1 The Lautenberg Center for General and Tumor Immunology, The Hebrew University-Hadassah Medical School, Jerusalem, 91120 Israel
2 Department of Immunology, The Weizmann Institute, Rehovot, 76100 Israel
3 Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, Karlsruhe D-76021, Germany
* Present address: Pharmacia & Upjohn, via Pasteur 10, 1-20014 Nerviano (Milan), Italy
Author for correspondence (e-mail: naord{at}md2.huji.ac.il)
Accepted June 25, 2001
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SUMMARY |
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Key words: Adhesion molecules, CD44, Cell motility, Cell rolling, Hyaluronic acid, lymphoma cells
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INTRODUCTION |
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The polymorphic nature of CD44 possibly contributes to its multifunctionality. For instance, CD44 molecules support cell migration (DeGrendele et al., 1997a), mediate T-cell signaling and activation (Taher et al., 1996; Lesley et al., 1993; Naor et al., 1997), as well as lymphocyte recirculation (Guo et al., 1994). CD44 is involved in cell-cell and cell-matrix interactions (Lesley et al., 1993; Naor et al., 1997) and in the presentation of cytokines and growth factors to high affinity receptors (Tanaka et al., 1993; Bennet et al., 1995). It is likely that the CD44 receptor interacts with many ligands, only some of which have been identified. The most solid evidence for CD44 ligands is that relating to glycosaminoglycans (GAGs), such as hyaluronic acid (HA). A single mutation at amino acid position 41 dramatically reduced the ligand binding capacity of the human CD44 receptor (Peach et al., 1993). The affinity of the CD44 receptor for HA appears to depend on post-translational modifications (i.e. N- or O-glycosylations and GAG attachments) (Lesley et al., 1995) as well as on specific structural changes in CD44 oligosaccharides (e.g. gain or loss of sialic acid) (English et al., 1998; Skelton et al., 1998). Cells carrying CD44 may bind HA constitutively, not interact with HA at all, or do so upon activation, depending on low, high or intermediate levels of receptor glycosylation, respectively (Lesley et al., 1995; Lesley et al., 1993; Naor et al., 1997).
The migration of many types of tumors is CD44-dependent. As CD44 is a diverse family of molecules, which includes CD44s as well as CD44v isoforms, specific isoform(s) might be involved in the support of tumor spread. Indeed, it has been found that CD44v molecules, especially those containing variant exon number 6 (v6), are predominantly expressed on some (but not all) progressive and metastatic neoplasms (Naor et al., 1997), including the non-Hodgkins lymphomas (Salles et al., 1993; Stauder et al., 1995). As mentioned above, it has been shown in an animal model that cell surface v6-containing CD44v supports the metastasis of a pancreatic adenocarcinoma cell line (Günthert et al., 1991). It was postulated that tumor cells, and especially lymphoma cells, resemble activated lymphocytes in their migratory properties and in the route of migration to the lymph nodes (Herrlich et al., 1993). In addition, it was reported that such activated lymphocytes express CD44v and its expression promotes accelerated lymphocyte proliferation following their stimulation (Moll et al., 1996). A possible mechanism for CD44v-promoted proliferation is the trapping of growth factors by this receptor (Bennett et al., 1995; Jones et al., 2000; Sherman et al., 1998). As mentioned above, CD44v seems to confer invasive properties (Günthert et al., 1991), perhaps, again, by capture of growth factors and their subsequent presentation to the relevant cell surface receptors (van der Voort et al., 1999) that activate the cytoskeleton.
All known CD44 isoforms carry a GAG binding motif. We previously showed that CD44v isoforms exhibit broader GAG binding specificity (which may reflect higher avidity) than CD44s (Sleeman et al., 1997), possibly due to the ability of CD44v to form cell surface clusters (Sleeman et al., 1996a). GAG binding may confer a number of different cellular functions, such as enhanced proliferation (Moll et al., 1996), or cell migration (DeGrendele et al., 1997a). To dissociate these effects, we decided to study how expression or lack of expression of CD44 alternatively spliced exons, which consitutively bind HA, influences the different tumor cell functions. To be independent of induced proliferation or induced alternative splicing, we transfected cDNAs driven by a constitutive promoter and selected clones expressing an equal level of the exogenous CD44v isoform. We report here that in the LB cell line, CD44v, but not CD44s, mediates GAG binding, rolling on HA substrate under physiological shear stress and migration of the tumor cells into lymphatic tissue upon subcutaneous (s.c.) injection. It should be emphasized, however, that in other cell lines CD44s can support cell rolling under shear stress (DeGrendele et al., 1997b). These properties disappear when the cell surface CD44v contains a mutation at the HA binding site located at the distal end of the molecules constant region. These findings show for the first time that the CD44 variant, but not standard CD44, supports the rolling of the lymphoma cells and suggest that the variable region of the cell surface CD44 receptor controls the ability of the constant region binding domain to support lymphoma cell migration in vitro and its accumulation in the lymph nodes in vivo.
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MATERIALS AND METHODS |
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Cell cultures
LB T-cell lymphoma cells derived from a spontaneous tumor of a BALB/c mouse (Ruggiero et al., 1985) were cultivated in LB medium, consisting of a 1:1 mixture of RPMI-1640 and DCCM-1 media (Sigma, St Louis, MO), supplemented with 5% heat-inactivated fetal calf serum (FCS) (Sigma), 2 mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes and 5x10-5 M ß-mercaptoethanol. Hybridomas obtained from American type culture collection (ATCC) were grown in RPMI-1640 medium enriched with 10% FCS, and expanded in nude mice. Cell cultures were incubated at 37°C in 5% humidified CO2. For FACS analysis, cells were stained with 3% FCS in PBS (FACS medium). The following antibiotics were used in the selective medium: 0.3 mg/ml Geneticin-G418 (GIBCO #41811; Life Technologies, Paisley, UK) and 0.5 mg/ml Zeomycin (ZeocinTM #R250-01; Invitrogen, Carlsbad, CA).
Monoclonal antibodies, enzymes and other reagents
The following rat anti-mouse pan CD44 monoclonal antibodies (mAbs) (constant region-specific) were produced from ATCC hybridomas: IM7.8.1 (IgG2b) (Trowbridge et al., 1982), KM81 (IgG2b) (Miyake et al., 1990) and KM114 (IgG1) (Miyake et al., 1990). The rat anti-mouse CD44v4 (10D1; IgG1) and rat anti-mouse CD44v6 (9A4; IgG1) were previously described (Weiss et al., 1997). The rat anti-mouse cell surface immunoglobulin idiotype (4D2; IgG2b), kindly provided by J. Haimovich, Tel Aviv University (Maloney et al., 1985), was used as an irrelevant isotype control. In addition, in the flow cytometry assay we used the following ATCC rat anti-mouse mAbs to analyze expression of cell adhesion molecules: anti-CD11a (M7/14; IgG2a) (Davignon et al., 1981), anti-CD11b (M1/70; Ig2a) (Springer et al., 1979), anti-CD18 (M18/2; IgG2a) (Sanchez-Madrid et al., 1983), MEL-14 (MEL-14.D54; IgG2a) (Gallatin et al., 1983) and anti-ICAM-1 (YN1/1.7; IgG2a) (Takei, 1985). The hybridomas were injected into the peritoneal cavity of nude mice and the mAbs were purified from the ascitic fluid by protein S-sepharose chromatography as described previously (Rochman et al., 2000). Biotin-conjugated anti-mouse CD44s was produced by biotinylation of KM81 mAb, as described (Yang et al., 1995). FITC-conjugated F(ab)2 goat anti-rat (goat IgG (H and L]) was obtained from Jackson Immunoresearch, West Grove, PA. HRP-peroxidase-conjugated streptavidine and R-phycoerythrine-conjugated streptavidine were obtained from Jackson Immunoresearch. Biotinylated anti-bromodeoxyuridine (BrdU) antibody was obtained from Biodesign International (Saco, Maine), CyTM5-conjugated streptavidin was obtained from Jackson Immunoresearch, BrdU (B5002) and propidium iodide were purchased from Sigma. The following GAGs and enzymes were obtained from Sigma: hyaluronic acid (H5388), chondroitin 4, 6-sulfate C (CS) (C4384), keratan sulfate (KS) (K3001), heparin (H3393), heparan sulfate (HS) (H7641), heparinase (H2519) and hyaluronidase (H3757).
Construction of CD44v4-v10 and CD44s expression plasmids
CD44v4-v10 cDNA was synthesized from 500 ng polyadenylated RNA prepared from mouse keratinocytes (KLN205 from ATCC), using the oligonucleotide primers ORF-3' and ORF-5', which overlap the open reading frame of the 3' (nucleotide positions 58-80) and 5' (nucleotide positions 1216-1244) constant regions of the CD44 DNA (Zhou et al., 1989), as described in Fig. 1A,B. ORF-5' includes the EcoRI cloning site. After 30 PCR cycles (95°C, 45 seconds; 60°C, 1 minute; 72°C, 3 minutes), the amplification products were purified, using a Qiagen PCR product purification kit. Purified products were cloned into the pZeoSV vector (#V855-01, Invitrogen, Carlsbad, CA) to generate the pZeoSVCD44v4-v10 plasmid. CD44s cDNA was prepared as described (Tölg et al., 1993), and cloned into the EcoRI site of pZeoSV, resulting in the pZeoSVCD44s plasmid. The correctness of the insert was confirmed by sequencing.
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Generation of CD44 stable transfectants
Logarithmically growing LB cells were harvested, washed with RPMI-1640 medium and then resuspended in LB medium containing 20% FCS. A quantity of 20 µg from the following expression vectors was added to 800 µl of cell suspension (4x106 cells per ml): empty pZeoSV vector (to generate transfectants designated LB-TRo cells), or pZeoSVCD44s, pZeoSVCD44v4-v10 and pZeoSVCD44v4-v10 mutated at the HA binding site (to generate transfectants designated LB-TRs, LB-TRv and LB-TRvM cells, respectively). Transfection was carried out by electroporation at 380 V and 500 µF, using a BIO-RAD, Gene Pulser (Bio Rad Laboratories, Hercules, CA). The cells were cultured in 6 ml LB medium containing 20% FCS, incubated for 24 hours and then grown in the same medium supplemented with 0.5 mg/ml Zeomycin to select for the transfected cells. Transfected cell clones were obtained by limiting dilution and cDNA expression was confirmed by exon-specific RT-PCR, by using the primers described previously (Rochman et al., 2000) and the primer orientations shown in Fig. 1A.
Generation of green fluorescence protein (GFP) stable transfectants
To detect dissemination of the lymphoma cells following their s.c. inoculation, LB, LB-TRs, LB-TRv and LB-TRvM cell lines were transfected with GFP-N1 expression vector (#6091-1, Clontech Laboratories, Palo Alto, CA), which contains jellyfish GFP cDNA (Chishima et al., 1997). The transfection was carried out as described above, except that 1 mg/ml G418 (GIBCO) was used for selection. Lymphoma cells expressing GFP were electronically sorted.
Flow cytometry
Lymphoma cell surface expression of CD44 was assessed by indirect immunofluorescence and analyzed by flow cytometry as described (Zahalka et al., 1993). Briefly, 1x106 cells were washed with FACS medium and incubated on ice for 45 minutes with 100 µl of 100 µg/ml mAbs. The cells were then washed with cold FACS medium and reincubated with 100 µl of 20 µg/ml FITC-conjugated goat anti-rat IgG (H and L chains). After 45 minutes on ice, the cells were washed and analyzed by flow cytometry using a FACStar (Becton Dickinson, Mountain View, CA). Cells incubated with the second antibody alone served as control.
HA and CS binding assay
Lymphoma cells (1x106) were incubated at 4°C with 20 µg/ml fluorescein-labeled HA (FL-HA) or 50 µg/ml fluorescein-labeled CS (FL-CS) in 100 µl FACS medium for 1 hour and analyzed by flow cytometry. The FL-HA and FL-CS were prepared as described (de Belder and Wik, 1975), except for FL-CS, where biotin-hydrazine was replaced by fluorescein hydrazide (Yang et al., 1995). The same cells incubated alone (autofluorescence) or with FL-HA or FL-CS and 1 mg/ml unlabeled HA or CS to competitively block ligand binding served as controls. To ascertain that the FL-HA and FL-CS binding was CD44-dependent, LB cells were also incubated with FL-HA or FL-CS in the presence of 100 µg/ml KM81 anti-CD44 mAb.
Shear assays on GAG-coated substrates
Cellular interactions on substrates coated with purified GAGs under shear flow were measured in a parallel-plate flow chamber as previously described (Lawrence et al., 1994; Clark et al., 1996). A polystyrene plate on which purified ligand had been adsorbed, was assembled in a parallel plate laminar flow chamber (260 µm gap) and mounted on the stage of an inverted phase-contrast microscope (Diaphot 300; Nikon, Japan). Hyaluronic acid (HA, 1 mg/ml or 0.5 mg/ml) or one of the other purified GAGs (1 mg/ml) was dissolved in PBS and adsorbed overnight at 4°C onto plastic petri dishes. The plates were blocked with 2% human serum albumin (Fraction V, Calbiochem, San Diego, CA) at 37°C for 2 hours. Flow was generated by an automated syringe pump (Harvard Apparatus, Natick, MA) attached to the outlet side of the flow chamber. Cultured cells were washed twice with cation-free H/H medium (HBSS containing 10 mM Hepes, (pH 7.4) and 2 mg/ml BSA (Sigma, Fraction V)), concentrated to 106 cells/ml and perfused into the flow chamber at the desired shear stress for 60 seconds. Cellular interactions in several fields (1-4 fields of 0.17 mm2 per data point) were videotaped and quantified directly from the monitor screen. Attachment of cells perfused under shear flow over an adhesive HA-coated substrate was considered stable if it was followed by rolling over a distance of at least 10 cell diameters or for at least 25 seconds. Detachment assays were performed using cells that had bound at stasis to ligand-coated plates for 30 seconds. After cell binding, wall shear stress was increased step-wise every 5 seconds (by a programmed set of flow rates delivered by the syringe pump) up to 15 dyn/cm2. At the end of each 5 second interval of increase in shear stress, the number of cells that remained bound relative to the number of cells that were attached at stasis was calculated. Background adhesive interactions of the cells with albumin-coated substrate were negligible, and essentially none of the transfectant cells remained bound to the control substrate above a shear stress of 0.1 dyn/cm2. The contribution of cells rolling into the observation field from upstream fields was minimized by locating the field at the upstream edge of the spot of adsorbed ligand. All assays were performed at room temperature (RT). For antibody blocking assays, 107 cells/ml were preincubated for 5 minutes at 4°C in binding medium with 100 µg/ml of different purified mAbs. The cells were diluted 1:10 in medium without washing out the antibodies and the suspension was perfused into the flow chamber. For inhibition with soluble GAGs, cells were suspended for 5 minutes at 24°C in binding medium in the presence of 1 mg/ml of the specified GAG and then perfused into the flow chamber. The GAG concentration was kept constant in the perfusate throughout the flow assay.
Co-precipitation of CD44 proteins with glycosaminoglycans by cetylpyridinium chloride
Cetylpyridinium chloride (CPC) precipitation (Scott, 1961; Lee et al., 1992) was used to analyze the binding of HA and other GAGs to the CD44 molecules of LB-TRv and LB-TRs cells. A quantity of 107 cells was washed three times in PBS, then lysed in cold PBS buffer containing 0.5% NP-40 and 1 mM phenylmethylsulfonyl fluoride (PMSF), and kept on ice for 30 minutes. The lysate was centrifuged at 10,000 g for 10 minutes to remove insoluble material. Aliquots containing 100 µl of the lysate supernatant were mixed with 50 µg of HA, CS, heparin, HS or KS. After incubation for 1 hour at RT, 350 µl of 1.43% CPC were added to the samples and the suspensions were stirred and incubated at RT for 1 hour. The suspensions were then centrifuged (Eppendorf Inc., Fermont, CA) at 10,600 g for 10 minutes. After rinsing three times with 1% CPC containing 30 mM NaCl, the pellets were dissolved in 50 µl sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromphenol blue). After fractionation on SDS-PAGE (7.5% polyacrylamide) CD44s and CD44v proteins present in the pellet, were identified by western blot, using KM81 anti-CD44 mAb.
Assessment of local tumor growth and lymph node invasion
A quantity of 3x106 lymphoma cells was injected s.c. into the left flank, close to the hind limb of female BALB/c mice (5-8 mice per group). Development of the tumor at the injection site was monitored by recording tumor diameter (in cm) over time. To determine lymphoma invasion into the peripheral axillary and brachial lymph nodes, the organs were removed at various intervals following s.c. inoculation of the fluorescent tumor cells (GFP-expressing cell lines designated LB-G, LB-TRs-G, LB-TRv-G and LB-TRvM-G cells; G=green). Five mice were killed at each time point. The fluorescence of the lymph node was measured by a computerized FujiFilm Fluorescence Image Analyzer FLA-2000 (Fuji Photo Film, Japan), using an excitation wavelength of 473 nm and an emission wavelength of 520 nm. Quantitation of the fluorescence in the organ is expressed as normalized emission intensity per area of the whole organ, using the following equation:
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where MFI is the mean fluorescence intensity of the whole organ and the MFI of a normal lymph node is a measure of background autofluorescence.
To evaluate the effect of specific treatment with antibody or enzyme on local tumor growth or lymph node invasion, PBS, 150 µg IM7.8.1 anti-CD44 mAb, 150 µg 4D2 isotype matched control mAb, 20 units of hyaluronidase or 20 units of heparinase (similar specific activity) were injected into BALB/c mice. The intraperitoneal (i.p.) injections were started 6 days after s.c. tumor inoculation and continued every other day for 12 days (a total of 6 injections). Local tumor growth and lymph node invasion were evaluated as indicated above.
Immunofluorescence analysis of lymphoma cell accumulation and proliferation in lymph nodes
A quantity of 0.5x106 green lymphoma cells (LB-TRv-G, LB-TRvM-G, LB-TRs-G or LB-G cells) was injected intra-venously (i.v.) into BALB/c mice. The lymph node-infiltrating tumor cells were analyzed by three-color flow cytometry on different days post-injection, to simultaneously assess their representation among the lymph node cell population and the proportion of dividing cells among the infiltrating cells, as described (Kastan et al., 1991). Axillary lymph nodes were removed 4, 8 and 12 days post tumor injection and lymph node cells were incubated with 10-5 M BrdU for 30 minutes at 37°C in a CO2 incubator. The cells were then harvested, fixed in cold 70% ethanol for 16 hours, and DNA was denatured in 2N HCl/0.5% Triton X-100 (30 minutes, RT). To determine the percentage of GFP-expressing tumor cells in the lymph node, cells of high green fluorescence were gated. GFP-positive cells from each cell line were analyzed for BrdU staining and DNA content. Incorporation of BrdU was detected by staining with biotin-conjugated anti-BrdU antibody followed by CyTM5-conjugated streptavidin, and total DNA was stained with propidium idodide, 5 µg/ml. The doubly treated cells were analyzed by flow cytometry, using a two laser FACSCaliber (Becton Dickenson). The percentage of BrdU-positive cells in the total GFP-positive population was recorded. This doubly fluorescent cell population represents dividing cells in the S-phase.
All the experiments described are representative of at least two, and in most cases, three trials all showing similar results.
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RESULTS |
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LB cells transfected with CD44v4-v10 (CD44v) cDNA were cultured in selective medium and cloned. Exon-specific RT-PCR analysis revealed that three representative clones of the transfected cells expressed the CD44v transcript (not shown). The total transfected cell population was designated LB-TRv. The individual clones were numbered (e.g. LB-TRv2, LB-TRv3). In parallel, LB cells were transfected with a control plasmid (designated LB-TRo) or with CD44s cDNA (designated LB-TRs) to overexpress this transcript. Parental LB cells, as well as LB-TRo, LB-TRs and LB-TRv cells, were analyzed for their ability to bind HA and other GAGs from the solution, to attach to GAGs under shear stress or to establish primary tumors and lymph node colonies in vivo.
LB cells, LB-TRs cells and six clones from two separate transfections (not shown), LB-TRv cells and two representative clones (TRv2 and TRv3) of six, from two separate transfections (Fig. 2A) or LB cells transfected with empty vector (LB-TRo cells; not shown), all expressed the CD44 constant epitope recognized by KM81 anti-pan CD44 mAb. By contrast, anti-CD44v4 and anti-CD44v6 mAbs stained only LB-TRv cells and their clones (Fig. 2A). The LB-TRv cells and their clones were also the only cells that bound FL-HA from the solution. Binding was blocked by an excess of non-labeled HA (Fig. 2B), or by KM81 anti-CD44 mAb (Fig. 2C), but not by irrelevant isotype-matched control mAb (4D2; data not shown), proving that the interaction is CD44-dependent. Note that LB, LB-RTo and LB-TRv cells expressed the pan CD44 epitope to an almost equal extent. The LB-TRs cells, like LB and LB-TRo cells, neither expressed v4- and v6-encoded epitopes nor bound fluorescein-labeled HA, although they were the only cells that overexpressed CD44s (Fig. 2A). This finding supports the notion that the acquisition of the HA binding capacity by LB cells is related to the expression of CD44v-encoded epitopes rather than to overexpression of CD44 molecules. Flow cytometry analysis of parental LB cells as well as of LB-TRv cells and their clones, revealed that transfection of CD44v4-v10 cDNA into LB cells did not markedly influence the logarithmic expression of three different constant CD44 epitopes detected with anti-KM114, anti-IM7.8.1 and anti-KM81 mAbs, or of other cell surface adhesion molecules detected with anti-ICAM-1, anti-CD18, anti-CD11a and anti-CD11b mAbs (data not shown). In addition, none of the cell lines and clones expressed MEL-14.
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A mutation in the HA-binding domain of cell surface CD44v inhibits its GAG binding capacity and interferes with its rolling attachments on HA substrate
The HA binding site of CD44v4-v10 was destroyed by replacing arginine with alanine at position 43 (analogous to position 41 in human CD44), and the construct was transfected into LB cells. The resulting transfectants, designated LB-TRvM, were cultured in selective medium and cloned. Flow cytometry analysis (Fig. 5A) revealed that, in comparison with LB-TRv cells, the total cell population of LB-TRvM cells, as well as six clones of two separate transfections (not shown), all expressing the mutated CD44v, lost their ability to bind soluble HA (Fig. 5A, right panels), whereas the level of their CD44 (pan and v6) (Fig. 5A, left panels) and CD18 adhesive proteins (not shown) remained unchanged. Also, CPC analysis (not shown) showed that LB-TRvM cells bound neither HA nor other GAGs (heparin, HS, CS and KS). The inability to bind CS by LB-TRvM cells was confirmed by flow cytometry (Fig. 5B). Furthermore, the mutated CD44v4-v10 isoform expressed in LB-TRvM cells did not exhibit any attachment and rolling capacity above the corresponding endogenous activities of the standard isoform expressed in LB-TRs cells (Fig. 4D), as shown under both physiological and hyperphysiological shear stress (data not shown).
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Cell lines and clones of nonfluoresceinated LB-TRv, LB-TRs and LB-TRvM, displayed local tumor development and lymph node invasion (8 mice in each group) that were only slightly faster (but statistically significant) than those of the corresponding fluoresceinated cells (derived from separate CD44 transfections) (data not shown), thereby confirming the growth advantage of cells expressing CD44v.
The above findings are supported by a subsequent experiment in which systemic injection of hyaluronidase 6 days after fluorescent LB-TRv lymphoma s.c. inoculation and then every other day for 12 days, markedly reduced local development of the tumor (Fig. 7A) and lymph node invasion (Fig. 7B). Administration of heparinase of the same specific activity, following an identical protocol, influenced neither local tumor growth nor lymph node invasion (Fig. 7A,B). In addition, injection of IM7.8.1 anti-CD44 mAb, but not of isotype-matched control mAb (4D2; to exclude Fc-mediated killing), adhering to the protocol used for enzyme treatment, suppressed local development of fluorescent LB-TRv cells (Fig. 7C) and their invasion of the lymph nodes (Fig. 7D). Note that IM7.8.1 anti-CD44 mAb interferes with HA binding to activated LB lymphoma cells (Vogt Sionov and Naor, 1997).
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DISCUSSION |
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Since the establishment of metastatic colonies in secondary organs may be dependent on the trafficking potential of the cancer cells, we focused our attention on the cell motility of LB cells. Our results show that CD44v, rather than CD44s, can provide the dynamic reversible bonds required for the interaction between migrating cells and their substrate (Duband et al., 1988). The finding that LB-TRv cells display CD44-dependent binding to HA under flow stress and roll under physiological shear stress, suggests that the CD44v-ligand pair rapidly establishes bonds that are sufficiently strong to maintain provisional cellular contacts with the substratum, but not strong enough to cause permanent cell arrest (Lauffenburger, 1991; DiMilla et al., 1993). However, these cellular contacts can be established by the interaction of CD44v with immobilized HA, but not with other immobilized GAGs (Fig. 4A). CD44v binding to non-HA GAGs is presumably not sufficiently strong to resist cell detachment from the substratum under physiological fluid shear stress.
LB-TRs and parental LB cells displayed weak rolling attachments on HA substrate under low shear stress (0.2-0.8 dyn/cm2), but failed to bind HA from the solution (Fig. 2). This dichotomy was also observed in certain cells whose cytoplasmic tail-truncated CD44 does not bind soluble HA, although it is still able to interact with immobilized HA (Lesley et al., 1992). These findings prove that under low shear stress (0.2-0.8 dyn/cm2), immobilized HA can weakly interact with cell surface CD44s, possibly due to its ability to crosslink cell surface receptors. This no longer holds true, however, when the shear stress approaches physiological levels. Under such conditions, only LB cells expressing CD44v can accumulate and roll on HA, possibly due to its ability to undergo oligomerization upon interacting with the multivalent HA ligand (Sleeman et al., 1996a). The oligomerization of CD44v may lead to the coalescence of the microdomains (Ilangumaran et al., 1999), resulting in the redirection of cytoskeleton actin bundles, which are associated with the microdomains in the cytoplasmic leaflets (Oliferenko et al., 1999). If this is the case, the association of CD44v, rather than of CD44s, with the detergent-resistant complexes in raft domains may explain how this isoform mediates HA-dependent rolling in LB-TRv cells.
Using a parallel plate flow chamber simulating physiological shear stress, it was previously demonstrated that the CD44 receptor can support HA-dependent rolling attachments (primary adhesion) of lymphoid cell lines (both activated and nonactivated) and of primary activated lymphocytes (DeGrendele et al., 1996; DeGrendele et al., 1997b; Mohamadzadeh et al., 1998), tonsillar lymphocytes (Clark et al., 1996; Estess et al., 1998), lymphocytes from patients with autoimmune diseases (Estess et al., 1998) and lymphoma cells (Clark et al., 1996). It is clear that in at least some cell types, such as the BW5147 cell line (DeGrendele et al., 1996), CD44s is involved in the cell rolling attachments under physiological shear, as in this cell line only the standard form of CD44 is expressed (Trowbridge et al., 1982; DeGrendele et al., 1997b). By contrast, we now show, for the first time, that cell surface CD44v (CD44v4-v10), rather than CD44s, is involved in LB lymphoma rolling attachment on HA substrate under physiological shear stress. It seems unlikely that integrins play an independent or mandatory role in the dynamic CD44-mediated binding of the LB lymphoma to HA substrate, as the level of these molecules on LB-TRv and LB-TRs cells is almost identical, yet only the former display efficient rolling. Selectins are definitely not involved in the rolling process on HA, as LB cells are deficient in these molecules, and also because rolling was independent of Ca2+, which is required for selectin function.
These in vitro rolling studies are matched by their in vivo counterparts. LB-TRv cells form local tumors and accumulate in the peripheral lymph nodes much more rapidly than do parental LB cells and LB-TRs cells. Furthermore, both the rolling of LB-TRv cells on HA substrate in vitro and lymph node accumulation of LB-TRv cells in vivo are dependent on the CD44-HA interaction, as these events are disrupted in LB-TRv cells expressing CD44v mutated at the HA binding site. These conclusions are supported by the observation that LB-TRv cells treated with anti-CD44 mAb (both in vitro and in vivo), an excess of soluble HA (in vitro), or the enzyme hyaluronidase (in vivo) lost their ability to roll on HA substrate in vitro and to form aggressive tumors in vivo. These correlated in vitro and in vivo findings suggest that a similar mechanism supports the migration of LB-TRv cells both in the flow chamber and in the intact animal. If this assumption is correct, the enhanced accumulation of LB-TRv cells in the peripheral lymph nodes, when compared with that of LB and LB-TRs cells, should be the result of their efficient migratory capacity rather than of intensive local proliferation in the invaded organ. In agreement with this premise, it was found that the proliferation rate of LB-TRv cells in culture and in the lymph nodes ex vivo (following i.v. injection) is similar to that of LB, LB-TRvM and LB-TRs cells, while the absolute number of LB-TRv cells detected in the same organ is far larger than that of the other cell lines. These findings suggest that cell surface CD44v supports efficient migration of the tumor cells into the lymph nodes, possibly following their previous localization in the lung or spleen. This could be the result of faster and/or more efficient migration (as shown in vitro in the flow chamber) or of larger numbers of migrating cells entering the lymph nodes, events that are sensitive to anti-CD44 mAb and hyaluronidase. The alternative explanation that LB-TRv cells proliferate at higher rates than the other cell lines, due to their preferential stimulation by lymph node-localized HA or growth factors, does not reconcile with the data of Fig. 8, which shows a similar proliferation rate for all the cell lines, despite the fact that LB-TRv cells exhibited faster accumulation. Furthermore, since LB-TRv cells were transfected with CD44v4-v10 cDNA, they do not express v3, an exon product known to bind HS-recognizing growth factors (Bennett et al., 1995; Jones et al., 2000). Hence, such factors are not involved in the stimulation of these tumor cells.
Note that the number of TRv-GFP cells from day 4 to day 12 increased by 6.5-fold (as percent cells in the lymph node), whereas the rest of the cell lines increased by 1.3 (TRvM-GFP cells) to 2.5-fold at that time. Taking into account that about 50-60% of all cell types are engaged in division, the number of cells (including the number of TRv-GFP cells) at day 12 would be expected to be higher. This phenomenon can be explained by the high death rate of cells accumulating in the lymph node as well as by the differential selective death between TRv-GFP cells and TRvM-GFP cells or the other cell lines. Our findings do not exclude accelerated proliferation of LB-TRv cells in the primary tumor growth (or in intermediate organs) and the consequent intensive release of mobile cells that migrate into the lymph nodes. However, to allow direct analysis of tumor cell accumulation in the lymph nodes, the various cell lines were injected intravenously rather than subcutaneously.
How can we explain the ability of anti-CD44 mAb or hyaluronidase to reduce local tumor growth and tumor accumulation in the lymph node when injected six days after lymphoma inoculation (Fig. 7)? At this point in time, inhibition of tumor migration by these agents can not explain the reduced tumor growth and we must, therefore, seek an alternative interpretation. It has been previously reported that interference with integrin- (Meredith and Schwartz, 1997) or CD44- (Tian et al., 2000) dependent cell attachment to substrate (e.g. by antibody) activates the signaling of programmed cell death. A similar mechanism of apoptosis may be valid after injection of anti-CD44 mAb or hyaluronidase into mice bearing LB lymphoma, as both agents could disrupt the tumor-substrate interaction in the primary growth and secondary organs, an interaction essential to adhesion-dependent survival. Consequently, the tumor cell reservoir in the local growth may be reduced and a smaller number of cells released and seeded in the lymph nodes. While this notion is not necessarily incompatible with the interpretation that anti-CD44 or hyaluronidase also interferes with tumor cell migration from the local growth to the lymph nodes, it provides a possible explanation for the failure of all cell lines other than TRvG cells to grow efficiently in the nodes following i.v. injection (Fig. 8). According, we suggest that most of the lymph node-infiltrating lymphoma cells that do not express an efficient HA-binding CD44 receptor, fail to interact with the substrate and undergo a process of apoptosis. Some of these cells survive and proliferate, perhaps due to their ability to attach to the ECM via integrins or other cell surface adhesion receptors. By contrast, LB-TRv cells have a relative survival advantage in the lymph node environment as they are the only cells that can establish efficient contacts with ECM via the CD44-HA interaction and, therefore, initiate DNA synthesis.
Although the suggestion that anti-CD44 mAb or hyaluronidase interfers with LB-TRv cell migration is based mostly on the correlation with the in vitro rolling studies, it clearly points to the significance of the CD44-HA interaction in lymphoma spread and to the practical implications of the finding (i.e. the potential use of CD44 or HA as therapeutic targets).
We suggest that the CD44-HA interaction is essential to lymph node invasion by the lymphoma cells. In this context, it should be noted that the lymph nodes and their afferent lymphatics contain hyaluronate (Fraser and Laurent, 1989; Aruffo et al., 1990), which may support the penetration of the tumor cells into these organs via the afferent lymphatics, as shown by us previously (Zahalka et al., 1995). Furthermore, as LB-TRv cells are deficient in L-selectin (Mel-14) they cannot enter the lymph nodes via the high endothelial venule (HEV) (Springer, 1994).
We previously showed that the HA9 cell line, a variant selected from the LB cell propulation by multiple cycles of binding to HA substrate, displayed enhanced expression of v4 and v6 CD44 epitopes and constitutively bound HA (Vogt Sionov and Naor, 1997). But, unlike LB-TRv cells, this cell line showed a reduced capacity to invade the lymph nodes when compared with the parental LB cells. Quantitative and/or qualitative differences in CD44 isoform expression may explain why the disseminational behavior of the tumors is at variance.
We would like to avoid generalizations at this time since the relationship between HA binding and tumor properties seems to be complex. For instance, in a pancreatic carcinoma model, CD44v expression was correlated with HA binding (Sleeman et al., 1996b) and metastatic behavior (Günthert et al., 1991). However, hyaluronidase overexpression on the surface of these cells, which efficiently abolished any intereaction with HA, did not alter their metastatic potency (Sleeman et al., 1996b). In human lymphoma and melanoma, HA binding was correlated with CD44s, not CD44v, overexpression (Sy et al., 1991; Bartolazzi et al., 1994; Bartolazzi et al., 1995) and tumor growth in vivo was supported by cell surface CD44s, but not by an HA-nonbinding CD44s mutant (Bartolazzi et al., 1994). As the human and mouse CD44 variable region shows only partial homology (65%) (Screaton et al., 1993), we suggest that its influence on the functional behavior of the entire molecule (including HA binding and the support of tumor progression) differs in the two species. However, because CD44 displays contradictory molecular functions, we need to know more about the mechanisms of CD44 action before venturing any further interpretation of these findings.
The same caution is warranted in evaluating the clinical data. Attempts to correlate cell surface expression of CD44 in general and CD44v in particular with the malignant status of human tumors have yielded conflicting results (Naor et al., 1997). Nevertheless, there is well-documented data on many human malignancies, e.g., renal epithelial cancer (de Alava et al., 1998), papillary thyroid carcinoma (Kurozumi et al., 1998), vulvar cancer (Tempfer et al., 1998), pancreatic cancer (Rall and Rustgi, 1995) and non-Hodgkins lymphomas (Salles et al., 1993; Stauder et al., 1995), showing that the more progressive or metastatic phenotypes express v6-containing CD44 isoforms (Naor et al., 1997).
Furthermore, we believe that this report makes a significant contribution to our understanding of cell migration in malignant diseases and perhaps in other diseases whose pathology is cell trafficking-dependent (as shown by us for inflammatory cell migration in insulin-dependent diabetes) (Weiss et al., 2000). It has been shown in an in vitro assay that the LB lymphoma uses CD44v for the rolling interaction with HA substrate. This activity may mimic in vivo adhesive and migratory cellular processes on the HA of the vasculature and ECM, essential to lymphoma dissemination. Furthermore, at a given level of expression, CD44v confers on LB cells three properties: GAG binding, rolling under physiological shear forces and enhanced accumulation in the lymph nodes. This strongly suggests that one and the same molecule is responsible for similar effects both in vitro and in vivo.
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