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Tumor Biology

In Vivo Induction of Insulin-like Growth Factor-I Receptor and CD44v6 Confers Homing and Adhesion to Murine Multiple Myeloma Cells

Kewal Asosingh, Ursula Günthert, Marleen H. C. Bakkus, Hendrik De Raeve, Eva Goes, Ivan Van Riet, Ben Van Camp and Karin Vanderkerken
DOI:  Published June 2000

Abstract

One of the main characteristics of multiple myeloma (MM) cells is their specific homing and growth in the bone marrow (BM). Differences between stroma-dependent and -independent MM cell lines may reveal key molecules that play important roles in their homing to the BM. We addressed this topic with a murine MM model, including the in vivo 5T33MM (5T33MMvv) stroma-dependent cell line and its in vitro stroma-independent variant (5T33MMvt). Fluorescence-activated cell-sorting analysis showed expression of insulin-like growth factor (IGF)-I receptor and CD44v6 on all 5T33MMvv cells but not on 5T33MMvt cells. Checkerboard analysis and adhesion assays revealed IGF-I-dependent chemotaxis toward BM-conditioned medium and involvement of CD44v6 in the adhesion to BM stroma of only 5T33MMvv cells. However, when 5T33MMvt cells were injected in vivo (5T33MMvt-vv), after 18 h the MM cells harvested from BM were IGF-I receptor and CD44v6 positive. This up-regulation was confirmed in 5T33MMvt-vv cells isolated from terminally diseased animals. These 5T33MMvt-vv cells exhibited IGF-I-dependent chemotaxis and CD44v6-dependent adhesion to BM stroma. In vitro culture of the 5T33MMvt-vv cells could completely down-regulate IGF-I receptor and CD44v6. In fact, we could show that direct contact of 5T33MMvt cells with BM endothelial cells is a prerequisite for IGF-I receptor and CD44v6 up-regulation. These data indicate that the BM microenvironment is capable of up-regulating molecules such as IGF-I receptor and CD44v6, which facilitate homing of MM cells to the BM and support their adhesion to BM stroma.

INTRODUCTION

MM 3 is a B-cell malignancy characterized by the expansion of plasma cells in the BM that secrete high levels of immunoglobulins and by the development of osteolytic lesions. Because the origin of MM cells is post germinal (1) , it is assumed that their localization in the BM implies migration from the vascular to the extravascular compartment of the BM. This migration to the BM is referred to as“ homing.” However, little is known about the mechanisms of this homing. Butcher and Picker (2) described the homing of lymphocytes as a process of several subsequent steps. After an initial, reversible rolling on the vascular endothelium, an activation-dependent arrest occurs, followed by a transendothelial migration. Depending on the homing site, different adhesion molecules and chemotactic factors, which are essential molecules in this process, are involved, making this homing highly specific.

The BM stroma provides the MM cells with a microenvironment that is essential for their survival and growth (3, 4, 5, 6, 7) . Several molecules have been reported to be involved in this complex process (6 , 8) , and most probably others are yet to be discovered.

Differences between stroma-dependent and -independent MM cells such as the 5T33MM cell line may reveal molecules that play important roles in the homing of MM cells to the BM. The 5TMM cell lines originated from spontaneously developed MM in elderly mice (9 , 10) and have since been propagated in young syngeneic animals by i.v. transfer of diseased BM cells harvested from tumor-bearing mice. This model is close to the human disease and is used as a model for human MM by several groups, including ours (11, 12, 13, 14, 15, 16, 17, 18) . Analogous to the human situation, the frequency of myeloma development in these mice is age related. Animals diseased by i.v. injection of malignant BM cells show expansion of monoclonal plasma cells in the BM, together with the development of osteolytic lesions, depressed concentrations of normal immunoglobulins in the serum, and a tumor load-related paraproteinemia. The 5T33MMvv experimental mouse model has been characterized in a previous study (9 , 19) . These in vivo-growing MM cells can be grown in vitro for a few weeks when cultured on a BM stroma feeder layer. From such cocultures, a BM stroma-independent variant of the 5T33MMvv cells, the 5T33MMvt cell line, has been obtained. The latter cells can be cultured and expanded in vitro limitlessly without a stromal feeder layer.

Recently, we reported IGF-I as a BM stroma-derived chemoattractant factor for 5T2MM cells (20) . This pleiotropic cytokine has high local concentrations in the BM and is produced by osteoblasts (21, 22, 23) , BM stromal cells (24 , 25) , and bone endothelial cells (26) . IGF-I has been reported to up-regulate the expression of CD44 splice variants (CD44v). Blockage of the IGF-I signaling pathway could inhibit this up-regulation, in particular that of CD44v6 (27) .

CD44 is an adhesion molecule known to be involved in the interaction between hematopoietic cells and BM stroma (28) . This molecule, engaged in the adhesion of murine (29) and human (5) MM cells, includes a family of broadly distributed cell surface glycoproteins with a variety of functions (30, 31, 32, 33) . The multitude of functions is most probably due to the existence of numerous CD44v isoforms. The CD44 gene contains 20 exons classified into two groups: standard exons (1 s to 10 s) and variant exons (1 v to 10 v). The standard exons encode for the common parts of the CD44 family. The CD44 splice variants can contain different combinations of the variant exons (1 v to 10 v; Refs. 32 ,, 34 ). The expression of these variant isoforms is highly restricted and is correlated with specific processes, such as leukocyte activation and malignant transformation (30) . In human MM, the expression of CD44v is correlated with different degrees of severity of this disease (35 , 36) .

In this study we compared the biological functions of the 5T33MMvv and 5T33MMvt cell lines. We demonstrated the clonal origin of both cell lines by their identical idiotypic immunoglobulin sequences. Expression of IGF-I receptor- and CD44v6-containing isoforms was demonstrated on the 5T33MMvv cells but not on the 5T33MMvt cell line. The 5T33MMvv but not the 5T33MMvt cells exhibited chemotaxis toward BM stroma-conditioned medium and IGF-I. When the 5T33MMvt cells were injected in mice, we could observe an up-regulation of the IGF-I receptor and CD44v6 on the 5T33MMvt cells in vivo (5T33MMvt-vv) harvested from the BM. The 5T33MMvt-vv cells migrated toward IGF-I and BM stroma-conditioned medium. In addition, their adhesion to BM stroma was also CD44v6 dependent. When these cells were cultured again in vitro, the expression of IGF-I receptor and CD44v6 was down-regulated. Transendothelial migration of the 5T33MMvt cells through a layer of BM endothelial cells resulted in up-regulation of both IGF-I receptor and CD44v6.

MATERIALS AND METHODS

Animals.

C57BL/KalwRij mice were obtained from Harlan CPB (Horst, The Netherlands). Male mice were 6–10 weeks of age when used. They were housed under conventional conditions and had free access to tap water and food. They were sacrificed by CO2 asphyxiation (License no. LA1230281).

Cell Lines.

The 5T33MMvv cell line originated from elderly C57BL/KalwRij mice that spontaneously developed MM (9 , 10) . The cells have since been expanded into young syngeneic animals by i.v. transfer of the diseased BM. Progression of MM in diseased animals was followed up by electrophoretic quantification of serum paraproteins (19) . Animals were sacrificed when a paraprotein concentration of 10 mg/ml was reached. MM cells were purified from the BM as described elsewhere (20) . Cell suspensions with at least 95% 5T33MM cells, as determined by FACS analysis, were obtained.

The 5T33MMvt cell line has been obtained by the Radl group, who developed the 5TMM experimental mouse model (9 , 10) . This cell line resulted spontaneously from cultured 5T33MMvv BM cells and grows in vitro independent of BM stroma. Cells were cultured and maintained in complete medium (DMEM supplemented with penicillin-streptomycin, glutamine and MEM; Life Technologies, Inc. Mérelbeke, Belgium) supplemented with 10% bovine serum (Hyclone, UT). For some experiments, in vivo-inoculated 5T33MMvt cells were used. We refer to these cells as 5T33MMvt-vv cells.

CD44v Abs.

The production of mAbs against mouse CD44v6 (mouse IgG2a; LN6.1), CD44v7 (mouse IgG1; LN7.1) and CD44v10 (rat IgG1; LN10.1) antigens is described elsewhere (37) . 4 An antimouse pan-CD44 (CD44s; rat IgG2b) Ab was obtained (Clone IM7.8.1; American Type Culture Collection, Rockville, MD). This clone of CD44s is known as a nonblocking Ab and was included in the adhesion assays as an additional control.

Flow Cytometry.

The percentage of 5T33MM cells in isolated 5T33MM BM cells and in trypsinized cells from adhesion assays (see below) was determined by FACS staining with anti-5T33MM idiotype-specific mAbs (19) , whereas rat antimouse IgG1-phycoerythrin (Becton Dickinson, Mountain View, CA) was used as a second step.

To assess the expression of IGF-I receptor, cells were stained with IGF-I receptor α- or β-chain Abs (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Donkey antirabbit IgG conjugated to FITC (Cruton Bioproducts, Brussels, Belgium) was used as a second step. Where indicated, cells were subsequently labeled with anti-idiotype-specific Abs.

Expression of CD44 splice variants and pan-CD44 antigens on the surface of 5T33MM cells was assessed by staining of the cells with biotinylated pan-CD44 (CD44s)- or CD44 variant (CD44v)-specific mAbs followed by streptavidin conjugated to phycoerythrin. For some experiments, double stainings were done for CD44v6 and anti-5T33MM idiotype. Cells were first labeled with CD44v6, with goat antimouse IgG2a-FITC (Sera-lab, Sussex, United Kingdom) as a second step; subsequent anti-idiotype labeling was performed as described above. For all stainings, isotype-matched irrelevant Abs were used as controls. Flow cytometric acquisitions were performed with a FACSort flow cytometer (Becton Dickinson).

In Vitro Migration Assays.

Migration of 5T33MM cells was measured by classical checkerboard analysis as described in detail previously (20) . Initial experiments showed optimal migration of 5T33MM cells through Transwell inserts with a pore size of 8 μm. Medium (300 μl) was added to the lower chambers, and 51Cr-labeled 5T33MM cells (20 × 10 3 in 200 μl) were added to the upper chambers. To assess the chemotactic activity of BM stroma-conditioned medium, it was concentrated 10-fold and diluted as described previously (20) . It was used at the following dilutions: 10× (concentrated), 5×, 2.5×, and 0× (control medium). Where indicated, neutralizing antihuman IGF-I Abs (PreproTech Inc., Rocky Hill, NY) were added at a concentration of 10 ng/ml. Isotype-matched irrelevant Abs were used as control. To study the chemotactic effect of IGF-I, concentrations of 10, 5, and 0 ng/ml (control medium) were used.

After an incubation for 2 h at 37°C and 5% CO2, Transwell inserts were removed and migrated cells in the lower chambers were harvested. Radioactivity was measured in a gamma counter. The ratio of migrated cells to total cells added was calculated.

In Vivo Migration Assays.

To study the migration of 5T33MM cells in vivo, 0.5 × 106 51Cr-labeled cells were injected i.v. into the lateral tail veins of naive mice. At 2- or 18-h time points, mice were sacrificed, EDTA-blood was collected, and all organs were removed. The radioactivity of each organ and 100 μl of blood was measured in a gamma counter.

Transendothelial Migration Assays.

For transendothelial migration experiments, the BM endothelial cell line STR-4 (kindly provided by Dr. Kobayashi, Japan) was grown on Transwell inserts (38) . A total of 20 × 10 3 5T33MMvt cells in 200 μl of medium was added to the upper chambers, and 300 μl of IGF-I (5 ng/ml) was used as chemoattractant in the lower chambers. After incubation for 4 h at 37°C and 5% CO2, the expression of IGF-I receptor and CD44v6 on the cells that migrated through the endothelial layer into the lower chamber was analyzed by FACS.

Adhesion Assays.

The adhesion of 5T33MM cells to BM stroma was measured by a flow cytometric adhesion assay (5 , 39) and by microscopic cell counting. 5T33MM cells were preincubated with different CD44 splice variants and a pan-CD44 mAb at a concentration of 10 μg/ml for 30 min at room temperature. Isotype-matched irrelevant mAbs were used as controls. Subsequently, 200-μl cell samples (0.1 × 10 6 cells/well) were incubated in quadruplicate for 2 h at 37°C and 5% CO2 on confluent BM stroma cultures in 96-well plates. After this incubation, weakly attached and floating cells were removed by three gentle washes with 200 μl of warm (37°C) complete medium. Subsequently, 100 μl of trypsin-EDTA (0.5 mg/ml; Sigma, St. Louis, MO) was added for 2 min (37°C and 5% CO2); plates were put on ice, and 20 μl of bovine serum was added to each well. Adherent 5T33MM cells and BM stromal cells were detached by vigorous resuspension. Complete detachment of the cells was checked by inversion microscopy. Cells were collected, counted, and stained for FACS analysis with anti-5T33MM idiotype-specific mAbs. Preliminary experiments showed that trypsinization as described above did not influence 5T33MM positivity, as measured by FACS: Math The percentage of adherent 5T33MM+ (anti-idiotype-positive) cells was determined by FACS. BM stromal cells were distinguished from 5T33MM cells by their forward and side scatter characteristics: Math where number of adherent 5T33MM cells = cell count after trypsinization × % 5T33MM+ (anti-idiotype-positive) cells; and number of input 5T33MM cells = (0.1 × 10 6 ) × % 5T33MM+ (anti-idiotype-positive) cells in the isolated 5T33MM BM cell population.

Histology and Radiology.

Six naive animals received injections of 0.5 × 10 6 5T33MMvt cells. The development of osteolytic lesions was examined by radiology as described previously (40) . Animals were sacrificed when a paraprotein concentration of 10 mg/ml was reached. Tissue blocks of all organs were fixed in 85% alcohol-40% acetic acid-4% formalin for 1 day. Bones were decalcified in FE10 (1% EDTA, 0.12% NaOH in 4% formalin). After embedding in paraffin, 5-μm sections were made and stained with H&E.

Immunoglobulin Sequence Analysis.

Total RNA was extracted from tumor cells (either BM or in vitro culture cells) by a guanidine isothiocyanate-acid-phenol modified method with TRIzol reagent (Life Technologies) and reversed transcribed using an oligo(dT) primer and the SuperScript Preamplification System (Life Technologies). The immunoglobulin heavy chain sequence expressed by the 5T33MM tumor cells has been published previously (13) . A tumor-specific primer in the CDR1 region (5T33-CDR1 sense primer, 5′-CAC-TAA-TTA-CTT-GAT-AGA-GTG-G-3′) was designed and used together with an isotype-specific Cγ primer (Cγ, 5′-GCG-AAT-TCC-CTT-GAC-CAG-GCA-TCC-3′) to amplify the tumor-specific immunoglobulin sequence. The amplification reaction was performed on 1μ l of first strand cDNA, corresponding to ∼100,000 cells, in a 50-μl reaction volume containing 1 mm Tris-HCl (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, 200 μm deoxynucleotide triphosphate, 10 pmol of each primer, and 0.625 units of Taq polymerase (Life Technologies). Normal C57BLKalwRij BM cDNA was used as negative control, and water was used as control for contamination.

Each PCR cycle consisted of heat denaturation at 94°C for 30 s and primer annealing at 55°C for 30 s, followed by primer extension at 72°C for 1 min. The first cycle was preceded by a 2-min denaturation step at 94°C, and the last elongation step was prolonged to 10 min to ensure full-length products. Forty cycles were performed in a Perkin-Elmer GeneAmp PCR system (Perkin-Elmer, Lennik, Belgium). The PCR-amplified DNA fragments were analyzed by standard agarose gel electrophoresis. Positive PCR samples were cloned into the pCRII vector (Invitrogen, Groningen, The Netherlands) and sequenced using the autoload solid-phase sequencing kit and the ALFexpress (Pharmacia, Roosemdaal, The Netherlands).

RESULTS

Clonal Origin of 5T33MM Cells.

To confirm the monoclonal origin of both cell lines, their immunoglobulin sequences were analyzed. Amplification products of cDNA derived from the 5T33MMvt cells and the 5T33MMvv BM cells with the CDR1 primer and the Cγ primer were cloned and sequenced. The 5T33MM VDJ sequence differed slightly (only three nucleotides) from the published sequence (Fig. 1 ⇓ ). The VDJ region was exactly the same in both the 5T33MMvv and the 5T33MMvt cells, which demonstrates that the latter cell line has derived from the original 5T33MMvv cells. Moreover, the hypermutation mechanism had stopped in these cells because no accumulation of further mutations in the VDJ region of the heavy chain immunoglobulin gene occurred during the long period of in vitro culture.

Fig. 1.
Fig. 1.

Clonal origin of the 5T33MM cells. Comparison between the nucleotide sequence of the 5T33MM VH gene published by Zhu et al. (13) and the amplified VH gene from 5T33MMvv BM cells and/or 5T33MMvt cells. Amplification was performed with a 5T33MM-CDR1-specific primer and different constant region primers. Primers are depicted in italics. Dashes represent identical nucleotides. Deviating nucleotides that result in amino acid replacements are underlined. CDR and CH1 indications are according to Kabat et al. (55) .

Differential Expression of IGF-I Receptor and CD44v6 on 5T33MM Cells.

The presence of IGF-I receptor α and β chains on the surfaces of 5T33MM cells was assessed by indirect double FACS staining with IGF-I receptor and 5T33MM anti-idiotype Abs (Fig. 2 ⇓ ). A clear shift of the whole tumor (5T33MM idiotype)-positive population toward a higher fluorescence intensity of IGF-I receptor α and β chains indicated that all 5T33MMvv cells expressed the IGF-I receptor. In contrast, the 5T33MMvt cell line showed no expression ofα or β chain.

Fig. 2.
Fig. 2.

Differential expression of IGF-I receptor on 5T33MM cells. Double stainings with anti-5T33MM idiotype and IGF-I receptorα - and β-chain Abs were performed. The 5T33MMvv cells, but not the 5T33MMvt cells, expressed IGF-I receptor α and β chains. Dot plots from one experiment, representative of three, are shown.

The expression of CD44 standard and splice variant antigens on the surfaces 5T33MM cells was also studied by flow cytometry (Fig. 3 ⇓ ). High expression of CD44s-, CD44v7-, and CD44v10-containing isoforms was found on both 5T33MM cell lines. CD44v6-containing isoforms were clearly expressed on the 5T33MMvv cells but only very faintly on the 5T33MMvt cell line.

Fig. 3.
Fig. 3.

Differential expression of CD44v6 isoforms on 5T33MM cells. 5T33MM cells were stained with biotinylated anti-CD44(v) mAbs. High expression of CD44v7, CD44v10, and CD44s isoforms was found. The 5T33MMvv cells (top) also expressed CD44v6-containing variant isoforms, in contrast to the 5T33MMvt cell line (bottom). Left panels indicate staining with control Abs. Results from one experiment, representative of three, are shown.

Chemotactic Activity of 5T33MM Cells Depends on IGF-I Receptor.

Does the absence of IGF-I receptor on the 5T33MMvt cells influence their ability to migrate? The chemotactic effect of IGF-I and BM stroma-conditioned medium on 5T33MM cells was studied by a classic checkerboard analysis (Fig. 4 ⇓ ). The 5T33MMvv cells exhibited a chemotactic activity with a chemokinetic component (latter not illustrated) toward IGF-I and BM stroma-conditioned medium. When neutralizing anti-IGF-I Abs were added to BM stroma-conditioned medium, the chemotactic activity was blocked. Pretreatment of the 5T33MMvv cells with IGF-I receptor function-blocking Ab (αIR-3) as an additional control could completely abolish chemotaxis, and thus directly demonstrated the involvement of the IGF-I receptor (results not shown).

Fig. 4.
Fig. 4.

5T33MM cells migrate in response to IGF-I. 51Cr-labeled 5T33MM cells were incubated in upper chambers of Transwell inserts with different concentrations of IGF-I or BM-conditioned medium in the lower chambers. Where indicated, neutralizing anti-IGF-I mAb was added. After an incubation time of 2 h, the transmigrated cells were quantified in a gamma counter. Results are expressed as the percentage of migration relative to the migration in control conditions. Data from one experiment, representative of three, are shown.

Consistent with the absence of IGF-I receptor on the 5T33MMvt cells, no migration was observed toward IGF-I and BM stroma-conditioned medium. Thus, IGF-I and BM stroma-conditioned medium attracted the 5T33MMvv cells but not the 5T33MMvt cell line.

Involvement of CD44v6 in the Adhesion of 5T33MM Cells to BM Stroma.

The difference in the expression of CD44v6 prompted us to investigate the adhesion of both 5T33MM cell lines. The involvement of CD44 splice variants in their adhesion to BM stroma was quantified as adhesion ratios and percentages of adhesion (Fig. 5 ⇓ ) as described in “Material and Methods.” The 5T33MMvv cells showed a spontaneous adhesion of ∼30%, which was significantly inhibited by mAbs against CD44v6 (mean inhibition, 43.7%). Results from adhesion ratios (not shown) were in a similar trend (mean inhibition, 41.9%). No significant decrease in adhesion was observed for the other variants when the respective Abs were used.

Fig. 5.
Fig. 5.

Involvement of CD44v6 in the adhesion of 5T33MM cells to BM stroma. 5T33MM cells were preincubated with different CD44v or pan-CD44 (CD44s) mAbs for 30 min and subsequently were incubated on BM stroma cells for 2 h. Percentage of adherent 5T33MM cells was determined by microscopic cell counting. Mean values of quadruplets of one experiment, representative of three, are shown; bars, SD. ∗∗ indicates significant difference (P < 0.01) compared with the control.

5T33MMvt cells showed no involvement of CD44v6 in their adhesion to BM stroma, which was in line with the faint expression of this variant. The spontaneous adhesion of 5T33MMvt cells was also lower (mean value, 18.5%) compared with the 5T33MMvv cell line. These data demonstrate involvement of CD44v6 in the adhesion of the 5T33MMvv cells, in contrast to the 5T33MMvt line.

CD44 binds to different extracellular matrix structures, including hyaluronan (mainly), laminin, fibronectin, collagen types I and IV, chondroitin sulfate, and other possible, as yet unidentified, ligands (30) . We tested the above-mentioned extracellular matrix elements, but none of these ligands for CD44s appeared to influence CD44v6-mediated adhesion of 5TMM cells to BM stroma (results not shown).

In Vivo Homing Kinetics of 5T33MM Cells.

Because the 5T33MMvt cells were not attracted by IGF-I or BM stroma-conditioned medium in vitro, we analyzed their initial homing kinetics by tracing 51Cr-labeled cells in vivo (Fig. 6 ⇓ ). Two h after i.v. injection, the majority of both 5T33MMvv and 5T33MMvt cells were found in the lungs and liver. After 18 h, the majority of the cells were found in the liver. 5T33MMvv and 5T33MMvt cells were also found in the spleen, BM, and in the blood circulation at both the 2- and 18-h time points. The kidneys were positive because of excretion of spontaneously released 51Cr in the urine. Homing to other organs, including testes, intestines, lymph nodes, stomach, heart, and thymus, was negligible for both cell lines. After 18 h, 70 and 50% of the total radioactivity injected was recovered for 5T33MMvv and 5T33MMvt cells, respectively.

Fig. 6.
Fig. 6.

Similar in vivo homing kinetics of 5T33MMvv and 5T33MMvt cells. 51Cr-labeled cells were injected i.v. After 2 and 18 h, radioactivity in the organs was measured and is expressed as percentage of total radioactivity injected. Bone marrow represents the sum of ribs, vertebrae, and fore and hind legs. For the calculation of radioactivity in the blood circulation, we assumed a total volume of 2 ml of blood per mouse. Mean values of six animals are shown; bars, SD.

These data demonstrate that despite different in vitro migratory behaviors, both the 5T33MMvv and the 5T33MMvt cell lines initially home to the BM.

Organ Involvement of 5T33MM Cells in Terminally Diseased Animals.

Organ involvement of 5T33MMvt cells in terminally diseased animals was studied by histology. BM infiltration was observed in all 5T33MMvt-diseased animals. Massive invasion of the vertebrae was found with extensions to the surrounding paravertebral tissue that gave rise to solid perivertebral tumors. The spleen was only focally invaded in two of the six animals. Subcapsular sinusoidal invasion of lymph nodes was observed in two animals. Other organs, including the liver, thymus, kidneys, intestine, stomach, testis, lungs, and heart, were not invaded in all mice examined. Osteolytic lesions as assessed by radiology were observed in some animals. Organ involvement of 5T33MMvv cells has been reported in a previous work (19) and includes mainly the BM, spleen, and liver. For the 5T33MMvv cells, osteolysis was also observed in some animals. These histological data confirm the restricted homing of 5T33MMvt cells to the BM.

De Novo Expression of IGF-I Receptor and CD44v6 on 5T33MMvt-vv Cells.

As it became clear that the 5T33MMvt cells, when injected in vivo, were able to enter and infiltrate the BM, we were interested in their IGF-I receptor and CD44v6 phenotypes 18 h after injection and in terminally diseased animals. The 5T33MMvt-vv cells were harvested from the BM, and surface expression was analyzed. The cells expressed IGF-I receptor α (Fig. 7 ⇓ a) and β chains (not shown) and were CD44v6-positive (Fig. 7b ⇓ ) both 18 h after injection and in terminally diseased animals. The high expression of CD44s, CD44v7, and CD44v10 (not shown) was not altered during the in vivo passage. Most likely, the BM environment induced expression of both IGF-I receptor and CD44v6 on the 5T33MMvt-vv cells. This phenotypic profile is similar to that of 5T33MMvv cells.

Fig. 7.
Fig. 7.

A, de novo expression of IGF-I receptor α chain on 5T33MMvt-vv cells. Naive mice were injected with 5T33MMvt cells. BM cells 18 h after the injection and from terminally diseased animals were harvested, stained with anti-5T33MM and -IGF-I receptor Abs. Shaded histogram (top) shows expression of IGF-I receptor on the cells in BM after 18 h. Overlapping open histograms show the level of expression before injection and staining with control Abs. Bottom panel illustrates the expression of the cells in BM of terminally diseased animals. Left histogram shows staining with control Abs. 5T33MMvt-vv cells isolated from the BM 18 h after injection were analyzed by a livegate on the anti-5T33MM idiotype-positive cells. Histograms of one experiment, representative of three experiments, are shown. B, de novo expression of CD44v6 on 5T33MMvt cells in vivo. Expression of CD44v6-containing isoforms on 5T33MMvt-vv cells in the BM 18 h after injections (left panel) and in terminally diseased animals (right panel) are shown. Double stainings with anti-5T33MM idiotype and CD44v6 Abs were performed. 5T33MMvt-vv cells isolated from the BM 18 h after infection were analyzed by a livegate on the anti-5T33MM idiotype-positive cells. Results from one experiment, representative of three experiments, are illustrated.

Chemotaxis and Adhesion of 5T33MMvt-vv Cells.

We tested whether the up-regulation of IGF-I receptor and CD44v6 on the 5T33MMvt-vv cells changed their in vitro migration and adhesion. The cells were harvested from BM of terminally diseased animal, and checkerboard analysis and adhesion assays were performed. In contrast to the situation before injection, the cells now showed clear chemotactic activity toward IGF-I (Fig. 8 ⇓ ) and BM stroma-conditioned medium, which could be neutralized by the addition of anti-IGF-I Abs (not shown).

Fig. 8.
Fig. 8.

Chemotaxis and adhesion of 5T33MMvt cells is re-established in vivo. 5T33MMvt-vv cells were harvested from terminally diseased animals, and checkerboard analysis and adhesion assays were performed. Left panel illustrates chemotactic activity of the cells toward IGF-I. Results are expressed as percentage of migration relative to the migration in control conditions. Right panel shows the involvement of CD44v6 in the adhesion of the 5T33MMvt-vv cells to BM stroma. Mean values of quadruplicate samples from one experiment, representative of three, are shown. Standard deviation is lower than 3% of the mean value. ∗∗ indicates significant difference (P < 0.01) compared with the control.

Adhesion assays showed an increased spontaneous adhesion of the 5T33MMvt-vv cells (mean adhesion, 28%) compared with the 5T33MMvt cells (mean adhesion, 18.5%). Anti-CD44v6 Abs inhibited this adhesion with 25% (mean inhibition; Fig. 8 ⇓ ). Adhesion ratios (not shown) showed a mean inhibition of 15%.

Down-Regulation of IGF-I Receptor and CD44v6 on 5T33MMvt-vv Cells ex Vivo.

To evaluate whether the up-regulation of IGF-I receptor and CD44v6 was dependent on the BM microenvironment or due to a clonal expansion of a few revertants, 5T33MMvt-vv cells were cultured, and surface expression was analyzed after 10 days of in vitro culture with or without BM stroma. A complete down-regulation of both IGF-I receptor and CD44v6 on the 5T33MMvt-vv cells was observed (Fig. 9 ⇓ ). These data indicate that when cultured in vitro, the 5T33MMvt-vv cells again obtain the phenotypic profile of the 5T33MMvt cells. Moreover, these results also suggest that cultured BM stroma, which consists mainly of fibroblasts, is not able to maintain the in vivo up-regulated expression.

Fig. 9.
Fig. 9.

Down-regulation of IGF-I receptor α chain and CD44v6 on 5T33MMvt-vv cells ex vivo. Double stainings of cultured 5T33MMvt-vv cells for IGF-I receptor α chain or CD44v6 and anti-5T33MM idiotype are shown. The cells are positive for 5T33MM idiotype but do not express IGF-I receptor or CD44v6. Results from one experiment, representative of three, are shown.

De Novo Expression of IGF-I Receptor and CD44v6 on 5T33MMvt Cells through Direct Contact with BM Endothelial Cells.

Are BM endothelial cells involved in the in vivo observed up-regulation of IGF-I receptor and CD44v6? When the 5T33MMvt cells were in direct contact, they migrated (30%) through the BM endothelial cell layer, and as a consequence, IGF-I receptor and CD44v6 were up-regulated (Fig. 10 ⇓ ). Thus, BM endothelial cells can induce up-regulation of IGF-I receptor and CD44v6 on the 5T33MMvt cells and increase their potential to migrate.

Fig. F10-9709.
Fig. F10-9709.

De novo expression of IGF-I receptor α chain and CD44v6 on 5T33MMvt cells after direct contact with endothelial cells. Direct contact between 5T33MMvt cells and BM endothelial cells resulted in transendothelial migration of the MM cells and induction of CD44v6 and IGF-I receptor α chain (right histograms). Left histograms indicate expression levels before migration. Histograms from one experiment, representative of three, are shown.

DISCUSSION

One of the distinguishing features of MM cells is their specific localization in the BM. This restricted presence could be due to (a) a random migration to several organs but a selective survival and growth only in the BM, or (b) a selective homing and survival in the BM. We recently have shown that MM cells selectively home to the BM (41) . In analogy to the model of lymphocyte homing, described by Butcher and Picker (2) , homing of myeloma cells to the BM is believed to be a multistep event. The mechanisms involved are, however, poorly understood. In this work, we have been able to contribute to the understanding of this process by investigating differences between stroma-dependent 5T33MMvv and stroma-independent 5T33MMvt cells. We demonstrated a differential expression of IGF-I receptor- and CD44v6-containing isoforms between the 5T33MMvv and 5T33MMvt cell lines. Initially, the idiotype immunoglobulin sequence was analyzed to demonstrate the clonal identity of both cell lines. The exact similarity of the VDJ region between the two cell lines demonstrated their common origin. The 5T33MMvv cell line expressed IGF-I receptor and CD44v6, in contrast to the 5T33MMvt cells. In view of their differential IGF-I receptor expression, their chemotactic response toward BM stroma-conditioned medium or IGF-I alone was analyzed. In line with their IGF-I receptor expression, only the in vivo cell line migrated, whereas the in vitro variant did not. These results suggested a different in vivo homing pattern. However, when injected in vivo, the 5T33MMvt cells were able to home to the BM. This ability appeared to be a consequence of an unexpected up-regulation of IGF-I receptor and CD44v6 detected on the 5T33MMvt-vv cells. This up-regulation was also observed on the tumoral cells present in the BM of terminally diseased mice.

Expression of CD44 and other adhesion molecules on MM cells have been reported by many groups, as reviewed by Van Riet et al. (6) and Helfrich et al. (8) . Fewer groups have performed functional studies whereby the involvement of CD44 in the adhesion of murine (29) and human (5) MM cells has been demonstrated. Another report demonstrated the binding of peripheral blood B cells from MM patients to BM stroma, showing that CD44 is involved (42) . Taken together, several reports have described the involvement of CD44 in the adhesion of MM cells to BM stroma. Little is known about the functional roles of CD44 variant isoforms in general and CD44v6 variant isoforms in particular. Correlations between altered CD44v expression and specific cancers, including hematological malignancies and carcinomas, have been reported (30 , 43) . Expression of CD44v3-, CD44v4-, CD44v6-, CD44v9-, and CD44v10-containing isoforms have been reported on BM biopsies from MM patients (35) . Van Driel et al. (36) reported expression of CD44v6-containing isoforms on MM cells from patients with stable disease. Expression of certain variants confers metastatic potential to rat tumor cells (44 , 45) . Our results show functional involvement of CD44v6-containing isoforms in the adherence of 5T33MM cells to BM stroma. Comparable results were obtained with the 5T2MM cell line (not shown) and in the in vitro human system. 5 Moreover, we observed up-regulation of CD44v6-containing isoforms by the 5T33MMvt-vv cells. They exhibited an increased adhesion to the BM stroma that could be inhibited by CD44v6 Abs, indicating an important role of this adhesion molecule in vivo. Because the localization of MM cells in the BM is important for their growth and survival, in vivo blocking of CD44v6-containing isoforms in 5TMM tumor-bearing mice may reveal potential in vivo roles of CD44v6 in the survival and growth of MM cells in the BM microenvironment.

The BM microenvironment is a complex structure of various extracellular components and many cell types, including, osteoblasts, osteoclasts, macrophages, hematopoietic progenitors, endothelial cells, and fibroblasts (46) . When the 5T33MMvt-vv cells were cultured in vitro, both IGF-I receptor and CD44v6 were down-regulated, emphasizing that the up-regulated expression of these receptors on the cell surface is dependent on the presence of the BM microenvironment. Coculture of these cells on cultured BM stroma, consisting mainly of fibroblasts, also could not maintain the up-regulated expression, suggesting that the in vivo observed up-regulation could be due to other in vivo interactions. Indeed, when the 5T33MMvt cells were allowed to migrate through a confluent BM endothelial cell layer, an up-regulated expression of CD44v6 and IGF-I receptor was observed. Because this in vitro up-regulation of IGF-I receptor and CD44v6 was less pronounced than that observed in vivo, we assume that other interactions in addition to those with the BM endothelial cells are involved. Nevertheless, BM endothelial cells are at least one of the cell types engaged. Important interactions between 5T33MMvt-vv cells and the BM vascular endothelium in vivo may thus induce up-regulation of IGF-I receptor and CD44v6, making MM cells accessible to the chemoattractive activity of IGF-I with subsequent transendothelial migration into the BM compartment. Moreover, IGF-I receptor appears to regulate the expression of matrix metalloproteinase-2 (47) and matrix metalloproteinase-9 (48) . Metalloproteinases are key proteinases in cancer cell invasion (49) and transendothelial migration (50) . CD44v has been reported to be associated with the active form of matrix metalloproteinase-9 on the cell surface of invading cancer cells (51 , 52) . Our data suggest a coexpression of CD44v6-containing isoforms and IGF-I receptor. IGF-I-induced up-regulation of CD44v6 via protein kinase C and phosphoinositide 3-kinase pathways has been reported (27) . In addition to the up-regulation of IGF-I receptor and CD44v6, interactions between 5T33MMvt cells and BM endothelial cells might also activate matrix metalloproteinase-2 and/or metalloproteinase-9, thus activating the whole machinery required for transendothelial migration into the BM compartment. Preliminary results indicate expression of active matrix metalloproteinase-9 by the 5T33MMvv and 5T33MMvt-vv cell lines but not by the 5T33MMvt cell line. Experiments with BM endothelial cells and 5TMM cells are in progress to elucidate the induction of matrix metalloproteinases in the process of transendothelial migration. Once the cells have entered the BM, CD44v6 variant isoforms may support their adhesion to BM stroma and IGF-I receptor may stimulate their proliferation via the binding of IGF-I, which is also a growth factor for human (53 , 54) and 5T33MM cells. 6

Our data clearly show that the presence of certain crucial molecules alters dependence on the local microenvironment. We have demonstrated that homing of MM cells is a dynamic process, involving transendothelial migration and induction of chemokine receptors and adhesion molecules, such as IGF-I receptor and CD44v6, which allow the MM cells to be attracted by the BM environment and adhere to stromal cells.

ACKNOWLEDGMENTS

We thank Drs. Stefan Mahlmann, Annegret de Baey (Basel, Switzerland), and Jiri Radl (TNO-PG, Leiden, The Netherlands) for critically reviewing the manuscript and helpful suggestions. We are also grateful to Dr. Kobayashi (Laboratory of Pathology, Cancer Institute Hokkaido University School of Medicine, Sapporo, Japan) for kindly providing the BM endothelial cell line.

Footnotes

  • 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.

  • ↵K. Vanderkerken and M. H. C. Bakkus are postdoctoral fellows of the “Fonds voor Wetenschappelijk Onderzoek-Vlaanderen” (FWO). The work was supported financially by the OZR-VUB, FWO, and Vlaamse Liga tegen tegen Kanker. The Basel Institute for Immunology was founded and is supported by Hoffmann-La Roche, Inc., Basel, Switzerland.

  • ↵To whom requests for reprints should be addressed, at Department of Hematology and Immunology, Free University Brussels, Laarbeeklaan 103, B-1090 Brussels, Belgium.

  • ↵The abbreviations used are: MM, multiple myeloma; BM, bone marrow; IGF, insulin-like growth factor; FACS, fluorescence-activated cell sorting; Ab antibody; mAb, monoclonal antibody.

  • ↵B.M. Wittig, et al. Abrogation of experimental colitis correlates with increased apoptosis in mice deficient for CD44v7. J. Exp. Med., in press, 2000.

  • ↵Andries Bloem, personal communication.

  • ↵K. Asosingh, K. Vanderkerken, and B. Van Camp, unpublished observations.

  • Received December 1, 1999.
  • Accepted April 4, 2000.

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June 2000
Volume 60, Issue 11

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In Vivo Induction of Insulin-like Growth Factor-I Receptor and CD44v6 Confers Homing and Adhesion to Murine Multiple Myeloma Cells
Kewal Asosingh, Ursula Günthert, Marleen H. C. Bakkus, Hendrik De Raeve, Eva Goes, Ivan Van Riet, Ben Van Camp and Karin Vanderkerken
Cancer Res June 1 2000 (60) (11) 3096-3104;
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