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Thrombospondin-induced attachment and spreading of human squamous carcinoma cells
Experimental
Cell Research 167 (1986) 376390
Thrombospondin-Induced Attachment and Spreading of Human Squamous Carcinoma Cells JAMES VARANI, ‘. * VISHVA M. DIXIT.’ SUZANNE E. G. FLIGIEL,4 PAUL E. McKEEVER’ and THOMAS E. CAREY’ ‘Department of Pathology, ‘Depurtmrnt c?f Otor~lino/ar~tl~(~l~)~~, Uniuersity Michigan Medicul School, Ann Arbor. MI 48109, ‘Department of Pathology, Division of Luborutory Medicine, Wushington Uniuersitx School of Medicine, St Louis, MO 631 IO. und 4Depurtment of‘Putholog~. A//en Purk VAMC Wuyne Stute University. Detroit. MI 48101, USA
of
Thrombospondin (TSP) induced the attachment and spreading of human squamous carcinoma cells on plastic culture dishes and dishes coated with type I or type IV collagen. Increased adhesion was detected as early as IS min after treatment. Dose-response studies indicated that l-5 ug of TSP per 35 mm (diameter) culture dish was sufficient to induce a response and that a half-maximal response occurred at 10 ug of TSPidish. The squamous carcinoma cells synthesized TSP as indicated by biosynthetic labeling experiments. TSP was secreted (or shed) into the culture medium by these cells and also became bound to the cell surface. TSP also promoted adhesion of human keratinocytex. fibroblasts and fibrosarcoma cells but did not induce attachment or spreading of human melanoma or glioma cells, 0 1986 Academic PRS. IW although these cells did respond to laminin.
Thrombospondin (TSP) is a high molecular weight (HMW) glycoprotein (M,=580 kD) which is released from the a-granules of platelets during activation [ 1, 21. In the presence of divalent cations, the released TSP becomes associated with the platelet surface and acts as a mediator of aggregation [3-51. TSP is also synthesized by various types of normal cells in culture [6-lo] and is deposited into the extracellular matrix produced by these cells. Since several other components of the extracellular matrix serve as adherence factors for cells [ 1I-161, it is possible that TSP may also be involved in cell-cell or cell-substratum adhesion. In the present study we examined TSP for its ability to promote cell-substrate adhesion of various human tumor cells. It stimulated adhesion of several squamous carcinoma and fibrosarcoma cell lines as well as human keratinocytes and fibroblasts. In contrast, TSP did not promote adhesion of human melanoma or glioma cells although these cell lines responded to laminin. MATERIALS
AND METHODS
Cells The human tumor cells examined in this study include eight squamous cell carcinoma lines. two tibrosarcoma lines, four melanoma lines and four glioma lines. The squamous cell carcinoma lines * To whom offprint
requests should be addressed. Copyright 0 1986 hy Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827186 $03.W
Thrombospondin-induced
adhesion
377
(designated UM-XC-) were derived from tumor specimens obtained from 5 different individuals. The UM-SCC-1 cell line was derived from a local recurrence of a tumor in the floor of the mouth of a 73year-old male, and the UM-XC-1 1A line from a biopsy specimen of laryngeal cancer in a previously untreated 65-year-old male patient. UM-XC-1lB was established from tumor tissue from a total laryngectomy performed 6 weeks after the biopsy. The UM-SCC-14C cell line was established from a tumor in the neck of a 64-year-old female patient. This tumor was a local recurrence of a tumor originating in the floor of the mouth. The UM-SCC-17A and 17B cell lines were both established from a persistent cancerous lesion of the larynx and a metastatic tumor in a cervical lymph node from a 48year-old female patient who underwent surgery after an unsuccessful attempt to control her primary cancer of the larynx with radiation therapy. Cell lines UM-SCC-22A and 22B were established respectively from a primary cancer of the hypopharynx and cervical lymph node metastasis in a 59year-old female patient. All of the squamous carcinoma lines formed tumors in nude mice. Histological examination of three tumors (those produced by UM-SCC-1 lB, UM-SCC-17B and UM-SCC-22B cells) showed that the UM-SCC-1 1B cells produced the least differentiated tumors and that the UMSCC-22B cells produced the best differentiated tumors. The tumors produced by the UM-SCCl7B cells were intermediately differentiated. The two tibrosarcoma lines, UM-FS-IA and UM-FS-lB, were established in culture, respectively, from a primary and recurrent malignant fibrosarcoma that arose in the facial musculature of a 14-yearold male. The cell lines have fibroblastic morphology, express non-polymorphic human HLA histocompatibility antigens and produce rapidly-growing tumors in nude mice that recreate the histology of the original neoplasms. One of the malignant melanoma lines (UM-MEL-l) was derived from a recurrent lesion in the neck of a 34-year-old male with a past history of malignant melanoma arising on the left auricle. A second melanoma line (UM-MEL-2) was obtained from a heavily pigmented mass that arose in the right supraclavicular region of a 19-year-old male in whom no primary melanoma was found. The isolation and characterization of the other two melanoma lines (SK-MEL-IO and SKMEL-28) have been described previously [17]. The isolation and characterization of the human glioma lines have also been previously described [18, 191. In addition to these tumor cell lines, primary human keratinocytes and fibroblasts obtained from explanted foreskins were used. The tibroblasts and keratinocytes were separated from each other by differential trypsinization as described by Kimmel & Carey [20]. In addition to these cells, human lung fibroblasts (MRC-S), obtained from Flow Laboratories (McLean, Va.) were also used. Finally, murine tibrosarcoma cells and murine melanoma cells were also examined for response to TSP. The fibrosarcoma cells were available from previous studies in our laboratory [21] and the melanoma cells (Bl6-BL6 line) were provided by Dr J. Niederkorn (Department of Ophthalmology, University of Texas; Dallas, Tex.). All of the human lines were grown in monolayer culture on Minimal Essential Medium of Eagle (MEM) supplemented with 15% fetal bovine serum (FBS), non-essential amino acids, 100 U/ml of penicillin and 100 @ml of streptomycin. The cells were grown at 37°C in the presence of 5 % COZ. The murine cells were maintained on MEM supplemented with 10% FBS and antibiotics.
Reagents TSP was purified from the supematant fluid of thrombin-activated platelets as described [l]. The purified TSP migrated as a single protein band with an apparent MW of 180 kD when examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The production and characterization of the monoclonal antibodies directed against TSP have been described previously [22-251. Monoclonal antibodies were purified from ascites fluid by ammonium sulfate precipitation followed by affinity chromatography on protein A Sepharose. Purity was determined by SDS-PAGE. The rabbit polyclonal antibody against TSP was purified by afftnity chromatography on protein A Sepharose. Specificity for TSP was confirmed by solid-phase radio-immunoassay performed as described [23] with immobilized antibodies and TSP iodinated with the Bolton-Hunter reagent or NA’*‘I and iodobeads. Laminin was used in certain experiments. The laminin was prepared from the Englebreth-HolmSwarm (EHS) tumor by the method of Timpl [26]. Purity of the laminin was assessed by SDS-PAGE and by enzyme-linked immunosorbent assay (ELISA) [21]. When examined on a 7% SDS-polyacrylamide gel under reducing conditions, only two protein bands (at 200 and 400 kD) were seen. The laminin reacted by ELISA with monospecific polyclonal anti-laminin antibodies at dilutions up to Exp Cell Res 167 (1986)
378 Varani et al. 1 : lo6 but did not react with polyclonal antibodies to type IV collagen or fibronectin. The laminin prepared in this manner stimulated adherence in a highly-sensitive line of murine fibrosarcoma cells at concentrations as low as 0.5 ug/35 mm (diameter) dish [21].
Adhesion Assay Cell attachment and spreading on plastic culture dishes and collagen-coated culture dishes was used as a measure of adhesion. The collagen-coated dishes were prepared by pretreating 35 mm (diameter) dishes with 50 ug of a Type I collagen preparation from bovine skin (Vitrogen-100; Collagen Carp, Palo Alto, Calif.) or 50 ug of a Type IV collagen preparation from the EHS sarcoma (Bethesda Research Laboratories; Gaithersburg, Md.). The dishes were incubated overnight with the collagen solutions in 0.1 N acetic acid. The collagen film was deposited onto the dish surface as the acid evaporated. The dishes were then washed twice in serum-free MEM and were ready for use. The adhesion assay was carried out by adding 1 ml of MEM supplemented with 200 ug/ml of bovine serum albumin (BSA) to each dish and incubating them for 15 min at 37°C. The cells were harvested from culture by trypsinization, washed two times in serum-free MEM and resuspended at 5x lo5 cells in 1 ml of MEM supplemented with 200 ug of bovine serum albumin per ml. The cells were mixed with the appropriate amount of TSP, incubated with the TSP in solution for 5 min and then added to the dishes. The dishes were incubated at 37°C. At various times later, the non-attached cells were removed and counted with an electronic particle counter. The dishes were washed and then flooded with 2% glutaraldehyde. The percentage of spread cells was determined by examining the dishes using a microscope with a calibrated grid in the eyepiece. In some experiments, the dishes were precoated with TSP. When this was done, the appropriate amount of TSP was added to the culture dishes in 1 ml of serum-free MEM and incubated at 37°C for 1 h. The dishes were then washed twice in serum-free MEM to remove the non-adsorbed TSP and used.
Biosynthetic Labeling The cells were grown in loo-mm (diameter) culture dishes to an approximate density of 1x 10’ cells per dish. The cells were washed and incubated for 30 min in methionine-free, serum-free minimal essential medium (Flow Laboratories; McLean, Va.) followed by a 60-min incubation in the same medium supplemented with 500 uCi per dish of [35S]methionine (1000-l 400 uCi/umole; NEN, Boston, Mass.). After the 60-min biosynthetic pulse, the cells were again washed and incubated for a further 4 h in complete medium. The 4-h chase cells were lysed in a solution of phosphate-buffered saline (PBS) containing three detergents (1% Triton X-100, 0.5 % sodium deoxycholate and 0.1% SDS; all obtained from Sigma Chemical Co.) and protease inhibitors including 20 mM EDTA, 5 mM N-ethyl maleimide, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 10 ul/lO ml of a protease inhibitor cocktail containing: leupeptin, 1 mg/ml; antipain, 2 mg/ml; benzamidine, 10 mg/ml; aprotinin, 10000 kallikrein-inactivating units/ml; chymostatin, 1 mgiml; and pepstatin, 1 mg/ml, as described by Ronnett et al. [27] in studies on the insulin receptor. All of the protease inhibitors were obtained from Sigma Chemical Co. The 4-h chase media were also supplemented with the detergent/ protease inhibitor mixture by adding one-fourth volume of a fivefold concentrated solution. The cell lysates and chase media were frozen at -8O”C, thawed, and clarified by ultracentrifugation (37000 g for 60 min). Immunoreactive TSP was precipitated with a 1 : 100 dilution of the rabbit anti-TSP and Protein A-Sepharose (Sigma Chemical Co.) according to the protocol of Ruddon et al. [28]. Affnitypurified anti-bovine serum albumin served as a control. The washed immunoprecipitates were divided in half and eluted with boiling (5 min) in twofold concentrated Laemmli SDS-PAGE sample buffer [29], either with or without 2 % 2-mercaptoethanol. The immunoprecipitated material was fractionated on a 3-10% polyacrylamide gel employing the Laemmli system. Radioactive bands were visualized by fluorography with En3Hance (NEN), exposing the dried gels to X-ray film (Kodak XAR-2) for 2 days.
Immunofluorescence The tumor cells were examined for anti-TSP binding by indirect immunofluorescence one day after plating. For this, 2.0x 10’ cells from each line were seeded onto 20x20 mm glass coverslips. The culture medium consisted of MEM supplemented with 10% normal rabbit serum. Rabbit serum was used in place of FBS to present possible cross-reactions between the rabbit anti-TSP and TSP present in FBS. At the time of staining, the culture medium was removed and the cells were gently washed Exp Cell Res 167 (1986)
Thrombospondin-induced
adhesion
379
three times in cold PBS. All subsequent steps were carried out at 4°C. The cells were stained initially with a 1 : 20 dilution of the rabbit anti-TSP or anti-bovine serum albumin (45 min) and then washed three times. They were then stained with a 1 : 50 dilution of fluorescein-conjugated swine anti-rabbit serum (45 min) obtained from Accurate Scientific and Chemical Company (Westbury, N. Y.). Following three additional rinses in PBS, the coverslips were placed on glass slides and viewed immediately by standard fluorescence microscopic techniques.
RESULTS Stimulation
of Human Squamous Carcinoma
Cell Adhesion
by TSP
The eight human squamous carcinoma cell lines were examined for adhesion to 35mm (diameter) plastic culture dishes which had been preincubated for 15 min with 1 ml of serum-free MEM supplemented with 200 ug of BSA. The addition of 20 ug of TSP to the cell suspensions before addition to the dishes stimulated attachment and spreading of all eight lines. Quantitative data is shown in table 1 and the response of one cell line is shown graphically in fig. I. Two of the cell lines (UM-SCC-1 and UM-SCC-1 1B demonstrated a dramatic response; in four of the lines (UM-SCC-llA, UM-SCC-14C, UM-SCC-17A and UM-SCC-17B), a moderate response was seen and in two lines (UM-SCC-22A and UM-SCC-22B), the response was minimal. It is interesting to note that there was a relationship between the percentage of cells which attached and spread in the absence of stimulation and the percentage of cells which responded to TSP (table 1). TSP also stimulated adhesion of the squamous carcinoma cells when it was preadsorbed onto the culture dishes. The cells responded somewhat faster under this condition (not shown), but the overall relationship among the cells was the same as that shown in table 1. When laminin was used as the stimulating agent in place of TSP, the squamous carcinoma cells were much less responsive or even nonresponsive (table 1). The ability of TSP to stimulate adhesion of the squamous carcinoma lines was not limited to plastic culture dishes. When dishes coated with 50 ug of either type I (bovine skin) or type IV (basement membrane) collagen were used as the substrate, the addition of 20 ug of TSP significantly increased the rate of attachment and spreading on these substrates as well. This is shown in fig. 2 with the UM-SCC-11B cells. The other squamous carcinoma lines responded similarly (not shown). Kinetic studies and dose-response studies were carried out next. As shown with the UM-SCC-1 1B cells in fig. 2, the effects of TSP were seen as early as 15 min after plating. Differences between the treated and control cells persisted for several hours, although the untreated cells also attached and spread after prolonged incubation (4-24 h). The time course of the response to TSP was similar with the other squamous carcinoma cell lines to that shown in fig. 2 with the UMSCC-11B cells. Dose-response studies indicated that 1.0-5.0 ug of TSP per reaction was sufficient to elicit a response and that a half-maximal response occurred at approx. 10 ug per reaction. This was true for all eight of the lines. Exp Cell Res 167 (1986)
380 Varani et al. Table 1. Stimulation of adhesion by thrombospondin Proportions attached and spread
Cell line Human squamous carcinoma UM-SCC-1 UM-XC-I IA UM-XC-1 IB UM-SCC-14A UM-SCC-17A UM-SCC-17B UM-SCC-22A UM-SCC-22B Normal keratinocytes Human fibrosarcoma UM-FS-IA UM-FS-1B Human foreskin tibroblasts Human lung tibroblasts (MRC-5) Human melanoma UM-MEL-l UM-MEL-2 SK-MEL-10 SK-MEL-28 Human glioma UMl U138MG LM UM6 Mouse fibrosarcoma Mouse melanoma
Untreated (%I
Thrombospondin (25 KS) ez)
(25 PCs)
Laminin
lOf3 12f3 1524 4*1 7f6 lOf2 If2 lf3 2+1
72+6 45+5 75+6 40f6 3023 48f4 12f6 to+2 3526
2+_1 2056 22+7 8fl 5+4 222 2*1 1+2 10+3
IO?1 61f-1 30f8 2426
79f8 7o+s 7026 89+8
75+6 80* 10 7215 86?8
5fl 0 35f4 40f5
0 0 0 15+_5
85+14 5Oi6 60+4 85+8
27+4 38f5 9+8 20* 10 0 75f6
0 24f3 14+6 18f7 0 5*1
8228 85+6 80f7 8528 12f5 602 10
(%z)
The percentage of cells attached and spread 1 h after plating was determined as described in Methods. The values represent averages + SDS based on four readings in each of duplicate dishes in a single experiment. Each cell type was examined two or more times with similar results.
Since the kinetics of the response to TSP and the dose responses were similar among the squamous carcinoma lines, the major difference was in the number of cells which ultimately responded. Rabbit polyclonal anti-TSP antibodies and mouse monoclonal antibodies to the various domains of TSP were examined for ability to inhibit TSP-induced attachment and spreading. In the first set of experiments 25 ug of TSP was mixed with 50 or 100 ug of the various antibody preparations and incubated for 1 h at 37°C in 35mm (diameter) plastic culture dishes (in a total volume of 0.35 ml). After the incubation period, the dishes were washed twice and the carcinoma cells (UMSCC-17B) were added in 1 ml of MEM supplemented with 200 ug/ml of bovine serum albumin. Two hours later, cell attachment and spreading were quantitated Exp Cell Res 167 (1986)
Thrombospondin-induced
adhesion
38 1
I. Phase-contrast photomicrographs of human squamous carcinoma cells CUM-XC-I IS) after 30 min of incubation on plastic culture dishes in the presence (A) and in the absence (B) of TSP. The photographs were taken after the dishes had been washed to remove the unattached cells and flooded with 2 % glutaraldehyde. x460.
Fig.
382 Varani et al. Type IV Collagen
Type I Collagen
?
;;F
i
IF
Plastic
EL
15 30 45
15 30 45
15 30 45
Tlme Imid
Tlme (mid
Tlme (mid
Fig. 2. Time course of TSP-induced adhesion of human squamous carcinoma cells (UM-SCC-11B) to type I collagen-coated dishes, type IV collagen-coated dishes, and plastic dishes. The values shown represent the average percentage attached and spread on the substrates in the absence of TSP (0-O) and in presence of 20 ug of TSP (O-O). The values are based on four readings in each of duplicate dishes in a single experiment. The SDS were with 15% of the averages. The experiment was repeated three times with similar results.
in the usual fashion. In the presence of 50 or 100 pg of the rabbit polyclonal antibody, attachment and spreading was reduced by 79+6 % and 81f6 % respectively. Monoclonal antibody A 6.1 (directed against the 70 kD trypsin-resistant core of the TSP molecule) also inhibited adhesion when added at 100 yg per reaction (882 1% inhibition). in contrast to these results, the antibodies directed against the heparin-binding domain (A2.5) and the platelet-binding domain (C6.7) failed to inhibit TSP-mediated adhesion of the squamous carcinoma cells. The antibodies that inhibited TSP-induced attachment and spreading (seen at 2 h) had no effect on the proportion of cells attached and spread when examined at 20 h. When a second squamous carcinoma cell line (UM-SCC-11A) was used, very similar results were obtained (not shown). In a second set of experiments, the TSP and the antibodies were pre-adsorbed onto the substrate sequentially. Initially, 2.5 ug of TSP was added in 1 ml of serum-free MEM to 35 mm (diameter) dishes and incubated for 1 h at 37°C. The non-attached TSP was removed and the dishes flooded with 1 ml of MEM containing 200 ug of bovine serum albumin per ml. The various antibodies (100 ug) were added next and incubated for an additional 1 h at 37°C. The excess antibodies were removed and the dishes washed twice in serum-free MEM. The UM-SCC-17B cells (5x 105)were then added to the dishes and the percentage of attached and spread cells determined in the normal manner. Again, the polyclonal antibody and monoclonal antibody A6.1 partially inhibited attachment and spreading (45+5 % and 34f6% inhibition respectively) while the other monoclonal antibodies did not. The same antibodies were next examined for ability to inhibit endogenous adhesion (in the absence of added TSP) of the UM-SCC-17B cells. For these experiments, 5x lo4 cells were preincubated for 1 h at 4°C in tubes containing 0.2 ml of serum-free MEM supplemented with 200 pg of BSA per ml and various Exp Cell Res 167 (1986)
Thrombospondin-induced adhesion
383
Table 2. Inhibition of squamous carcinoma cell attachment and spreading by pretreatment of the cells with antibodies to TSP” Treatment
Percent attached and spreadf
None 10 ng rabbit polyclonal antibody 25 ng rabbit polyclonal antibody 1 ug monoclonal antibody A6.1 b 10 ug monoclonal antibody A6.1 1 ug monoclonal antibody C6.7’ 10 ng monoclonal antibody C6.7 1 ug monoclonal antibody A2Sd 10 ng monoclonal antibody A 2.5 25 ug monoclonal antibody A 2.5 10 ug monoclonal antibody D4.6’ 40 ng monoclonal antibody D4.6 75 ng monoclonal antibody D4.6
72+6 45+5 1052 751t3 2726 68+4 10&l 70+-l 35+8 10+4 85f3 80+6 40+3
” UM-XC-17B cells were used in this experiment, They were harvested from culture, washed in serum-free MEM and incubated for 30 min at 4°C in serum-free MEM supplemented with 200 rig/ml BSA and the appropriate amount of each antibody. After the preincubation, 150 ul (5X lo4 cells) from each tube was added to duplicate microtiter wells coated with 10 ng of type IV collagen. Attachment and spreading were determined 30 min later. b Antibody A6.1 is directed against the trypsin-resistant 70 kD core of the TSP molecule. ’ Antibody C6.7 is directed against the platelet-binding domain of the TSP molecule. d Antibody A2.5 is directed against the heparin-binding domain of the TSP molecule. ’ Antibody D4.6 is directed against Ca2+-dependent structures of the TSP molecule. f The values shown are averages of four readings in each of duplicate dishes in a single experiment + SDS. The experiment was carried out twice, with similar results.
amounts of the antibodies. At the end of the preincubation period, the cells were transferred to microtiter wells coated with 10 pg of type IV collagen and incubated at 37°C. The percentage of attached and spread cells was assessed in the normal manner. Under this condition, monoclonal antibodies C6.7 and A6.1 were the most effective in inhibiting cell attachment and spreading (table 2). Monoclonal antibody A2.5 and the polyclonal antibodies were less effective and a fourth monoclonal antibody (D4.6) was the least effective of all. Effects of TSP on Other Cell Types In addition to the eight squamous carcinoma lines, TSP was also examined for ability to stimulate adhesion of other cell types (table 1). Cells that responded to TSP include keratinocytes (normal counterpart of squamous carcinoma cells), normal fibroblasts and the two tibrosarcoma cell lines. The human melanoma cells and the human glioma cells did not respond to this agent. In fact, TSP actually inhibited the attachment and spreading of these cells. Although they did not respond to TSP, the melanoma and glioma lines were all highly responsive to Exp CeNRes167(1986)
384 Varani et al.
laminin (table 1). The fibrosarcoma cells and tibroblasts responded to both TSP and laminin (table 1). In addition to the human cells, two mouse tumor lines were also examined for response to TSP. One of the lines (derived originally from a carcinogen-induced fibrosarcoma) attached but did not spread on plastic culture dishes when plated under serum-free conditions. Addition of TSP had no effect on this line although it was responsive to laminin (table 1). The second line (B16BL6 melanoma) attached rapidly and spread when plated under the same conditions. Like the human melanoma cells, the spreading of these cells was dramatically inhibited in the presence of TSP (table 1). Production
of TSP by Squamous Cnrcinoma
Cells
TSP production by the UM-SCC-1 1B cells was evaluated by incorporation of [35S]methionine into immunoreactive forms that were precipitated from both the cell lysates and chase media and analysed by SDS-PAGE (fig. 3). Analysis of the immunoprecipitates under non-reducing conditions revealed HMW bands in the chase media (fig. 3, lane A) and in cell lysates (fig. 3, B). These bands were not present when anti-bovine serum albumin was used in place of the anti-TSP (fig. 3, C). The HMW forms co-migrated with authentic TSP [l]. Upon reduction with 2-mercaptoethanol (fig. 3, D, E), the HMW forms were no longer present in either the cell lysates or the chase media. They were replaced by sharp bands with an apparent M, of 180 kD, corresponding to the TSP subunits [I]. These bands were not present when anti-BSA was used in place of anti-TSP (fig. 3,F). In a subsequent series of experiments, we compared three of the squamous carcinoma lines for biosynthesis of TSP. The cell lines examined include the UMSCC-11B (which was highly responsive to exogenous TSP), the UM-SCC-17B (which was moderately responsive) and the UM-SCC-22B (the least responsive of the eight lines). All three lines synthesized similar immunoreactive forms of TSP as indicated by SDS-PAGE analysis of the immunoprecipitates from the cell lysates under non-reducing conditions (fig. 4). However, quantitative differences between the three lines were suggested by the fluorograms. The UM-SCC-11B cells appeared to synthesize more TSP than the UM-SCC-17B cells which in turn appeared to synthesize more than the UM-SCC-22B cells. Similar differences were observed on the gels run under reducing conditions (not shown). To confirm the apparent differences between the three cell lines, we compared the amount of radioactivity in the cell lysates and chase media that was precipitated with anti-‘I’SP with the total amount precipitated with 10% trichloroacetic acid (TCA). Although all three lines incorporated similar amounts of [3’Slmethionine into TCA-precipitable material, the amount precipitated with anti-TSP was greatest in the UM-SCC-11B cells, followed by the UM-SCC-17B cells and then the UM-SCC-22B cells (table 3). Immunofluorescence studies were carried out next. For these, the cells were grown on glass coverslips for one day and then stained with the rabbit polyclonal anti-TSP in the viable state. When examined by fluorescence microscopy and Exp
Cdl
Res
167 (1986)
Thrombospondin-induced adhesion A8
C
DE
F A8
580Kd
385
CD
EF
580 Kd
ep & *
I
3
4
3. Analysis by SDS-PAGE of TSP immunoprecipitates from biosynthetically labeled UM-SCC1lB squamous carcinoma cells. Onex 10’ cells in loo-mm dishes were pulsed for 1 h with [sSS]methionine and chased for 4 h in the absence of labeled methionine. Cell lysates and chase media were divided in half and immunoprecipitated either with anti-TSP or with anti-BSA as a control. Half of each immunoprecipitate was reduced with 2-mercaptoethanol(2 %) and half was not. The immunoprecipitates were then analysed by SDS-PAGE on a 3-10% polyacrylamide gradient slab gel. Radioactive bands were visualized by fluorography after a 2-day exposure. Lanes A, B and Care non-reduced gels; D, E, Fare reduced gels. A, Chase medium precipitated with anti-TSP; B, cell lysate precipitated with anti-TSP; C is the cell lysate precipitated with anti-bovine serum albumin; D, Chase medium precipitated with anti-TSP; E, F, Cell lysates precipitated with anti-TSP (I?) and with anti-BSA (F). Fig. 4. Analysis by SDS-PAGE of TSP immunoprecipitates from biosynthetically labeled UM-SCCllB, UM-SCC-17B and UM-SCC-22B cells. Onex 10’ cells in loo-mm dishes were pulsed for 1 h with [35S]methionine and chased for 4 h in the absence of labeled methionine. Cell lysates were divided in half and immunoprecipitated with either anti-TSP or anti-BSA as a control. The immunoprecipitates were then analysed by SDS-PAGE under non-reducing conditions on a 3-10% polyacrylamide gradient slab gel. Radioactive bands were visualized by fluorography after a 2-day exposure. Lanes A, B are lysates of UM-SCC-1lB cells precipitated with anti-TSP (A) and anti-BSA (B). C, D, Lysates of UM-SCC-17B cells precipitated with anti-TSP (C) and anti-BSA (D). E, F, Lysates from UM-SCC22B cells precipitated with anti-TSP (0 and anti-BSA (F). Fig.
Table 3. Quantitative analysis of TSP immunoprecipitates from biosynthetically labeled squamous carcinoma cells Proportion TSP Cell line
Cell lysate (So)
Chase medium (96)
UM-SCC-1lB UM-SCC-17B UM-SCC-22B
0.09F0.01 0.01+0.01 Not detected
2.60+0.22 0.41f0.09 0.08iO.02
Onex 10’ cells in 100 mm dishes were pulsed for 1 h with [35S]methionine and chased for 4 h in the absence of labeled methionine. Cell lysates and chase media were divided in three parts. One part was precipitated with 10% (final) TCA. The other two parts were immunoprecipitated with anti-TSP and anti-BSA. The amount of radioactivity precipitated with anti-BSA was subtracted from the amount precipitated with anti-TSP. The remaining value was compared with the total amount precipitated with 10% TCA and expressed as a percentage of the total. Exp Cd! Res 167 (IYR6)
Fig. 5. Immunofluorescence staining of the UM-SCC-1IB cells in the viable state. Cells grown on coverslips for one day were gently washed in PBS and then stained at 4°C with a 1 : 20 dilution of antiTSP (panel A) or a 1 : 20 dilution of anti-BSA (B), followed by a 1 : 50 dilution of fluorescein isothiocyanate conjugated swine anti-rabbit IgG. Photographs are 5 min exposures at X400.
simultaneously by phase-contrast microscopy, it could be seen that more than 95 % of the UM-SCC-11B cells stained with the rabbit polyclonal anti-TSP (table 4). Staining appeared punctate and distributed evenly over the entire surface of the cell (fig. 5). There was little variability from area to area on the coverslip. The UM-SCC-17B cells also stained with the anti-TSP antibodies. The pattern of staining was similar to that seen with the UM-SCC-I IB cells except that a smaller proportion of the cells stained (table 4). Likewise, the staining pattern of the UMSCC-22B was similar to that of the other two squamous carcinoma lines. Howev-
Table 4. Immunojluorescence staining of human tumor cells in the viable state with anti-TSP
Cell line Squamous carcinoma UM-XC-1lB UM-SCC-17B UM-SCC-22B Fibrosarcoma UM-SCC-IA Melanoma UM-MEL-l SK-MEL-10 SK-MEL-28
Proportion staining cells” (%I
92+10 36+5 5+1 87+6 4 4 <5
’ The cells were examined by phase-contrast microscopy for total cells and by fluorescence microscopy for fluorescent cells. Values are averages f SDS based on five fields per cell line in a single experiment. Each line was examined three or more times, with similar results. Exp CellRes 167(1986)
Thrombospondin-induced
adhesion
387
ABCDEF
580 Kd Fig. 6. Analysis by SDS-PAGE of TSP immunoprecipitates from biosynthetically-labeled UM-WC-1 lB, UMMEL-l and SK-MEL-28 cells. Onex 10’ cells in 100 mm dishes were pulsed for 1 h with [‘5S]methionine and chased for 4 h in the absence of labeled methionine. Cell lysates were divided in half and immunoprecipitated either with anti-TSP or anti-BSA as a control. The immunoprecipitates were then analysed by SDS-PAGE under non-reducing conditions on a 3-10 % polyacrylamide gradient slab gel. Radioactive bands were visualized by fluorography after a f-day exposure. Lanes A, B, lysates of UM-SCC-1lB cells precipitated with anti-TSP (A) and anti-BSA (B). C, D, Lysates of UM-MEL-l cells precipitated with anti-TSP (C) and anti-BSA (D). E, F, Lysates from SK-MEL-28 cells precipitated with anti-TSP (,I?)and anti-BSA (F).
er, only a very small proportion of the cells (5%) stained (table 4). None of the three lines showed any staining when affinity-purified anti-BSA was used instead of the anti-TSP. Examination
of Melanoma
Cells for TSP Production
Two of the human melanoma cell lines (UM-MEl-1 and SK-MEL-28) were examined for TSP biosynthesis. When these cells were examined under exactly the same conditions as the squamous carcinoma cells, there was no evidence of TSP production (fig. 6). The inability to detect TSP biosynthesis was not due to a failure of the melanoma cells to incorporate [35S]methionine. These cells incorporated about the same amount of the labeled methionine per cell as did the squamous carcinoma cells. Furthermore, when cell lysates and chase media from [35Slmethionine-labeled melanoma cells and squamous carcinoma cells were examined for laminin, comparable amounts were seen (not shown). Three of the melanoma lines were stained with the polyclonal anti-TSP. With none of these lines was there evidence of distinct staining, though an occasional faintly-stained cell could be seen (table 4). Nor did the melanoma cells stain when anti-bovine serum albumin was used instead of the anti-TSP, but these cells demonstrated bright punctate staining when affinity-purified anti-laminin was used as the primary reagent. Thus, the lack of staining observed with the antiTSP was probably not due to any innate inability of these cells to bind rabbit antibodies. In contrast, the fibrosarcoma cells resembled the squamous carcinoma cells in that a large proportion of these cells stained brightly with anti-TSP (table 4). Exp Cell Res 167 (1986)
388 Varani et al.
DISCUSSION A number of extracellular matrix components including laminin, fibronectin and collagens of various types are known to function as cell attachment factors [ll-161. The data presented here indicate that TSP, another large extracellular matrix glycoprotein [3-51, is an adhesion factor for human squamous carcinoma cells. It induced attachment and spreading of several cell lines on plastic culture dishes and on dishes coated with 50 ug of collagen (including a preparation of type I collagen from bovine skin and a preparation of type IV ‘basement membrane’ collagen obtained from the EHS sarcoma). While the in vivo function of TSP has yet to be demonstrated, the finding that it can promote attachment and spreading of cells in vitro suggests that it may perform a similar function in vivo. The ability of TSP to promote adhesion was not unique to squamous carcinoma cells. It also promoted adhesion of human keratinocytes (the normal counterpart cell of the squamous carcinomas) as well as normal human fibroblasts and malignant fibrosarcoma cells. In contrast to its effect on these cells, TSP did not stimulate adhesion in a number of human melanoma or glioma cell lines. In fact, TSP actually inhibited attachment and spreading in several of these lines. It is interesting that the human melanoma and glioma lines responded in the adhesion assay when laminin was used as the stimulating agent, while the squamous carcinoma cells were much less responsive or non-responsive to this protein. Thus, there appears to be specificity in the adherence response to the large glycoprotein matrix components. The squamous carcinoma cells not only responded to exogenously added TSP, but also synthesized it. The synthesized TSP was secreted into the culture medium and bound to the cell surface. The TSP synthesized by the squamous carcinoma cells may serve as an endogenous adhesion factor for these cells. Rabbit polyclonal antibodies and mouse monoclonal antibodies to TSP were able to partially inhibit cell attachment to collagen-coated dishes when the cells were pretreated with the antibodies. Furthermore, there was a direct relationship between the amount of TSP synthesized by a given cell line and the adhesiveness of that line in the absence of exogenous TSP (compare table 1 with fig. 4 and tables 3 and 4). It is interesting that among the three squamous carcinoma lines examined in detail, the cell line that synthesized the greatest amount of TSP and was the most adhesive (UM-SCC-11B) also formed the most undifferentiated tumors when injected into athymic mice. The cell line which synthesized the least amount of TSP and was the least adhesive (UM-SCC-22B), formed the most welldifferentiated tumors when injected into athymic mice. How TSP facilitates cell-substrate adhesion is not known at present and a number of possibilities exist. It may be that the secreted TSP binds to the extracellular matrix and then interacts with the cell through TSP-binding moieties on the cell surface. Specific receptors for TSP have not yet been described but it has been shown that TSP can bind to sulfated glycolipids, which are present in cell membranes [30]. Other cell surface proteins such as fibronectin, which are Em
Cell
Res
I67 (IYR6j
Thrornbospondin-induced
adhesion
389
known to interact with TSP [31], may also serve as binding sites for the secreted TSP. If this is correct, then differences in responsiveness to TSP may result from differences in fibronectin synthesis or surface expression. Alternatively, the TSP synthesized by the squamous carcinoma cells may be inserted directly into the cell surface and then facilitate interactions with the substratum. While we cannot distinguish between the various possibilities at present, the fact that certain cell lines (notably, the UM-SCC-17B) are heterogeneous with regard to surface TSP expression and with responsiveness to exogenous TSP may allow us to probe these questions. Studies are currently in progress to separate and clone variant populations from the UM-SCC-17B line. These populations will then be used in an effort to determine if the cells that synthesize TSP are, in fact, the same cells that respond to it. Likewise, studies are underway to determine if there is a correlation between synthesis of TSP by the various squamous carcinoma lines and synthesis of other surface/extracellular matrix proteins such as tibronectin and laminin. These studies should help to determine how TSP acts to facilitate cell-substrate interactions. In summary, these studies demonstrate that TSP stimulates attachment and spreading in a number of cell lines derived from human squamous cell carcinomas. TSP thus joins a number of other large glycoproteins of the extracellular matrix which are known to serve as adhesion factors for various types of cells. This study was supported in part by grants CA36132. CA28564 and 2 SO7 RR05384-25 from the USPHS and by grants BC-512 and IM-432 from the American Cancer Society. T. E. C. is the recipient of USPH RCDA No CA0062 I.
REFERENCES 1. Dixit, V M, Grant, G A, Santoro, S A & Frazier, W A, J biol them 259 (1984) 10000. 2. Coligan, J E & Slayter, H S, J biol them 259 (1984) 3944. 3. Baenziger, N L, Brodie, G N & Majerus, P W, J biol them 247 (1972) 2723. 4. Phillips, D R, Jennings, L K & Prasanna, H R, J biol them 255 (1980) 11629. 5. Gartner, T K, Gerrard, J M, White, J G & Williams, D C, Blood 5 (1981) 153. 6. McPherson, J, Sage, H & Bornstein, P, J biol them 256 (1981) 11330. 7. Mosher, D F, Doyle, M J & Jaffe, E A, J cell biol 93 (1982) 343. 8. Raugi, G J, Mumby, S M, Abbott-Brown, D & Bomstein, P, J cell biol 95 (1982) 351. 9. Jaffe, E A, Ruggiero, J T, Leung, L L K, Doyle, M J, McKeown-Longo, P J & Mosher, D F. Proc natl acad sci US 80 (1983) 998. 10. Sage, H, Farin, F M, Striker, G E & Fisher, A B, Biochemistry 22 (1983) 2148. 11. Pearlstein, E, Gold, L I & Garcia-Pardo, A, Mol cell biochem 29 (1980) 103. 12. Terranova, V P, Rohrbach, D & Martin, G R, Cell 22 (1980) 719. 13. Vlodavsky, I & Gospodarowicz, D, Nature 289 (1981) 304. 14. ROOS, E, Biochim biophys acta 738 (1984) 263. 15. Grinnell, F & Bennett, M H, J cell sci 48 (1981) 19. 16. Schor, S L, Schor, A M & Bazill, G W, J cell sci 49 (1981) 299. 17. Carey, T E. Takahashi, T, Resnick, L A, Oettgen. H F & Old, L J, Proc natl acad sci US 73 (1976) 3278. 18. Black, P M, Komblith, P L, Davison, P F, Liszczak, T M, Merk, L P, Smith, B H, McKeever, P E & Quindlen, E A, J neurosurg 56 (1982) 62. 19. McKeever. P E, Fligiel, S E G, Varani, J, Hudson, J L, Smith. D, Castle, R L & McCoy, J P. Intj cancer 37 (1986) 867. 20. Kimmel, K A & Carey, T E, Cancer res 46 (1986) 3614.
390 Varani et al. 21. Varani, J, Lovett, E J, McCoy, J P, Shibata, S, Maddox, D, Goldstein, I J & Wicha, M, Am j path01 111 (1983) 27. 22. Dixit, V M, Haverstick, D M, O’Rourke, K M, Hennessy, S W, Grant, G A, Santoro, S A & Frazier, W A, Proc natl acad sci US 82 (1985) 3844. 23. - Biochemistry 24 (1985) 4270. 24. Galvin, N J, Dixit, V M, O’Rourke, K M, Santoro, S A, Grant, G A & Frazier, W A, J cell biol 101 (1985) 1434. 25. Dixit, V M, O’Rourke, K M, Grant, G A, Santoro, S A & Frazier, W A, J biol them 261 (1986) 1962. 26. Timpl, R, Rohde, H, Robey, P G, Rennard, S 1, Foidart, J M & Martin, G R, J biol them 254 (1979) 9933. 27. Ronnett, G V, Knutson, V P, Kohnski, R A, Simpson, T L & Lane, M D, J biol them 259 (1984) 4566. 28. Ruddon, R W, Hanson, C A & Addison, N J, Proc natl acad sci US 76 (1979) 5173. 29. Laemmli, U K, Nature 227 (1970) 680. 30. Roberts, D D, Haverstick, D M, Dixit, V M, Frazier, W A, Santoro, S A & Ginsburg, V, J biol them 260 (1985) 9405. 31. Leung, L L K & Nachman, R L, J clin invest 70 (1982) 542. Received April 1, 1986 Revised version received June 16, 1986
Exp Cell Res 167 (19861
Prmted
I” Sweden
Cell Research 167 (1986) 376390
Thrombospondin-Induced Attachment and Spreading of Human Squamous Carcinoma Cells JAMES VARANI, ‘. * VISHVA M. DIXIT.’ SUZANNE E. G. FLIGIEL,4 PAUL E. McKEEVER’ and THOMAS E. CAREY’ ‘Department of Pathology, ‘Depurtmrnt c?f Otor~lino/ar~tl~(~l~)~~, Uniuersity Michigan Medicul School, Ann Arbor. MI 48109, ‘Department of Pathology, Division of Luborutory Medicine, Wushington Uniuersitx School of Medicine, St Louis, MO 631 IO. und 4Depurtment of‘Putholog~. A//en Purk VAMC Wuyne Stute University. Detroit. MI 48101, USA
of
Thrombospondin (TSP) induced the attachment and spreading of human squamous carcinoma cells on plastic culture dishes and dishes coated with type I or type IV collagen. Increased adhesion was detected as early as IS min after treatment. Dose-response studies indicated that l-5 ug of TSP per 35 mm (diameter) culture dish was sufficient to induce a response and that a half-maximal response occurred at 10 ug of TSPidish. The squamous carcinoma cells synthesized TSP as indicated by biosynthetic labeling experiments. TSP was secreted (or shed) into the culture medium by these cells and also became bound to the cell surface. TSP also promoted adhesion of human keratinocytex. fibroblasts and fibrosarcoma cells but did not induce attachment or spreading of human melanoma or glioma cells, 0 1986 Academic PRS. IW although these cells did respond to laminin.
Thrombospondin (TSP) is a high molecular weight (HMW) glycoprotein (M,=580 kD) which is released from the a-granules of platelets during activation [ 1, 21. In the presence of divalent cations, the released TSP becomes associated with the platelet surface and acts as a mediator of aggregation [3-51. TSP is also synthesized by various types of normal cells in culture [6-lo] and is deposited into the extracellular matrix produced by these cells. Since several other components of the extracellular matrix serve as adherence factors for cells [ 1I-161, it is possible that TSP may also be involved in cell-cell or cell-substratum adhesion. In the present study we examined TSP for its ability to promote cell-substrate adhesion of various human tumor cells. It stimulated adhesion of several squamous carcinoma and fibrosarcoma cell lines as well as human keratinocytes and fibroblasts. In contrast, TSP did not promote adhesion of human melanoma or glioma cells although these cell lines responded to laminin. MATERIALS
AND METHODS
Cells The human tumor cells examined in this study include eight squamous cell carcinoma lines. two tibrosarcoma lines, four melanoma lines and four glioma lines. The squamous cell carcinoma lines * To whom offprint
requests should be addressed. Copyright 0 1986 hy Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827186 $03.W
Thrombospondin-induced
adhesion
377
(designated UM-XC-) were derived from tumor specimens obtained from 5 different individuals. The UM-SCC-1 cell line was derived from a local recurrence of a tumor in the floor of the mouth of a 73year-old male, and the UM-XC-1 1A line from a biopsy specimen of laryngeal cancer in a previously untreated 65-year-old male patient. UM-XC-1lB was established from tumor tissue from a total laryngectomy performed 6 weeks after the biopsy. The UM-SCC-14C cell line was established from a tumor in the neck of a 64-year-old female patient. This tumor was a local recurrence of a tumor originating in the floor of the mouth. The UM-SCC-17A and 17B cell lines were both established from a persistent cancerous lesion of the larynx and a metastatic tumor in a cervical lymph node from a 48year-old female patient who underwent surgery after an unsuccessful attempt to control her primary cancer of the larynx with radiation therapy. Cell lines UM-SCC-22A and 22B were established respectively from a primary cancer of the hypopharynx and cervical lymph node metastasis in a 59year-old female patient. All of the squamous carcinoma lines formed tumors in nude mice. Histological examination of three tumors (those produced by UM-SCC-1 lB, UM-SCC-17B and UM-SCC-22B cells) showed that the UM-SCC-1 1B cells produced the least differentiated tumors and that the UMSCC-22B cells produced the best differentiated tumors. The tumors produced by the UM-SCCl7B cells were intermediately differentiated. The two tibrosarcoma lines, UM-FS-IA and UM-FS-lB, were established in culture, respectively, from a primary and recurrent malignant fibrosarcoma that arose in the facial musculature of a 14-yearold male. The cell lines have fibroblastic morphology, express non-polymorphic human HLA histocompatibility antigens and produce rapidly-growing tumors in nude mice that recreate the histology of the original neoplasms. One of the malignant melanoma lines (UM-MEL-l) was derived from a recurrent lesion in the neck of a 34-year-old male with a past history of malignant melanoma arising on the left auricle. A second melanoma line (UM-MEL-2) was obtained from a heavily pigmented mass that arose in the right supraclavicular region of a 19-year-old male in whom no primary melanoma was found. The isolation and characterization of the other two melanoma lines (SK-MEL-IO and SKMEL-28) have been described previously [17]. The isolation and characterization of the human glioma lines have also been previously described [18, 191. In addition to these tumor cell lines, primary human keratinocytes and fibroblasts obtained from explanted foreskins were used. The tibroblasts and keratinocytes were separated from each other by differential trypsinization as described by Kimmel & Carey [20]. In addition to these cells, human lung fibroblasts (MRC-S), obtained from Flow Laboratories (McLean, Va.) were also used. Finally, murine tibrosarcoma cells and murine melanoma cells were also examined for response to TSP. The fibrosarcoma cells were available from previous studies in our laboratory [21] and the melanoma cells (Bl6-BL6 line) were provided by Dr J. Niederkorn (Department of Ophthalmology, University of Texas; Dallas, Tex.). All of the human lines were grown in monolayer culture on Minimal Essential Medium of Eagle (MEM) supplemented with 15% fetal bovine serum (FBS), non-essential amino acids, 100 U/ml of penicillin and 100 @ml of streptomycin. The cells were grown at 37°C in the presence of 5 % COZ. The murine cells were maintained on MEM supplemented with 10% FBS and antibiotics.
Reagents TSP was purified from the supematant fluid of thrombin-activated platelets as described [l]. The purified TSP migrated as a single protein band with an apparent MW of 180 kD when examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The production and characterization of the monoclonal antibodies directed against TSP have been described previously [22-251. Monoclonal antibodies were purified from ascites fluid by ammonium sulfate precipitation followed by affinity chromatography on protein A Sepharose. Purity was determined by SDS-PAGE. The rabbit polyclonal antibody against TSP was purified by afftnity chromatography on protein A Sepharose. Specificity for TSP was confirmed by solid-phase radio-immunoassay performed as described [23] with immobilized antibodies and TSP iodinated with the Bolton-Hunter reagent or NA’*‘I and iodobeads. Laminin was used in certain experiments. The laminin was prepared from the Englebreth-HolmSwarm (EHS) tumor by the method of Timpl [26]. Purity of the laminin was assessed by SDS-PAGE and by enzyme-linked immunosorbent assay (ELISA) [21]. When examined on a 7% SDS-polyacrylamide gel under reducing conditions, only two protein bands (at 200 and 400 kD) were seen. The laminin reacted by ELISA with monospecific polyclonal anti-laminin antibodies at dilutions up to Exp Cell Res 167 (1986)
378 Varani et al. 1 : lo6 but did not react with polyclonal antibodies to type IV collagen or fibronectin. The laminin prepared in this manner stimulated adherence in a highly-sensitive line of murine fibrosarcoma cells at concentrations as low as 0.5 ug/35 mm (diameter) dish [21].
Adhesion Assay Cell attachment and spreading on plastic culture dishes and collagen-coated culture dishes was used as a measure of adhesion. The collagen-coated dishes were prepared by pretreating 35 mm (diameter) dishes with 50 ug of a Type I collagen preparation from bovine skin (Vitrogen-100; Collagen Carp, Palo Alto, Calif.) or 50 ug of a Type IV collagen preparation from the EHS sarcoma (Bethesda Research Laboratories; Gaithersburg, Md.). The dishes were incubated overnight with the collagen solutions in 0.1 N acetic acid. The collagen film was deposited onto the dish surface as the acid evaporated. The dishes were then washed twice in serum-free MEM and were ready for use. The adhesion assay was carried out by adding 1 ml of MEM supplemented with 200 ug/ml of bovine serum albumin (BSA) to each dish and incubating them for 15 min at 37°C. The cells were harvested from culture by trypsinization, washed two times in serum-free MEM and resuspended at 5x lo5 cells in 1 ml of MEM supplemented with 200 ug of bovine serum albumin per ml. The cells were mixed with the appropriate amount of TSP, incubated with the TSP in solution for 5 min and then added to the dishes. The dishes were incubated at 37°C. At various times later, the non-attached cells were removed and counted with an electronic particle counter. The dishes were washed and then flooded with 2% glutaraldehyde. The percentage of spread cells was determined by examining the dishes using a microscope with a calibrated grid in the eyepiece. In some experiments, the dishes were precoated with TSP. When this was done, the appropriate amount of TSP was added to the culture dishes in 1 ml of serum-free MEM and incubated at 37°C for 1 h. The dishes were then washed twice in serum-free MEM to remove the non-adsorbed TSP and used.
Biosynthetic Labeling The cells were grown in loo-mm (diameter) culture dishes to an approximate density of 1x 10’ cells per dish. The cells were washed and incubated for 30 min in methionine-free, serum-free minimal essential medium (Flow Laboratories; McLean, Va.) followed by a 60-min incubation in the same medium supplemented with 500 uCi per dish of [35S]methionine (1000-l 400 uCi/umole; NEN, Boston, Mass.). After the 60-min biosynthetic pulse, the cells were again washed and incubated for a further 4 h in complete medium. The 4-h chase cells were lysed in a solution of phosphate-buffered saline (PBS) containing three detergents (1% Triton X-100, 0.5 % sodium deoxycholate and 0.1% SDS; all obtained from Sigma Chemical Co.) and protease inhibitors including 20 mM EDTA, 5 mM N-ethyl maleimide, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 10 ul/lO ml of a protease inhibitor cocktail containing: leupeptin, 1 mg/ml; antipain, 2 mg/ml; benzamidine, 10 mg/ml; aprotinin, 10000 kallikrein-inactivating units/ml; chymostatin, 1 mgiml; and pepstatin, 1 mg/ml, as described by Ronnett et al. [27] in studies on the insulin receptor. All of the protease inhibitors were obtained from Sigma Chemical Co. The 4-h chase media were also supplemented with the detergent/ protease inhibitor mixture by adding one-fourth volume of a fivefold concentrated solution. The cell lysates and chase media were frozen at -8O”C, thawed, and clarified by ultracentrifugation (37000 g for 60 min). Immunoreactive TSP was precipitated with a 1 : 100 dilution of the rabbit anti-TSP and Protein A-Sepharose (Sigma Chemical Co.) according to the protocol of Ruddon et al. [28]. Affnitypurified anti-bovine serum albumin served as a control. The washed immunoprecipitates were divided in half and eluted with boiling (5 min) in twofold concentrated Laemmli SDS-PAGE sample buffer [29], either with or without 2 % 2-mercaptoethanol. The immunoprecipitated material was fractionated on a 3-10% polyacrylamide gel employing the Laemmli system. Radioactive bands were visualized by fluorography with En3Hance (NEN), exposing the dried gels to X-ray film (Kodak XAR-2) for 2 days.
Immunofluorescence The tumor cells were examined for anti-TSP binding by indirect immunofluorescence one day after plating. For this, 2.0x 10’ cells from each line were seeded onto 20x20 mm glass coverslips. The culture medium consisted of MEM supplemented with 10% normal rabbit serum. Rabbit serum was used in place of FBS to present possible cross-reactions between the rabbit anti-TSP and TSP present in FBS. At the time of staining, the culture medium was removed and the cells were gently washed Exp Cell Res 167 (1986)
Thrombospondin-induced
adhesion
379
three times in cold PBS. All subsequent steps were carried out at 4°C. The cells were stained initially with a 1 : 20 dilution of the rabbit anti-TSP or anti-bovine serum albumin (45 min) and then washed three times. They were then stained with a 1 : 50 dilution of fluorescein-conjugated swine anti-rabbit serum (45 min) obtained from Accurate Scientific and Chemical Company (Westbury, N. Y.). Following three additional rinses in PBS, the coverslips were placed on glass slides and viewed immediately by standard fluorescence microscopic techniques.
RESULTS Stimulation
of Human Squamous Carcinoma
Cell Adhesion
by TSP
The eight human squamous carcinoma cell lines were examined for adhesion to 35mm (diameter) plastic culture dishes which had been preincubated for 15 min with 1 ml of serum-free MEM supplemented with 200 ug of BSA. The addition of 20 ug of TSP to the cell suspensions before addition to the dishes stimulated attachment and spreading of all eight lines. Quantitative data is shown in table 1 and the response of one cell line is shown graphically in fig. I. Two of the cell lines (UM-SCC-1 and UM-SCC-1 1B demonstrated a dramatic response; in four of the lines (UM-SCC-llA, UM-SCC-14C, UM-SCC-17A and UM-SCC-17B), a moderate response was seen and in two lines (UM-SCC-22A and UM-SCC-22B), the response was minimal. It is interesting to note that there was a relationship between the percentage of cells which attached and spread in the absence of stimulation and the percentage of cells which responded to TSP (table 1). TSP also stimulated adhesion of the squamous carcinoma cells when it was preadsorbed onto the culture dishes. The cells responded somewhat faster under this condition (not shown), but the overall relationship among the cells was the same as that shown in table 1. When laminin was used as the stimulating agent in place of TSP, the squamous carcinoma cells were much less responsive or even nonresponsive (table 1). The ability of TSP to stimulate adhesion of the squamous carcinoma lines was not limited to plastic culture dishes. When dishes coated with 50 ug of either type I (bovine skin) or type IV (basement membrane) collagen were used as the substrate, the addition of 20 ug of TSP significantly increased the rate of attachment and spreading on these substrates as well. This is shown in fig. 2 with the UM-SCC-11B cells. The other squamous carcinoma lines responded similarly (not shown). Kinetic studies and dose-response studies were carried out next. As shown with the UM-SCC-1 1B cells in fig. 2, the effects of TSP were seen as early as 15 min after plating. Differences between the treated and control cells persisted for several hours, although the untreated cells also attached and spread after prolonged incubation (4-24 h). The time course of the response to TSP was similar with the other squamous carcinoma cell lines to that shown in fig. 2 with the UMSCC-11B cells. Dose-response studies indicated that 1.0-5.0 ug of TSP per reaction was sufficient to elicit a response and that a half-maximal response occurred at approx. 10 ug per reaction. This was true for all eight of the lines. Exp Cell Res 167 (1986)
380 Varani et al. Table 1. Stimulation of adhesion by thrombospondin Proportions attached and spread
Cell line Human squamous carcinoma UM-SCC-1 UM-XC-I IA UM-XC-1 IB UM-SCC-14A UM-SCC-17A UM-SCC-17B UM-SCC-22A UM-SCC-22B Normal keratinocytes Human fibrosarcoma UM-FS-IA UM-FS-1B Human foreskin tibroblasts Human lung tibroblasts (MRC-5) Human melanoma UM-MEL-l UM-MEL-2 SK-MEL-10 SK-MEL-28 Human glioma UMl U138MG LM UM6 Mouse fibrosarcoma Mouse melanoma
Untreated (%I
Thrombospondin (25 KS) ez)
(25 PCs)
Laminin
lOf3 12f3 1524 4*1 7f6 lOf2 If2 lf3 2+1
72+6 45+5 75+6 40f6 3023 48f4 12f6 to+2 3526
2+_1 2056 22+7 8fl 5+4 222 2*1 1+2 10+3
IO?1 61f-1 30f8 2426
79f8 7o+s 7026 89+8
75+6 80* 10 7215 86?8
5fl 0 35f4 40f5
0 0 0 15+_5
85+14 5Oi6 60+4 85+8
27+4 38f5 9+8 20* 10 0 75f6
0 24f3 14+6 18f7 0 5*1
8228 85+6 80f7 8528 12f5 602 10
(%z)
The percentage of cells attached and spread 1 h after plating was determined as described in Methods. The values represent averages + SDS based on four readings in each of duplicate dishes in a single experiment. Each cell type was examined two or more times with similar results.
Since the kinetics of the response to TSP and the dose responses were similar among the squamous carcinoma lines, the major difference was in the number of cells which ultimately responded. Rabbit polyclonal anti-TSP antibodies and mouse monoclonal antibodies to the various domains of TSP were examined for ability to inhibit TSP-induced attachment and spreading. In the first set of experiments 25 ug of TSP was mixed with 50 or 100 ug of the various antibody preparations and incubated for 1 h at 37°C in 35mm (diameter) plastic culture dishes (in a total volume of 0.35 ml). After the incubation period, the dishes were washed twice and the carcinoma cells (UMSCC-17B) were added in 1 ml of MEM supplemented with 200 ug/ml of bovine serum albumin. Two hours later, cell attachment and spreading were quantitated Exp Cell Res 167 (1986)
Thrombospondin-induced
adhesion
38 1
I. Phase-contrast photomicrographs of human squamous carcinoma cells CUM-XC-I IS) after 30 min of incubation on plastic culture dishes in the presence (A) and in the absence (B) of TSP. The photographs were taken after the dishes had been washed to remove the unattached cells and flooded with 2 % glutaraldehyde. x460.
Fig.
382 Varani et al. Type IV Collagen
Type I Collagen
?
;;F
i
IF
Plastic
EL
15 30 45
15 30 45
15 30 45
Tlme Imid
Tlme (mid
Tlme (mid
Fig. 2. Time course of TSP-induced adhesion of human squamous carcinoma cells (UM-SCC-11B) to type I collagen-coated dishes, type IV collagen-coated dishes, and plastic dishes. The values shown represent the average percentage attached and spread on the substrates in the absence of TSP (0-O) and in presence of 20 ug of TSP (O-O). The values are based on four readings in each of duplicate dishes in a single experiment. The SDS were with 15% of the averages. The experiment was repeated three times with similar results.
in the usual fashion. In the presence of 50 or 100 pg of the rabbit polyclonal antibody, attachment and spreading was reduced by 79+6 % and 81f6 % respectively. Monoclonal antibody A 6.1 (directed against the 70 kD trypsin-resistant core of the TSP molecule) also inhibited adhesion when added at 100 yg per reaction (882 1% inhibition). in contrast to these results, the antibodies directed against the heparin-binding domain (A2.5) and the platelet-binding domain (C6.7) failed to inhibit TSP-mediated adhesion of the squamous carcinoma cells. The antibodies that inhibited TSP-induced attachment and spreading (seen at 2 h) had no effect on the proportion of cells attached and spread when examined at 20 h. When a second squamous carcinoma cell line (UM-SCC-11A) was used, very similar results were obtained (not shown). In a second set of experiments, the TSP and the antibodies were pre-adsorbed onto the substrate sequentially. Initially, 2.5 ug of TSP was added in 1 ml of serum-free MEM to 35 mm (diameter) dishes and incubated for 1 h at 37°C. The non-attached TSP was removed and the dishes flooded with 1 ml of MEM containing 200 ug of bovine serum albumin per ml. The various antibodies (100 ug) were added next and incubated for an additional 1 h at 37°C. The excess antibodies were removed and the dishes washed twice in serum-free MEM. The UM-SCC-17B cells (5x 105)were then added to the dishes and the percentage of attached and spread cells determined in the normal manner. Again, the polyclonal antibody and monoclonal antibody A6.1 partially inhibited attachment and spreading (45+5 % and 34f6% inhibition respectively) while the other monoclonal antibodies did not. The same antibodies were next examined for ability to inhibit endogenous adhesion (in the absence of added TSP) of the UM-SCC-17B cells. For these experiments, 5x lo4 cells were preincubated for 1 h at 4°C in tubes containing 0.2 ml of serum-free MEM supplemented with 200 pg of BSA per ml and various Exp Cell Res 167 (1986)
Thrombospondin-induced adhesion
383
Table 2. Inhibition of squamous carcinoma cell attachment and spreading by pretreatment of the cells with antibodies to TSP” Treatment
Percent attached and spreadf
None 10 ng rabbit polyclonal antibody 25 ng rabbit polyclonal antibody 1 ug monoclonal antibody A6.1 b 10 ug monoclonal antibody A6.1 1 ug monoclonal antibody C6.7’ 10 ng monoclonal antibody C6.7 1 ug monoclonal antibody A2Sd 10 ng monoclonal antibody A 2.5 25 ug monoclonal antibody A 2.5 10 ug monoclonal antibody D4.6’ 40 ng monoclonal antibody D4.6 75 ng monoclonal antibody D4.6
72+6 45+5 1052 751t3 2726 68+4 10&l 70+-l 35+8 10+4 85f3 80+6 40+3
” UM-XC-17B cells were used in this experiment, They were harvested from culture, washed in serum-free MEM and incubated for 30 min at 4°C in serum-free MEM supplemented with 200 rig/ml BSA and the appropriate amount of each antibody. After the preincubation, 150 ul (5X lo4 cells) from each tube was added to duplicate microtiter wells coated with 10 ng of type IV collagen. Attachment and spreading were determined 30 min later. b Antibody A6.1 is directed against the trypsin-resistant 70 kD core of the TSP molecule. ’ Antibody C6.7 is directed against the platelet-binding domain of the TSP molecule. d Antibody A2.5 is directed against the heparin-binding domain of the TSP molecule. ’ Antibody D4.6 is directed against Ca2+-dependent structures of the TSP molecule. f The values shown are averages of four readings in each of duplicate dishes in a single experiment + SDS. The experiment was carried out twice, with similar results.
amounts of the antibodies. At the end of the preincubation period, the cells were transferred to microtiter wells coated with 10 pg of type IV collagen and incubated at 37°C. The percentage of attached and spread cells was assessed in the normal manner. Under this condition, monoclonal antibodies C6.7 and A6.1 were the most effective in inhibiting cell attachment and spreading (table 2). Monoclonal antibody A2.5 and the polyclonal antibodies were less effective and a fourth monoclonal antibody (D4.6) was the least effective of all. Effects of TSP on Other Cell Types In addition to the eight squamous carcinoma lines, TSP was also examined for ability to stimulate adhesion of other cell types (table 1). Cells that responded to TSP include keratinocytes (normal counterpart of squamous carcinoma cells), normal fibroblasts and the two tibrosarcoma cell lines. The human melanoma cells and the human glioma cells did not respond to this agent. In fact, TSP actually inhibited the attachment and spreading of these cells. Although they did not respond to TSP, the melanoma and glioma lines were all highly responsive to Exp CeNRes167(1986)
384 Varani et al.
laminin (table 1). The fibrosarcoma cells and tibroblasts responded to both TSP and laminin (table 1). In addition to the human cells, two mouse tumor lines were also examined for response to TSP. One of the lines (derived originally from a carcinogen-induced fibrosarcoma) attached but did not spread on plastic culture dishes when plated under serum-free conditions. Addition of TSP had no effect on this line although it was responsive to laminin (table 1). The second line (B16BL6 melanoma) attached rapidly and spread when plated under the same conditions. Like the human melanoma cells, the spreading of these cells was dramatically inhibited in the presence of TSP (table 1). Production
of TSP by Squamous Cnrcinoma
Cells
TSP production by the UM-SCC-1 1B cells was evaluated by incorporation of [35S]methionine into immunoreactive forms that were precipitated from both the cell lysates and chase media and analysed by SDS-PAGE (fig. 3). Analysis of the immunoprecipitates under non-reducing conditions revealed HMW bands in the chase media (fig. 3, lane A) and in cell lysates (fig. 3, B). These bands were not present when anti-bovine serum albumin was used in place of the anti-TSP (fig. 3, C). The HMW forms co-migrated with authentic TSP [l]. Upon reduction with 2-mercaptoethanol (fig. 3, D, E), the HMW forms were no longer present in either the cell lysates or the chase media. They were replaced by sharp bands with an apparent M, of 180 kD, corresponding to the TSP subunits [I]. These bands were not present when anti-BSA was used in place of anti-TSP (fig. 3,F). In a subsequent series of experiments, we compared three of the squamous carcinoma lines for biosynthesis of TSP. The cell lines examined include the UMSCC-11B (which was highly responsive to exogenous TSP), the UM-SCC-17B (which was moderately responsive) and the UM-SCC-22B (the least responsive of the eight lines). All three lines synthesized similar immunoreactive forms of TSP as indicated by SDS-PAGE analysis of the immunoprecipitates from the cell lysates under non-reducing conditions (fig. 4). However, quantitative differences between the three lines were suggested by the fluorograms. The UM-SCC-11B cells appeared to synthesize more TSP than the UM-SCC-17B cells which in turn appeared to synthesize more than the UM-SCC-22B cells. Similar differences were observed on the gels run under reducing conditions (not shown). To confirm the apparent differences between the three cell lines, we compared the amount of radioactivity in the cell lysates and chase media that was precipitated with anti-‘I’SP with the total amount precipitated with 10% trichloroacetic acid (TCA). Although all three lines incorporated similar amounts of [3’Slmethionine into TCA-precipitable material, the amount precipitated with anti-TSP was greatest in the UM-SCC-11B cells, followed by the UM-SCC-17B cells and then the UM-SCC-22B cells (table 3). Immunofluorescence studies were carried out next. For these, the cells were grown on glass coverslips for one day and then stained with the rabbit polyclonal anti-TSP in the viable state. When examined by fluorescence microscopy and Exp
Cdl
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3. Analysis by SDS-PAGE of TSP immunoprecipitates from biosynthetically labeled UM-SCC1lB squamous carcinoma cells. Onex 10’ cells in loo-mm dishes were pulsed for 1 h with [sSS]methionine and chased for 4 h in the absence of labeled methionine. Cell lysates and chase media were divided in half and immunoprecipitated either with anti-TSP or with anti-BSA as a control. Half of each immunoprecipitate was reduced with 2-mercaptoethanol(2 %) and half was not. The immunoprecipitates were then analysed by SDS-PAGE on a 3-10% polyacrylamide gradient slab gel. Radioactive bands were visualized by fluorography after a 2-day exposure. Lanes A, B and Care non-reduced gels; D, E, Fare reduced gels. A, Chase medium precipitated with anti-TSP; B, cell lysate precipitated with anti-TSP; C is the cell lysate precipitated with anti-bovine serum albumin; D, Chase medium precipitated with anti-TSP; E, F, Cell lysates precipitated with anti-TSP (I?) and with anti-BSA (F). Fig. 4. Analysis by SDS-PAGE of TSP immunoprecipitates from biosynthetically labeled UM-SCCllB, UM-SCC-17B and UM-SCC-22B cells. Onex 10’ cells in loo-mm dishes were pulsed for 1 h with [35S]methionine and chased for 4 h in the absence of labeled methionine. Cell lysates were divided in half and immunoprecipitated with either anti-TSP or anti-BSA as a control. The immunoprecipitates were then analysed by SDS-PAGE under non-reducing conditions on a 3-10% polyacrylamide gradient slab gel. Radioactive bands were visualized by fluorography after a 2-day exposure. Lanes A, B are lysates of UM-SCC-1lB cells precipitated with anti-TSP (A) and anti-BSA (B). C, D, Lysates of UM-SCC-17B cells precipitated with anti-TSP (C) and anti-BSA (D). E, F, Lysates from UM-SCC22B cells precipitated with anti-TSP (0 and anti-BSA (F). Fig.
Table 3. Quantitative analysis of TSP immunoprecipitates from biosynthetically labeled squamous carcinoma cells Proportion TSP Cell line
Cell lysate (So)
Chase medium (96)
UM-SCC-1lB UM-SCC-17B UM-SCC-22B
0.09F0.01 0.01+0.01 Not detected
2.60+0.22 0.41f0.09 0.08iO.02
Onex 10’ cells in 100 mm dishes were pulsed for 1 h with [35S]methionine and chased for 4 h in the absence of labeled methionine. Cell lysates and chase media were divided in three parts. One part was precipitated with 10% (final) TCA. The other two parts were immunoprecipitated with anti-TSP and anti-BSA. The amount of radioactivity precipitated with anti-BSA was subtracted from the amount precipitated with anti-TSP. The remaining value was compared with the total amount precipitated with 10% TCA and expressed as a percentage of the total. Exp Cd! Res 167 (IYR6)
Fig. 5. Immunofluorescence staining of the UM-SCC-1IB cells in the viable state. Cells grown on coverslips for one day were gently washed in PBS and then stained at 4°C with a 1 : 20 dilution of antiTSP (panel A) or a 1 : 20 dilution of anti-BSA (B), followed by a 1 : 50 dilution of fluorescein isothiocyanate conjugated swine anti-rabbit IgG. Photographs are 5 min exposures at X400.
simultaneously by phase-contrast microscopy, it could be seen that more than 95 % of the UM-SCC-11B cells stained with the rabbit polyclonal anti-TSP (table 4). Staining appeared punctate and distributed evenly over the entire surface of the cell (fig. 5). There was little variability from area to area on the coverslip. The UM-SCC-17B cells also stained with the anti-TSP antibodies. The pattern of staining was similar to that seen with the UM-SCC-I IB cells except that a smaller proportion of the cells stained (table 4). Likewise, the staining pattern of the UMSCC-22B was similar to that of the other two squamous carcinoma lines. Howev-
Table 4. Immunojluorescence staining of human tumor cells in the viable state with anti-TSP
Cell line Squamous carcinoma UM-XC-1lB UM-SCC-17B UM-SCC-22B Fibrosarcoma UM-SCC-IA Melanoma UM-MEL-l SK-MEL-10 SK-MEL-28
Proportion staining cells” (%I
92+10 36+5 5+1 87+6 4 4 <5
’ The cells were examined by phase-contrast microscopy for total cells and by fluorescence microscopy for fluorescent cells. Values are averages f SDS based on five fields per cell line in a single experiment. Each line was examined three or more times, with similar results. Exp CellRes 167(1986)
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ABCDEF
580 Kd Fig. 6. Analysis by SDS-PAGE of TSP immunoprecipitates from biosynthetically-labeled UM-WC-1 lB, UMMEL-l and SK-MEL-28 cells. Onex 10’ cells in 100 mm dishes were pulsed for 1 h with [‘5S]methionine and chased for 4 h in the absence of labeled methionine. Cell lysates were divided in half and immunoprecipitated either with anti-TSP or anti-BSA as a control. The immunoprecipitates were then analysed by SDS-PAGE under non-reducing conditions on a 3-10 % polyacrylamide gradient slab gel. Radioactive bands were visualized by fluorography after a f-day exposure. Lanes A, B, lysates of UM-SCC-1lB cells precipitated with anti-TSP (A) and anti-BSA (B). C, D, Lysates of UM-MEL-l cells precipitated with anti-TSP (C) and anti-BSA (D). E, F, Lysates from SK-MEL-28 cells precipitated with anti-TSP (,I?)and anti-BSA (F).
er, only a very small proportion of the cells (5%) stained (table 4). None of the three lines showed any staining when affinity-purified anti-BSA was used instead of the anti-TSP. Examination
of Melanoma
Cells for TSP Production
Two of the human melanoma cell lines (UM-MEl-1 and SK-MEL-28) were examined for TSP biosynthesis. When these cells were examined under exactly the same conditions as the squamous carcinoma cells, there was no evidence of TSP production (fig. 6). The inability to detect TSP biosynthesis was not due to a failure of the melanoma cells to incorporate [35S]methionine. These cells incorporated about the same amount of the labeled methionine per cell as did the squamous carcinoma cells. Furthermore, when cell lysates and chase media from [35Slmethionine-labeled melanoma cells and squamous carcinoma cells were examined for laminin, comparable amounts were seen (not shown). Three of the melanoma lines were stained with the polyclonal anti-TSP. With none of these lines was there evidence of distinct staining, though an occasional faintly-stained cell could be seen (table 4). Nor did the melanoma cells stain when anti-bovine serum albumin was used instead of the anti-TSP, but these cells demonstrated bright punctate staining when affinity-purified anti-laminin was used as the primary reagent. Thus, the lack of staining observed with the antiTSP was probably not due to any innate inability of these cells to bind rabbit antibodies. In contrast, the fibrosarcoma cells resembled the squamous carcinoma cells in that a large proportion of these cells stained brightly with anti-TSP (table 4). Exp Cell Res 167 (1986)
388 Varani et al.
DISCUSSION A number of extracellular matrix components including laminin, fibronectin and collagens of various types are known to function as cell attachment factors [ll-161. The data presented here indicate that TSP, another large extracellular matrix glycoprotein [3-51, is an adhesion factor for human squamous carcinoma cells. It induced attachment and spreading of several cell lines on plastic culture dishes and on dishes coated with 50 ug of collagen (including a preparation of type I collagen from bovine skin and a preparation of type IV ‘basement membrane’ collagen obtained from the EHS sarcoma). While the in vivo function of TSP has yet to be demonstrated, the finding that it can promote attachment and spreading of cells in vitro suggests that it may perform a similar function in vivo. The ability of TSP to promote adhesion was not unique to squamous carcinoma cells. It also promoted adhesion of human keratinocytes (the normal counterpart cell of the squamous carcinomas) as well as normal human fibroblasts and malignant fibrosarcoma cells. In contrast to its effect on these cells, TSP did not stimulate adhesion in a number of human melanoma or glioma cell lines. In fact, TSP actually inhibited attachment and spreading in several of these lines. It is interesting that the human melanoma and glioma lines responded in the adhesion assay when laminin was used as the stimulating agent, while the squamous carcinoma cells were much less responsive or non-responsive to this protein. Thus, there appears to be specificity in the adherence response to the large glycoprotein matrix components. The squamous carcinoma cells not only responded to exogenously added TSP, but also synthesized it. The synthesized TSP was secreted into the culture medium and bound to the cell surface. The TSP synthesized by the squamous carcinoma cells may serve as an endogenous adhesion factor for these cells. Rabbit polyclonal antibodies and mouse monoclonal antibodies to TSP were able to partially inhibit cell attachment to collagen-coated dishes when the cells were pretreated with the antibodies. Furthermore, there was a direct relationship between the amount of TSP synthesized by a given cell line and the adhesiveness of that line in the absence of exogenous TSP (compare table 1 with fig. 4 and tables 3 and 4). It is interesting that among the three squamous carcinoma lines examined in detail, the cell line that synthesized the greatest amount of TSP and was the most adhesive (UM-SCC-11B) also formed the most undifferentiated tumors when injected into athymic mice. The cell line which synthesized the least amount of TSP and was the least adhesive (UM-SCC-22B), formed the most welldifferentiated tumors when injected into athymic mice. How TSP facilitates cell-substrate adhesion is not known at present and a number of possibilities exist. It may be that the secreted TSP binds to the extracellular matrix and then interacts with the cell through TSP-binding moieties on the cell surface. Specific receptors for TSP have not yet been described but it has been shown that TSP can bind to sulfated glycolipids, which are present in cell membranes [30]. Other cell surface proteins such as fibronectin, which are Em
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known to interact with TSP [31], may also serve as binding sites for the secreted TSP. If this is correct, then differences in responsiveness to TSP may result from differences in fibronectin synthesis or surface expression. Alternatively, the TSP synthesized by the squamous carcinoma cells may be inserted directly into the cell surface and then facilitate interactions with the substratum. While we cannot distinguish between the various possibilities at present, the fact that certain cell lines (notably, the UM-SCC-17B) are heterogeneous with regard to surface TSP expression and with responsiveness to exogenous TSP may allow us to probe these questions. Studies are currently in progress to separate and clone variant populations from the UM-SCC-17B line. These populations will then be used in an effort to determine if the cells that synthesize TSP are, in fact, the same cells that respond to it. Likewise, studies are underway to determine if there is a correlation between synthesis of TSP by the various squamous carcinoma lines and synthesis of other surface/extracellular matrix proteins such as tibronectin and laminin. These studies should help to determine how TSP acts to facilitate cell-substrate interactions. In summary, these studies demonstrate that TSP stimulates attachment and spreading in a number of cell lines derived from human squamous cell carcinomas. TSP thus joins a number of other large glycoproteins of the extracellular matrix which are known to serve as adhesion factors for various types of cells. This study was supported in part by grants CA36132. CA28564 and 2 SO7 RR05384-25 from the USPHS and by grants BC-512 and IM-432 from the American Cancer Society. T. E. C. is the recipient of USPH RCDA No CA0062 I.
REFERENCES 1. Dixit, V M, Grant, G A, Santoro, S A & Frazier, W A, J biol them 259 (1984) 10000. 2. Coligan, J E & Slayter, H S, J biol them 259 (1984) 3944. 3. Baenziger, N L, Brodie, G N & Majerus, P W, J biol them 247 (1972) 2723. 4. Phillips, D R, Jennings, L K & Prasanna, H R, J biol them 255 (1980) 11629. 5. Gartner, T K, Gerrard, J M, White, J G & Williams, D C, Blood 5 (1981) 153. 6. McPherson, J, Sage, H & Bornstein, P, J biol them 256 (1981) 11330. 7. Mosher, D F, Doyle, M J & Jaffe, E A, J cell biol 93 (1982) 343. 8. Raugi, G J, Mumby, S M, Abbott-Brown, D & Bomstein, P, J cell biol 95 (1982) 351. 9. Jaffe, E A, Ruggiero, J T, Leung, L L K, Doyle, M J, McKeown-Longo, P J & Mosher, D F. Proc natl acad sci US 80 (1983) 998. 10. Sage, H, Farin, F M, Striker, G E & Fisher, A B, Biochemistry 22 (1983) 2148. 11. Pearlstein, E, Gold, L I & Garcia-Pardo, A, Mol cell biochem 29 (1980) 103. 12. Terranova, V P, Rohrbach, D & Martin, G R, Cell 22 (1980) 719. 13. Vlodavsky, I & Gospodarowicz, D, Nature 289 (1981) 304. 14. ROOS, E, Biochim biophys acta 738 (1984) 263. 15. Grinnell, F & Bennett, M H, J cell sci 48 (1981) 19. 16. Schor, S L, Schor, A M & Bazill, G W, J cell sci 49 (1981) 299. 17. Carey, T E. Takahashi, T, Resnick, L A, Oettgen. H F & Old, L J, Proc natl acad sci US 73 (1976) 3278. 18. Black, P M, Komblith, P L, Davison, P F, Liszczak, T M, Merk, L P, Smith, B H, McKeever, P E & Quindlen, E A, J neurosurg 56 (1982) 62. 19. McKeever. P E, Fligiel, S E G, Varani, J, Hudson, J L, Smith. D, Castle, R L & McCoy, J P. Intj cancer 37 (1986) 867. 20. Kimmel, K A & Carey, T E, Cancer res 46 (1986) 3614.
390 Varani et al. 21. Varani, J, Lovett, E J, McCoy, J P, Shibata, S, Maddox, D, Goldstein, I J & Wicha, M, Am j path01 111 (1983) 27. 22. Dixit, V M, Haverstick, D M, O’Rourke, K M, Hennessy, S W, Grant, G A, Santoro, S A & Frazier, W A, Proc natl acad sci US 82 (1985) 3844. 23. - Biochemistry 24 (1985) 4270. 24. Galvin, N J, Dixit, V M, O’Rourke, K M, Santoro, S A, Grant, G A & Frazier, W A, J cell biol 101 (1985) 1434. 25. Dixit, V M, O’Rourke, K M, Grant, G A, Santoro, S A & Frazier, W A, J biol them 261 (1986) 1962. 26. Timpl, R, Rohde, H, Robey, P G, Rennard, S 1, Foidart, J M & Martin, G R, J biol them 254 (1979) 9933. 27. Ronnett, G V, Knutson, V P, Kohnski, R A, Simpson, T L & Lane, M D, J biol them 259 (1984) 4566. 28. Ruddon, R W, Hanson, C A & Addison, N J, Proc natl acad sci US 76 (1979) 5173. 29. Laemmli, U K, Nature 227 (1970) 680. 30. Roberts, D D, Haverstick, D M, Dixit, V M, Frazier, W A, Santoro, S A & Ginsburg, V, J biol them 260 (1985) 9405. 31. Leung, L L K & Nachman, R L, J clin invest 70 (1982) 542. Received April 1, 1986 Revised version received June 16, 1986
Exp Cell Res 167 (19861
Prmted
I” Sweden