer Research. MOTILITY OF MALIGNANT MESOTHEL1OMA CELLS extracellular matrix particularly in areas where cells formed a multi 25 /u,g,respectively. Substratum-bound laminin was weakly haptotac layer but was also seen around individual cells. In areas where cells tic for mesothelioma cells with peak activity at 12.5 jag. The biphasic grew sparsely spread on the substratum there was relatively weak motile response of STAV cells to ECM components parallels findings fibronectin immunoreactivity. Laminin immunoreactivity was present in B16 and A2058 malignant melanoma cells (22, 24). as a matrix in areas of multilayer formation and as a diffuse staining Coating of the lower surface of the filter with BSA 1-1000 ju.gor throughout the culture. Type IV collagen immunoreactivity was also serum-free RPMI did not induce translocation of mesothelioma cells. most pronounced in areas were cells formed multilayers and was In several triplicate experiments merely 0-1 cell migrated to any BSA distinctly located as a matrix on and between individual cells. As with concentration or to serum-free RPMI coating of the lower filter sur the fibronectin matrix, the intensity of the type IV collagen immuno face. Coating of filters with ECM components 1-200 fig on both sides reactivity varied in different areas of the culture, indicating that local induced migration of only 0-5 cells to the lower filter surface (not environmental factors such as cell density and multilayer formation shown). probably play a role for production and assembly of these molecules. In order to study if the tripeptide arginine-glycine-aspartic acid Adhesion and Motility of STAY Cells to ECM Components. (ROD) (37), known to constitute a cell-binding site in three of the STAV cells exhibited adhesion and spreading on ECM components. tested ECM components, was necessary for motile responses of me The highest number of attached cells was observed when Petri dishes sothelioma cells in the Boyden chamber experiments described above, were coated with fibronectin and type IV collagen with 90 and 85% we pretreated the cells with different concentrations of GRGDS or the attached cells, while only 30% cells adhered to laminin. Virtually all control peptide GROES (Fig. 7). Chemotactic (25 ^g/ml) and hapto attached cells showed pronounced cytoplasmic spreading. tactic migration to fibronectin (100 ;u.g)was inhibited by GRGDS in The motility of STAV cells was tested in multiwell chemotaxis concentrations 500-31 ju.g/ml, whereas different concentrations of chambers. Relatively long incubation times were required since no or GRGES had no effect. These observations indicate that the ROD very few cells translocated during initial experiments when the incu sequence is an important binding site for mesothelioma cells migrat bation times were 4â€”6h.The optimal time necessary for STAV cells ing to soluble or insoluble fibronectin. No significant inhibition of to respond to attractants was 15 h. It has previously been reported that laminin or type IV collagen-stimulated motility was observed when tumor cells may need different incubation times in order to translocate mesothelioma cells were incubated with the pentapeptides (not through filter pores (36). STAV cells migrated in a concentration- shown), indicating that the ROD sequence present in these molecules dependent fashion toward soluble fibronectin, laminin, and type IV does not comprise a major binding site necessary for migration. collagen (chemotaxis) (Fig. 6A). The most potent chemoattractant for mesothelioma cells was fibronectin, which at a concentration of 1.5 DISCUSSION /xg/ml caused cell migration as compared with BSA controls. Peak stimulation was observed at 25 jug/ml whereas higher fibronectin This study shows that the human malignant pleural mesothelioma concentrations resulted in gradually weaker motile responses. Soluble cell line STAV PCS produces fibronectin, laminin, and type IV col type IV collagen also stimulated mesothelioma cell migration with a lagen as detected by biosynthetic incorporation of [1;iS]methionine, peak motile response at 100 jag/ml. A similar concentration-dependent Western blotting, and immunocytochemistry. The results further show stimulation of STAV cell motility was observed with laminin, al that these ECM components in an autocrine fashion can induce ad though laminin induced a weaker chemotactic response than fibro hesion, spreading, and chemotactic and haptotactic responses in the nectin and type IV collagen. producer cells. STAV cells also migrated toward step-density gradients of ECM In addition, we have detected an activity with apparent M, 67,000 components bound to the lower surface of filters (Fig. 6B). Fibronec which is both chemotactic and chemokinetic and thus similar to the tin and type IV collagen were the most potent haptotactic agents with autocrine motility factors previously described (14, 38), but different maximal stimulation of motility at a coating concentration of 100 and from ECM components which are chemotactic and haptotactic.

80- 80-

70-

60-

50-

40- 8 3-. 30- Ã¤ g 20- 10-

10 100 1000 10 1000

ECM cone ug/ml Coating cone ng Fig. 6. Chemotactic (A) and haptotactic (B) migration of STAVcells in response to ECM components. In chemotactic experiments increasing concentrations of fibronectin (A), collagen type IV ( 0 ), and laminin (O) were diluted into serum-tree RPMI and added to the lower wells. In the determination of haptotaxis the Nucleopore filters were precoated with a step gradient of increasing concentrations of fibronectin (A), collagen type IV ( 0 ). and laminin (O). Migration to any concentration of control protein BSA was 0-1 cell/high power field (HPF). 4380

80- 80-

70- 70-

60- x 60- * 50- ^ 50 -

40- I 40- O 30- Â¡ 30-

00 20- Â§ 20-

10- 10-

0 1 0 10 100 1000 10 100 1000

Peptideconc (Hg/ml) Peptide cone (u,g/ml) Fig. 7. Effects of peplidcs GRGDS (U) and GROES ( 0 ) on chcmolaclic and haptotactic migration of STAV cells to fihronectin. In these experiments STAV cells were preincubated for l h with indicated concentrations of peptidcs and allowed to migrate to cither soluble fibronectin (25 fig/ml) (A ) or to fibroncctin (10(1 fig/ml) that was coated on the distal surface of the filters (ÃŸ).The peptides were allowed to remain with the cells during the assay. HPF. high power field.

STAV cells secrete a large number of biosynthetically labeled pro previously described for Schwanoma and human melanoma cells (21, teins into the medium. Three of the proteins were characterized as 22). Furthermore, when STAV SFCM was separated on a FPLC Su disulfide-linked ECM molecules located in the high molecular weight perÃ³se 6 column, high molecular weight fractions containing ECM region on SDS-PAGE gels. The largest molecule (A/r 850,000 before components were found to stimulate both chemotaxis and haptotaxis, and M, 200,000 and M, =400,000 after reduction) was identified as indicating that these molecules have the ability to stimulate mesothe- laminin. Laminin is known to have a M, of 850,000 and after reduc lioma cell motility in an autocrine fashion. Control experiments with tion of Sâ€”S bonds to form a Mr =400,000 a chain and two ÃŸchains BSA or serum-free medium alone unequivocally showed that ECM components are "true" chemotactic and haptotactic agents. with M, 230,000 and 240,000, respectively, which often form a single broad band (39). The a chain is usually difficult to detect on Western Human mesothelioma cells in vivo are often imbedded in a diffuse blots and often yields fainter staining than the ÃŸchains or may even collagenous matrix (1,2). The tumor and the surrounding matrix form be absent (39). The next high molecular weight component secreted flat sheets that spread along serosal surfaces. Thus, malignant meso by STAV cells was identified as type IV collagen. The monoclonal thelioma matrix has a different distribution than desmoplasia, an ex anti-type IV collagen antibody used did not detect reduced forms of cessive matrix formation that encapsulates certain solid tumors (42). this molecule, probably due to the fact that it is prepared for identi The possible origin of increased matrix formation that surrounds some fication of the intact molecule in tissue sections rather than in Western tumors is a subject of controversy. It has been suggested that the blotting. After reduction the polyclonal type IV collagen antibody invasive breast carcinoma cells secrete the extracellular matrix (43), recognized M, 230,000 and MT 180,000 bands. The M, 180,000 com but it has also been argued that source of desmoplasia were host cells ponent corresponds to the previously described reduced form of type (44). Due to a different distribution of the matrix surrounding some IV collagen (28). The M, 230,000 component is probably insuffi tumors such as breast carcinomas, which are often encapsulated, and ciently reduced collagen or a contaminant. The third high molecular that seen in mesotheliomas, we consider it likely that mesothelioma weight band detected by fluorography had a molecular weight of cells may use self-secreted matrix as a substrate for invasion. 440,000 and after reduction of disulfide bonds a molecular weight of Pleural fluid of patients with mesothelioma contains high concen 220,000. These bands were detected on Western blots with anti- trations of fibronectin. Our data supports this evidence (5) that fibro fibronectin monoclonal antibody and are in good agreement with nectin is locally secreted and indicate that the source is the malignant previously described staining patterns of this molecule (40). mesothelioma tumor itself. STAV cells also produced an insoluble matrix of fibronectin, lami An additional observation in the present study is the partial char nin, and type IV collagen associated with the cell surface, as detected acterization of M, 67,000 FPLC SuperÃ³se6 fractions that induced both by immunocytochemistry. This matrix of fibronectin, type IV colla chemotactic and chemokinetic responses in mesothelioma cells. This gen, and laminin immunoreactivity was associated with areas of high activity might be a serum contaminant that remained on the cells and cell density and multilayer formation. This observation is consistent in the culture dish despite extensive washing with PBS. However, with previous reports of presence of fibronectin preferentially at the different dilutions of serum containing medium did not stimulate the surface of confluent cells as compared to sparse cultures (reviewed by motility of STAV cells. Therefore we conclude that the motility factor Hynes, Ref. 41). In addition to the matrix formation in some areas in M, 67,000 fractions must be produced by STAV cells. An important laminin showed a homogeneous distribution throughout the culture. difference which distinguishes the M, 67,000 factor from the high From these data we conclude that STAV cells produce both soluble molecular weight peak of locomotor activity is the absence of hapto and insoluble forms of fibronectin, laminin, and type IV collagen. tactic activity, and the presence of a chemokinetic activity. The mo- The data presented in this paper demonstrate a direct role of ECM tility-inducing activity of pooled M, 67,000 fractions was heat labile components in mediating adhesion, spreading, and chemotactic and and degradable by trypsin. When STAV cells were pretreated with haptotactic responses of STAV cells in concentrations similar to those nanogram concentrations of pertussis toxin the motility toward M, 4381

67,000 fractions was lost, indicating that G proteins are involved in 17. Kataoka, H., WickstrÃ¶m, B., Klominek. J.. Gay, R. E., Gay. S., and Hjerpe, A. signal transduction while motility toward high molecular weight ECM Immunocytochemical demonstration of collagen types I and IV in cells isolated from malignant mesothelioma and in lung cancer cell lines. Lung Cancer, 6: 16-27, 1990. components became largely unaffected. From this observation we 18. Klominek. J.. Robert. K-H., Hjerpe, A.. WickstrÃ¶m. B., and Gahrton. G. Serum- conclude that the M, 67,000 fractions contain a protein with properties dependent growth patterns of two, newly established human mesothelioma cell lines. similar to a previously described autocrine motility factor (14, 38, 45). Cancer Res., 49: 6118-6122. 1989. 19. Mayer, K., and Chaffee. E. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1993 American Association for Cancer Research. Chemotaxis and Haptotaxis of Human Malignant Mesothelioma Cells: Effects of Fibronectin, Laminin, Type IV Collagen, and an Autocrine Motility Factor-like Substance

Julius Klominek, Karl-Henrik Robért and Karl-Gösta Sundqvist

Cancer Res 1993;53:4376-4382.

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