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(CANCER RESEARCH 51. 6660-6667. December 15. I99I| Isolation and Characterization of Human Melanoma Cell Variants Expressing High and Low Levels of CD44 Mary Birch, Stephen Mitchell, and Ian R. Hart1 Imperial Cancer Research Fund Laboratories, Lincoln 's Inn Fields, London H C2A 3PX, United Kingdom ABSTRACT Variants of the human melanoma cell line LT5.1 were selected for high and low expression of the \1, 90,000 CD44 glycoprotein by using the Hermes-3 monoclonal antibody combined with fluorescence-activated cell sorting. Cells were single cell cloned and clones of CD44 highexpressing and CD44 low-expressing phenotype were isolated. The var iants, which exhibited up to a 7-fold difference between high and low expression, have maintained a stable phenotype over a period of 3 months in tissue culture. Northern blot analysis of mRNA from the different clones showed correlation of levels of transcripts with fluorescenceactivated cell sorting analysis data. Wound migration assays, utilizing the different clones, showed that the low-expressing clones manifested less motility than did cells showing high levels of CD44. Homotypic aggregation of cells was increased in those cells expressing high levels of CD44, and these variants were also better able to adhere to hyaluronate substrates. All of these activities were inhibited by the presence of antiCD44 antibody. When injected i.v. into nu/nu BALB/c mice, the lowexpressing clones gave significantly fewer lung nodules than the highexpressing clones, although the two variant types did not differ in their capacity to form s.c. tumors in similar mice. These results suggest that the CD44 molecule, possibly as a function of its activities as a hyaluronate receptor, may play a vital role in determining the fate of hematogenously disseminating melanoma cells. INTRODUCTION CD44 is a widely distributed integral cell membrane glyco protein that exists in a variety of forms, the most prevalent being M, 85,000-95,000 in size, arising from posttranslational modifications of a M, 37,000 core protein (1, 2). Monoclonal antibodies raised against the Hermes antigen (3), polymorphic glycoprotein Pgp-1 antigen (4), extracellular matrix receptor ECM-III (5), and the human CD44 antigen all recognize the same molecule (6), and suggest that this glycoprotein has a pleiotropic role in various cell processes. One of the major functions of CD44 appears to be in regulating lymphocyte adhesion to the cells of high endothelial venules during lym phocyte migration (7, 8), a process which has many similarities to the metastatic dissemination of solid malignancies (9). Cloning of CD44 cDNA revealed significant homology in the NH2-terminal domain of the molecule with the tandem repeat domains of the cartilage link and proteoglycan core proteins (10, 11), suggesting that the molecule might have a more ubiquitous role in cell adhesion than simply determining lym phocyte-high endothelial venule interactions. This possibility was underscored by the recent demonstration that CD44 is the principal cell surface receptor for hyaluronate (12, 13). It is known that this glycosaminoglycan plays a major role in diverse cellular processes including cell-cell adhesion, embryonic de velopment, and cell migration and angiogenesis (14, 15). Many tumors are characterized by the production and accu mulation of high levels of hyaluronate and neoplastic cells often Received 4/11/91; accepted 10/4/91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed. exhibit substantial capacity to bind to this glycosaminoglycan (16, 17). Accordingly, it was of interest to note that elevated levels of CD44 were detected in carcinomas relative to normal epithelium, a finding which was thought to be consistent with the possibility that the molecule could play a part in regulating the invasive and metastatic process (11). In the present study we have sought to investigate the putative involvement of CD44 in the hematogenous spread of melanoma cells by isolating a series of stable CD44-deficient and CD44sufficient variants from a single human melanoma cell line. The technique used was that of iterative fluorescence-activated cell sorting by using monoclonal anti-CD44 antibodies, followed by single cell cloning, as described by Schreiner et al. (18) for the isolation of fibronectin receptor-deficient variants. Isolated clones have then been assessed for tumorigenic potential, ca pacity to colonize the lungs of athymic mice after i.v. injection, growth characteristics, cell adhesion and migration abilities, and mRNA levels for the CD44 molecule. MATERIALS AND METHODS Cell Culture. The DX3.LT5.1 human melanoma line (19) was grown on plastic in Dulbecco's modification of Eagle's minimal essential medium supplemented with L-glutamine and 10% heat-inactivated fetal calf serum. Cultures were maintained at 37°Cin a humidified atmos phere of 95% air, 5% CO2. Variant Selection. DX3.LT5.1 cells were harvested following a brief incubation with 0.25% trypsin, washed once in DEM,2 followed by two washes in ice-cold PBS containing 1% BSA. Cells were stained by direct fluorescence with the use of anti-CD44 antibody (Hermes-3, 10 Mg/ml; generously provided by Dr. E. Butcher, Stanford University, Stanford, CA) and fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (1:40 dilution; Dako Immunoglobulins F232). Background controls were established by omitting the primary antibody. Cells were separated by fluorescence-activated cell sorting on 2 Facstar Plus (Becton Dick inson) flow cytometer equipped with an argon ion 488 nm laser. Cells were run through three selection cycles, where the brightest and dim mest 5% of the population were selected and amplified between each cycle, before the respective bright and dim populations were plated directly to 96-well plates at a concentration of 0.5 cells/well and grown to confluence. The individual clones were then harvested with trypsin and replicate dishes were established which were then screened by using the Hermes-3 antibody (10 ^g/ml) in the enzyme-linked immunosorbent assay technique as detailed by Durbin and Bodmer (20). The four clones expressing the highest level of CD44 expression (high clones 1, 2, 6, and 7) and the four clones expressing the lowest level of CD44 expression (low clones 3, 5, 6, and 7) were then expanded for further analysis from the original unassayed duplicate 96-well dish. These clones were rescreened regularly over a period of 4 months, to ensure the stability of CD44 expression, using both fluorescence-activated cell sorting and enzyme-linked immunosorbent assays. Evaluation of VLA3«chain expression, an integrin molecule unrelated to CD44, was performed by utilizing the monoclonal antibody J143 (saturation dilu tion; kindly provided by Dr. M. Horton, ICRF Dominion House, St. Bartholemew's Hospital). 2The abbreviations used are: DEM. Dulbecco's modified Eagle's medium: BSA. bovine serum albumin: PBS. phosphate-buffered saline; SDS. sodium dodecyl sulfate: cDNA, complementary DNA. 6660 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1991 American Association for Cancer Research. CD44 VARIANTS FROM A HUMAN MELANOMA Cell Aggregation Assay. The aggregation assay described by St. John et al. (21) was used. Briefly, adherent cells were harvested with trypsin solution as described above and washed once in DEM. Cells were resuspended to a concentration of IO6 cells/ml in PBS containing I mg/ml BSA and 40 mm piperazine-iY,/V"-bis(2-ethanesulfonic acid) (pH 7.0). Three hundred n\ of this single cell suspension (as determined by microscopic examination) were placed into each well of a 24-well tissue culture plate which was then rotated on a gyratory shaker at 4°Cfor 1 h. After this time cell mixtures were fixed by the addition of 1 ml of PBS containing 2% paraformaldehyde. Aggregation was assessed by counting cells in a Neubauer chamber. Cells in three independent fields were counted, including those in aggregates and those present as single cells. The degree of aggregation was determined as the fraction of cells in clumps of a size greater than five cells. The effects of anti-CD44 antibodies on cell aggregation was examined by incubating cells with Hermes-3 (10 Mg/ml) for l h at 0°Cprior to addition to the plates. Adhesion Assay. A modified version of the assay utilized by Miyake et al. (13) was used to determine cellular adhesion. Ninety-six-well flexible plates (Falcon 3912 Assay Plate. Becton Dickinson) were treated for 2 h at 37°Cwith hyaluronate solution (5 mg/ml in PBS) before being washed with PBS and blocked with PBS/1% BSA. Cells were labeled with Na"CrO4 (Amersham International, Little Chalfont, United Kingdom). Specific activity. 250-500 mCi/mg chromium, as detailed previously (22). Labeled cells (5 x IO4) in 100-/¿1 aliquots (PBS/0.02% BSA) were added to each well and incubated at 37°Cfor 70 min before unbound cells were removed by gentle washing (3 times) with PBS/0.02% BSA. The flexible plates were then cut up into individual wells and the radioactivity associated with each well was counted by using a gamma counter (LKB 1261 Multigamma). The percentage of attached cells was calculated from the formula. lane), and blotted onto a filter (Hybond N; Amersham Inc., Little Chalfont). Filters were hybridized at 42°C50% formamide, 5 x Denhardt's solution [0.1% (w/v) BSA, 0.1% (w/v) Ficoll, 0.1% (w/v) polyvinyl pyrrolidone], 5 x 0.9 M NaCl, 50 mM sodium phosphate, pH 7.7, 5 mM sodium EDTA), 0.5% SDS, and 10% dextran sulfate with a [-"PjdCTP nick-translated CD44 cDNA probe (11) which was gener ously supplied by Dr. B. Seed, Massachusetts General Hospital, Boston, MA. Filters were washed at 65°Cto a final stringency of 0.2 x 0.9 M NaCl, 50 mM sodium phosphate, pH 7.7, 5 mM sodium EDTA, and 0.2% SDS. In y ivo Behavior of Selected Variants. Specific pathogen-free athymic nude mice (BALB/c background) were obtained from the Animal Breeding Unit, ICRF, Clare Hall, South Mimms, United Kingdom. Animals were housed within plastic isolators and were used when 810 weeks old. To assess tumorigenic capacity of the clones, cells were harvested, adjusted to a concentration of 5 x IO6 cells/ml PBS, and mice were given injections s.c. in the flank region of an inoculum of 0.2 ml (1 x IO6cells). Animals were examined three times a week for the appearance of palpable tumors and were killed when the size of this lesion ap proached 1.5 cm at the greatest diameter. In order to determine the lung colonization capacity of the clones, cells were harvested as above, adjusted to a concentration of 1 x IO6 cells/ml, and the viability was assessed by trypan blue exclusion. All cell suspensions had viabilities of 90% or greater. Mice received inoc ulum volumes of 0.2 ml via the lateral tail vein and were killed 8 weeks later when the lungs were removed and assessed for pulmonary tumor nodule formation under a dissecting microscope. RESULTS Residual cpm x 100 % of attached cells = Input cpm Variant Selection and Growth Characteristics. In the study of Schreiner et al. (18) the isolation of Chinese hamster ovary cell Cell Migration Assay. The wound assay of Goodman et al. (23) was variants deficient in fibronectin receptor expression was achieved by a protocol on which the present investigation was used to determine the migratory capacity of the various clones. Har vested cells (5 x IO1cells/ml) in DEM were plated into the wells of 24based. These workers found that a single flow cytometric sort well plates and cultured at 37°Cfor 24-48 h until the cultures were resulted in a population of cells with unstable levels of receptor subconfluent. A wound track (approximately 5 mm in size) was scored expression (18). Consequently in our experiments we utilized in each well with the use of a plastic scraper. Replicate wells were three batch sorts, followed by limiting dilution cloning, to terminated at 16. 24, and 48 h after wounding by fixing and staining obtain variants which expressed different levels of CD44 at the cell cultures with 1% crystal violet in methanol. The stained cells their cell surface. Fluorescence intensity histograms of four were then examined under an inverted microscope. high-expressing and four low-expressing clones are presented Immunoprecipitation. Cells from the individual clones were surface in Fig. \A. The population means of log-integrated green fluo labeled by the lactoperoxidase iodination procedure (24). Harvested cells were suspended (0.2-1 x IO7 cells/ml) in PBS to which were rescence for the high clones ranged from 40 to 60 fluorescent unitsv whereas those of the low clones varied from 7 to 10 added, in sequence, 5 /jl sodium iodide (Sigma, United Kingdom; 5 x 10~4 M), 10 ß\glucose (0.5 M), 5 /¿Ilactoperoxidase (Calbiochem, fluorescent units, indicating a 4- to 6-fold difference in levels Cambridge Bioscience, Cambridge, United Kingdom; 1.8 mg/ml PBS) of expression between the two populations. 100 fiCi sodium |'-sl]iodide (Amersham, United Kingdom), and 5 n\ These differences were very stable over an 8-week period in glucose oxidase (20 units/ml); the suspension was mixed and incubated tissue culture and have continued to be maintained over 4 for 15 min at room temperature and then 10 n\ sodium iodide (0.1 M) months of continuous culture (data not shown). were added. Cells were washed 3 times with PBS and then extracted All clones, whether low or high CD44 expressers, showed twice in Penman's buffer (20 mM 4-(2-hydroxyethyl)-l-piperazineethcomparable levels of VLA-3« chain on their cell surface as anesulfonic acid, 3 mM MgCl;, 50 mM NaCl, 1 mM CaCI2, 300 mM detected by the monoclonal antibody J143 as illustrated in Fig. sucrose, 0.5% Nonidet P-40, 1 m,Mphenylmethylsulfonyl fluoride, 0.5% IB. sodium-azide). Paniculate debris was removed by centrifugation; ali quots of lysate were incubated, on ice, with Hermes-3 antibody (10 ¿ig/ Growth curves, using both the incorporation of tritiated ml) for 45-60 min, followed by 5 ß\rabbit antiserum to mouse IgG thymidine into DNA and cell counts as the parameter meas (DAKO Corp., Santa Barbara, CA) for a further 45-60 min. Complexes ured, did not identify any difference in growth rates, with all were then removed with 50-100 M' protein A-Sepharose [50% (w/v) clones exhibiting population doubling times of 12-14 h. There suspension] for 60 min [Pharmacia (GB) Ltd., Milton Keynes, United is therefore no correlation between level of receptor expression Kingdom). The bound complexes were washed as described elsewhere and growth capacity. (25) and then boiled for 10 min in Laemmli buffer (26). and analyzed Immunoprecipitation Analysis. The result of surface iodinatby SDS-polyacrylamide gel electrophoresis (8% gel). Gels were dried ing cells from CD44 high-expressing and CD44 low-expressing and autoradiographed with Kodak XAR-5 film at -70°C. clones and immunoprecipitating the Nonidet P-40 extracted Northern Blot Analysis. Total RNA was prepared by the guanidinium isothiocyanate technique (27), and polyadenylated RNA isolated by lysates with the Hermes-3 antibody is presented in Fig. 2A. From molecular weight standards it was clear that the immuoligodeoxythymidylate cellulose (type 7, Pharmacia, Uppsala, Sweden), then electrophoresed through a 1% formaldehyde-agarose gel (10 Mg/ noprecipitated CD44 was of the M, 80,000-90,000 species. 6661 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1991 American Association for Cancer Research. CD44 VARIANTS FROM A HUMAN MELANOMA HK3HSORTS LOW SORTS AACLONE LT5.1 CD44 LOW LT5.1 CD44 HIGH +3 90kD II.* B CLONE ABCD I- CLONE *6 CLONE CLONE f7 CLONE *7 RELATIVE FLUORESCENCE LOW 3 RELATIVE FLUORESCENCE EFGH Fig. 2. Immunoprccipitation of representative LT5. l CD44 high- and lowexpressing clones. A. immunoprecipitations of CD44 by Hermes-3 monoclonal antibody (10 Mg/ml) were performed as described in "Materials and Methods," using I2'l-labeled cell lysates. Equal cpm «ereimmunoprecipitated and each cell type was loaded on to an 8rr acrylamide gel and run at 50 V overnight. Gels »ere fixed in acetic acid/glycerol before drying and were autoradiographed for 24 h. Lanes A-D, CD44 low-expressing clones 3, 5, 6, and 7; Lanes E-H CD44 highexpressing clones I. 2. 6. and 7. B, immunoprecipitation of same clones with JI43. and SDS-polyacrylamide gel electrophoresis under reducing conditions, reveals approximately equal expression of VLA-3«.Ordinale, molecular weight in thousands. Since all lysates contained equal amounts of radioactivity (cpm) the clones have variable amounts of labeled species at their cell surface, with the high clones having approximately 8-fold higher levels than the low-expressing clones as determined by densitometric scanning. Immunoprecipitations of the same clones with control antibody J143 and analysis under reducing conditions revealed the expected band at a molecular weight of about 130,000 («,and i3¡), showing approximately equal expres sion by high- and low-expressing clones for this unrelated surface molecule (Fig. 2B). Northern Blot Analysis. RNA blot hybridization revealed three message sizes of approximately 1.6, 2.2, and 5.5 kilobases (Fig. 3) as reported for other melanoma cell lines elsewhere (10, 11). The 5.5-kilobase transcript, scarcely discernible in the example shown, always was expressed to a lesser degree than the 1.6- and 2.2-kilobase size messages. Verification that equal loading had been achieved was obtained by stripping the nylon filters (30 min boiling in 0.1% standard saline citrate) and reprobing with a ['-PjdCTP nick-translated actin cDNA probe (Oncor probe, Hybaid Ltd., Middlesex, United Kingdom). From densitometric measurements it was calculated that the high-expressing clones had up to a 7-fold increase in steady state mRNA levels for CD44 relative to the low-expressing clones (Fig. 3). Cell Aggregation. When single cell suspensions were incu bated at 4°Cfor set time periods the CD44 high-expressing clones self-aggregated to a much greater extent than the lowexpressing clones. Thus, whereas this latter group did show the occasional cluster consisting of approximately 2-6 cells, the high-expressing groups regularly formed large aggregates cornFig. 1. A, selection of LT5.1 CD44 high- and low-expressing clones. LTS.l cells were surface labeled with anti-CD44 monoclonal antibody (Hermes-3, 10 ng/ml). followed by fluorescein isothiocyanate-labeled secondary antibody, batch sorted three times, and finally single sorted into 96-well plates, as described in "Materials and Methods." The four highest and four lowest CD44-expressing clones were grown to confluency after enzyme-linked immunoabsorbent assay analysis of duplicate plates. For each panel 5000 cells were analyzed. Full scale in the >'-axis is 400 cells, with hatch marks indicating every 100 cells. The axis spans four logs relative fluorescence intensity. B. expression of VLA-3a (detected by J143) by both CD44 high- and low-expressing clones. 6662 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1991 American Association for Cancer Research. CD44 VARIANTS FROM A HUMAN MELANOMA LT5.1 CD44 5.5kb —¿ 2.2kb —¿ 1.6kb —¿ B ACTIN « LOW LT5.1 CD44 HIGH •¿Mill •¿IV* B D E H Fig. 3. Northern blot analysis of LT5.1 CD44 RNA. A, RNA was prepared from LT5.1 CD44 high- and low-expressing clones as described in "Materials and Methods." Ten ¿igof mRNA/lane were loaded onto an 0.8r< agarose gel, electrophoresed. transferred to Hybond N membranes (Amersham. Inc.. Buck inghamshire. United Kingdom) and hybridized with "P-labeled CDW44 probes. The transcripts seen are 5.5. 2.2, and 1.6 kilobases (kh). Lanes A-l), low CD44 expressing clones 3, 5, 6. and 7: Lanes K-H, high-expressing clones I, 2. 6. and 7. /?, filter was striped and reprobed with actin to verify equal loading. posed of 20-40 cells. Representative fields illustrating this phenomenon with high clone 7 and low clone 3 are presented in Fig. 4. The observed self-aggregation was abrogated by the presence of anti-CD44 antibody (Hermes-3, saturation dilu tion). The degree of aggregation observed, defined by the frac tion of cells in clumps larger than 5 cells, in the presence and absence of monoclonal antibody, is presented in Fig. 5. Highexpressing clones I and 7 manifested aggregation of 52 and 50%, respectively, whereas the low-expressing clones 3 and 5 only had percentage aggregation values of 13 and 10%. Aggre gation of the high-expressing clones could be reduced to the same value as that of the low-expressing clones by the addition of Hermes-3 antibody, although comparable treatment of the low-expressing clones had no effect on aggregation character istics (Fig. 5). Control antibody (J143, saturation dilution) had no effect on the self-aggregation phenomenon (data not shown). Cell Migration. The migration of the different clonal lines was analyzed by using the in vitro "wound" system. Wounds of approximately 5 mm were made in subconfluent monolayers of (+/- ANTI-CD44 LTS.l CD44 CLONES MONOCLONAL ANTMiODY) Fig. 5. Quantitative analysis of aggregation of LTS.l CD44 high- and lowexpressing clones. Data from aggregation assays using high clones 1 and 7 low clones 3 and 5 are presented. Aggregaties containing five cells or more were counted in triplicate samples. Addition of anti-CD44 antibody (ah) (Hermes 3, 10 ^g/ml) to cells followed by incubation at 4°Cfor l h prior to initiation of assay abrogated aggregation. All standard deviations were less than 10%. the different clones and cells were allowed to migrate into the cell-free area over a 24-h period. Representative experiments using four clones are illustrated in Fig. 6. Here the two clones which expressed low levels of CD44 migrated less than did the two clones expressing high levels of CD44. The results from the assay, which was repeated at least 3 times with virtually identical results, indicate a correlation between CD44 expres sion and the migrating ability of DX3.LTS.l melanoma cells. Quantitation of data obtained from a single experiment with a representative high and low clone (Fig. 7) showed that, in the 24-h period examined, roughly 3 times the number of cells from the high-expressing clones migrated into the cleared area. Moreover, incubation in the presence of anti-CD44 antibody (Hermes-3) reduced the migratory activity of the high clone to that of the low clone (Fig. 7). Cell Adhesion. When two of the high-expressing clones (1 and 7) and two of the low-expressing clones (3 and 5) were assessed for their ability to bind to a hyaluronate substrate, it was found that there was a substantial difference in the adhesive Fig. 4. Aggregation of LTS.l CD44 highand low-expressing clones. Representative fields of cells from one of a number of similar aggregation assays are presented. A, a clone of LT5. l CD44 high-expressing cells (high clone 7) showing extensive homotypic cell aggrega tion; B, a clone of LT5.1 CD44 low-expressing cells (low clone 3): C. the high clone 7 in the presence of anti-CD44 monoclonal antibody (Hermes 3, 10 ng/ml): lì,low clone 3 in pres ence of Hermes 3 antibody. Testicular hyaluronidase (5000 units/ml) also inhibited cellular aggregation (data not shown). Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1991 American Association for Cancer Research. CD44 VARIANTS FROM A HUMAN MELANOMA o, Fig. 6. Migratory ability of the LT5.1 C"D44 high- und low-expressing clones. Subconfluent monolayers of representative LT5. l CD44 high and low clones were "wounded" at time 0 as described in "Materials and Methods." The cells were allowed to migrate into the cell-free area for 24 h then fixed and stained with crystal violet. A and B. CD44 low-expressing clones 3 and 5; C and D. CD44 high-expressing clones I and 7. weeks after s.c. injection large progressively growing tumors were apparent in virtually all animals (Table 1) with no obvious difference in take or growth rate apparent between those re sulting from the high-expressing clones or low-expressing clones. When lungs were removed from animals which had been killed after being given injections i.v. of 5 x IO5 cells of any of three of the CD44 low-expressing clones (clones 3, 6, and 7) or with cells from any of three of the CD44 high-expressing clones (clones 1, 2, and 7) a very different picture was apparent. Of a total of 18 animals given injections of cells from the highexpressing clones, 16 showed gross evidence of lung tumor Fig. 7. Quantitation of cell migration in LT5.1 CD44 high- and low-expressing cells. A, wound assay was carried out as described in "Materials and Methods." The columns show the number of cells that had migrated into a 5-mm wound over a 16-h period in the presence or absence of Hermes 3 antibody (10 /jg/ml). The results are derived from three randomly chosen areas of the wound. Bars, SD. A, high-expressing clone 7: B, low-expressing clone 3; (, high-expressing clone 7 in the presence of antibody: D, low-expressing clone 3 in presence of antibody. 60i u u at £ Q capabilities of these variants (Fig. 8). Thus, clones 1 and 7 achieved attachment values of 50% or greater within the culture period, whether sulfated or nonsulfated hyaluronate was uti lized as substrate, whereas clones 3 and 5 only exhibited less than 20% adherence. The addition of testicular hyaluronidase (Sigma Chemco., Poole, Dorset) to the wells (Fig. 8) or the addition of anti-CD44 antibodies (data not shown) virtually abolished all adherence (to 10% or less) of both the variant populations. In Vivo Behavior of Variant Lines. The tumorigenic capacity of six clonal lines was assessed in athymic nude mice. By 8 50- HYALURONATE (sulfated) 40 - HYALURONATE 30- HYALURONATE -f hyaluronidase 20 - PBSA 10 - LOW 3 LOW 5 (LTS.l CD44 HIGH 1 HIGH 7 CLONES) Fig. 8. Adhesion of LT5.1 CE44 high- and low-expressing clones to hyaluron ate. Hyaluronate. either sulfonated or nonsulfonated (5 mg/ml), was coated onto the surface of 96-well plates. Radiolabeled LT5.I CD44 high- and low-expressing clones were added to the wells as described in "Materials and Methods." The columns represent the percentage of cells added which adhered to the 96-well plates. Standard deviation of quadruplicate determinations was lO^r or less. The results shown arc representative of three separate experiments. 6664 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1991 American Association for Cancer Research. CD44 VARIANTS FROM A HUMAN MELANOMA Fig. 9. Lung colonizing capacity of LT5.1 CD44 high- and low-expressing clones. Gross appearance of representative lungs obtained from groups of BALB/c nude mice given injec tions of 5 x 10' LT5.1 CD44 high-expressing cells (high clone 7) (A) and LT5.1 CD44 lowexpressing cells (low clone 3) (B) 8 weeks previously. Arrows indicate pulmonary tumor colonies which stand out in contrast to the lung parenchyma. B Table I Tumor incidence resulting from injection ofLT5.l-CD44highLT5.I-CD44 low-expressing clones and of lung no. of lung incidence"10010010010010060% colonization*838310033400Mean CloneHighlHigh nodules ±SD43 2High?Low ±1315 ±717 ±92 ±0.51 ±0.70 3Low 6Low 7%oftumor " Five mice/group were given injections s.c. of IxlO6 cells/animal. Animals were killed when tumors reached 1.5 cm. 'Six mice/group were given injections i.v. of 5x10* cells/animal. Animals were killed 8 weeks later. nodules (median number of nodules, 30; range, 15-45), whereas of 17 animals given injections of cells from the low-expressing clones, only 4 showed evidence of what was a lower level of pulmonary involvement (median number of tumor nodules, 1; range, 0-2). Not only the number of nodules varied between the two populations of DX-3.LT5.1 cells, but also the size of the neoplastic lesions, as can be seen in Fig. 9. Thus the highexpressing clones produced large, very evident lung nodules, whereas those produced by the low-expressing clones were markedly smaller, being scarcely discernible by the naked eye. DISCUSSION Metastatic dissemination of solid tumors is a complex pathophysiological process consisting of several steps (28). Many of these steps, such as entrance into small vascular or lymphatic channels, survival during circulation, arrest at distant sites, and extravasation, would seem to be very comparable to the steps involved in lymphocyte migration (9, 29). It was these apparent similarities of function that caused us to wonder whether cell surface molecules shown to have a major role in determining the trafficking of normal lymphocytes also had a part to play in regulating the dissemination of cells from solid tumors. CD44 was characterized as an adhesion molecule determining the interaction of lymphocytes with specialized endothelium in lymphoid tissue (1, 30); antibodies against varied epitopes of this "homing receptor" were able to abrogate selectively this on the analogy outlined above, CD44 was a good candidate molecule to examine for a possible contribution to metastatic dissemination; a feeling strengthened by the fact that CD44 expression is not limited to cells of the hematolymphoid series (10,11,32). Our approach has been to utilize the fluorescence-activated cell sorter to select out populations of cells, from a single parental line, which vary in the levels of CD44 expressed at the cell surface. Like Schreiner et al. (18), who isolated variants lacking the fibronectin receptor, we found that variations in surface expression were a reflection of variations in steady-state mRNA levels (Fig. 3), and that the selected clones exhibited a stable phenotype. Upon injection into immunoincompetent athymic nude mice there were substantial differences between the CD44 high- and CD44 low-expressing clones such that the high-expressing clones produced substantially more, and dra matically larger, pulmonary nodules than did the low-express ing clones (Fig. 9). That each of the three clones from either category manifested similar behavior made it unlikely that these differences were simply a random consequence of préexistent cellular heterogeneity (33), and this fact was underlined by the precise duplication of these findings when similar variants from the murine B16 melanoma system were obtained and analyzed.1 These results suggest strongly that, as predicted, the CD44 molecule may play an important part in affecting the dissemi native capacity of the DX3.LT5.1 melanoma cells. The determination that CD44 functions as the principal cell surface receptor for the glycosaminoglycan, hyaluronate (12, 13) offers several possibilities for explaining the mechanistic basis of this observed increase in lung-colonizing capacity. Tumors of many types have been characterized as exhibiting high levels of hyaluronate accumulation (16), and transformed cells frequently manifest high levels of hyaluronate-binding activity (17). Hyaluronate is widely distributed throughout the body but has been found to be present in greatest amounts in lung tissue (34), raising the possibility that the observed de crease in pulmonary colonization by the low-expressing clones was a reflection of decreased attachment of i.v. introduced tumor cells. Certainly in in vitro assays the clones selected for higher levels of CD44 expression were better able to adhere to binding propensity (31). Accordingly, we reasoned that, based ' Unpublished observations. 6665 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1991 American Association for Cancer Research. CD44 VARIANTS FROM A HUMAN MELANOMA hyaluronate substrata than their low-expressing counterparts (Fig. 8). Equally, it has long been known that hyaluronate plays an important role in mediating cell to cell interaction (35-37). Interestingly, when St. John et al. (21) transfected mouse Lcells with CD44 cDNA they found an increase in self-aggrega tion in high-expressing clones. It was felt highly likely that this aggregation phenomenon probably was a consequence of the Lcells expressing at least one ligand for the transfected primate CD44 (21). Whether the interaction observed in our studies results from a receptor-ligand-receptor interaction or a homophilic receptor-receptor interaction is not known but, indisput ably, the clones expressing greater amounts of CD44 aggregated to a considerably greater extent than did those expressing low levels of CD44 (Fig. 5). Since homotypic clumping has been shown to increase the experimental metastatic capacity of tu mor lines (38), it seems quite probable that this effect could have contributed to the in vivo results presented here. Finally, it has been known that the hyaluronate receptor is linked to the actin filaments (39) and Pgpl has been associated with cell movement of fibroblasts (5, 40), making it seem likely that the CD44 molecule may facilitate the locomotory behavior of cells. In our wound migration assay we have noted that the high-expressing clones are indeed more motile than the lowexpressing clones (Fig. 7). Given the importance of cellular motility in the invasive and metastatic process (41), it is tempt ing to speculate that this characteristic also may well have contributed to the in vivo behavior of the various clones. The binding of lymphocytes to high endothelial venules of lymphoid tissue is regulated by more homing receptors than just the CD44 molecule (42). Both the "selectin" LECAM-1 and the integrin VLA-4 molecules are known to play an impor tant role in determining lymphocyte adhesion, and it is probable that the binding behavior of melanoma cells also is a conse quence of coordinate regulation of a variety of surface receptors (43). Interestingly, therefore, the results obtained with the J143 antibody suggest that the selection procedure utilized herein did not result in variation of all cellular adhesion molecules. 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The relationship of embolie homogeneity number size and viability to the incidence of experimental metastasis. Eur. J. Cancer, 9: 223227, 1973. 39. Lacy, B. E., and Underbill, C. B. The hyaluronate receptor is associated with 34^: 425-434, 1990. 43. R|ce, G. E., and Bevilacqua, M. P. An inducible endothelial cell surface glycoprotein mediates melanoma adhesion. Science (Washington DC), 246: 1303-1306, 1989. 6667 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1991 American Association for Cancer Research. Isolation and Characterization of Human Melanoma Cell Variants Expressing High and Low Levels of CD44 Mary Birch, Stephen Mitchell and Ian R. Hart Cancer Res 1991;51:6660-6667. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/51/24/6660 Sign up to receive free email-alerts related to this article or journal. 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