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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4727-4737
A gp130 Interleukin-6 Transducer-Dependent SCID Model of
Human Multiple Myeloma
By
Cosette Rebouissou,
John Wijdenes,
Patrick Autissier,
Karin Tarte,
Valerie Costes,
Janny Liautard,
Jean-Francois Rossi,
Jean Brochier, and
Bernard Klein
From the Institute for Molecular Genetics, CNRS, Montpellier, France;
the Diaclone, Besançon, France; INSERM U475, Montpellier, France;
and the Service des Maladies du Sang B, CHU Montpellier, Hôpital
Lapeyronie, Montpellier, France.
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ABSTRACT |
Agonist antihuman gp130 transducer monoclonal antibodies (MoAbs)
were used in SCID mice to grow myeloma cells whose survival and
proliferation is dependent on gp130 transducer activation. The agonist
anti-gp130 MoAbs neither bound to murine gp130 nor activated murine
cells and, as a consequence, did not induce interleukin-6 (IL-6)-related toxicities in mice. They have a 2-week half-life in
vivo when injected in the peritoneum. The agonist antibodies made
possible the in vivo growth of exogenous IL-6-dependent human myeloma
cells as well as that of freshly explanted myeloma cells from 1 patient
with secondary plasma cell leukemia. Tumors occurred 4 to 10 weeks
after myeloma cell graft and weighed 3 to 5 g. They grew as solid
tumors in the peritoneal cavity and metastasized to the different
peritoneal organs: liver, pancreas, spleen, and intestine. Tumoral
cells were detected in blood and bone marrow of mice grafted with the
XG-2 myeloma cells. Tumoral cells grown in SCID mice had kept the
phenotypic characteristics of the original tumoral cells and their in
vitro growth required the presence of IL-6 or agonist anti-gp130 MoAbs.
Myeloma cells from 4 patients with medullary involvement persisted for
more than 1 year as judged by detectable circulating human Ig. However,
no tumors were detected, suggesting a long-term survival of human
myeloma cells without major proliferation. These observations
paralleled those made in in vitro cultures as well as the tumor growth
pattern in these patients. This gp130 transducer-dependent SCID model
of multiple myeloma should be useful to study various therapeutical
approaches in multiple myeloma in vivo.
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INTRODUCTION |
ANIMAL MODELS OF human tumors are
essential to validate the development of novel therapies and especially
that of immunotherapies. Tumoral samples taken from some patients with
multiple myeloma (MM) may persist for several months in SCID mice, as
judged by the presence of circulating human Ig without any significant
tumor growth.1,2 A myeloma SCID model has been described
using the ARH77 cell line,3 but this cell line is an
Epstein-Barr virus (EBV)-infected B-cell line and not a myeloma cell
line.4 Recently, several autonomously growing myeloma cells
have been shown to grow close to femoral bones5 or in the
bone marrow6 in SCID mice. One difficulty of getting a SCID
model of MM might be due to the myeloma cell growth dependence on gp130
interleukin-6 (IL-6) transducer-activating cytokines.7,8
Because murine gp130 cytokines do not activate human gp130
transducer,9 there is a need either to implant human
stromal cells producing these cytokines or to inject human gp130
cytokines, in particular IL-6. However, these cytokines have a short
half-life in vivo (20 to 60 minutes for IL-6)10,11 that
necessitates a daily infusion. Another difficulty is that human IL-6
binds to murine IL-6R and activates murine gp130 transducer. As a
consequence, human IL-6 may induce the toxicities reported for IL-6 in
vivo, mainly an inflammatory and cachectic syndrome.12-14
In the present study, we have developed a SCID model of human MM,
taking into account the myeloma cell growth dependence on gp130
cytokines and avoiding IL-6 toxicities. We used agonist monoclonal
antibodies (MoAbs) to human gp130 transducer.15,16 These
antibodies support the long-term growth of IL-6-dependent myeloma cell
lines and the short-term proliferation of primary myeloma cells in
vitro (unpublished results).
We show that these antibodies neither recognized nor activated murine
gp130 IL-6 transducer and that they have a 2-week half-life in vivo.
These antibodies made it possible to grow IL-6-dependent human cell
lines as solid tumors in SCID mice. In addition, we have been able to
grow primary myeloma cells from 1 patient with terminal MM and to
further obtain a gp130-cytokine-dependent myeloma cell line.
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PATIENTS, MATERIALS, AND METHODS |
Patients.
Tumor samples were obtained from 5 patients with MM (median age, 57 years) after written informed consent was received. According to the
Durie-Salmon classification, 4 patients were of stage IIIB and 1 of
stage IIIA. Two patients had IgG MM, and 3 had IgG MM. One
patient had a secondary plasma cell leukemia.
Reagents.
Recombinant IL-6 was provided by Dr A. Ytier (Ares Serono,
Geneva, Swiss). The MI15 anti-syndecan-1 and the M91 anti-IL-6R MoAbs
were obtained by our group.17,18 MoAbs against CD3, CD11a, CD18, CD19, CD28, CD80, CD38, CD40, CD45, CD54, CD56, CD58, MUC-1, HLA-DR, HLA-ABC antigens, fluorescein isothiocyanate (FITC)-conjugated (Fab )2 fragments of goat antibodies to mouse IgG and
phycoerythrin (PE)-conjugated (Fab)2 fragments of
goat antibodies to mouse IgG were purchased from Immunotech (Marseille,
France). IgG1 and IgG2 control murine Igs (recognizing no human
antigens) were purchased from Sigma (St Louis, MO), biotin-conjugated
goat antimouse Ig and streptavidin-horseradish peroxidase from Amersham
(Ulis, France), and Annexin-V-Fluos from Boehringer Mannheim (Meylan,
France).
Agonist anti-gp130 antibodies.
Two different pairs of agonist anti-gp130 MoAb were used. B-S12 and
B-P8 murine IgG1 MoAbs were cooperatively obtained by our group and the
Diaclone Co (Besançon, France).15,19 B1 and I2 murine IgG1 MoAbs were produced by our group and recognized epitopes different from those recognized by B-S12 and B-P8
MoAbs.20 Both mixtures of MoAbs (used at 1/1 ratio) were
proven to support long-term growth of IL-6-dependent myeloma cell
lines.15,16 Most of the experiments were performed using
the mixture of B-S12 and B-P8 MoAbs.
Isolation of primary myeloma cells.
Patients' myeloma cells were purified using the antimyeloma cell MI15
MoAb and Dynal magnetic beads (Dynal M450; Dynal, Oslo, Norway) coated
with sheep antimouse IgG as previously described in
detail.21 The MI15 MoAb recognizes syndecan-1, which is
present only on myeloma cells in bone marrow samples.17,22
Purified myeloma cells were resuspended in RPMI 1640 medium
supplemented with 10% of fetal calf serum (FCS).
Cell lines.
Two human myeloma cell lines (HMCL) were used: XG-1 and XG-2. They had
cytoplasmic Ig, expressed plasma cell antigens (CD38 and syndecan-1),
and lacked the usual B-cell antigens (CD19 and CD20). Their growth was
dependent on addition of exogenous IL-6. Detailed characteristics of
these lines have been reported elsewhere.23 The
IL-6-dependent B9 murine hybridoma cells were a generous gift of Dr L. Aarden (CLB, Amsterdam, NL).
Cell culture.
XG cells were cultured in RPMI 1640 medium supplemented with 10% FCS,
2 mmol/L L-glutamine, 5 × 10 5 mol/L
2-mercaptoethanol and with 3 ng/mL of IL-6 or 10 µg/mL of a pair of
agonist anti-gp130 antibodies. The B9 cells were cultured in the same
medium containing 100 pg of IL-6.
Proliferation assay of myeloma cell lines.
To investigate the effects of agonist anti-gp130 MoAbs or IL-6 on the
proliferation of XG or B9 cells, cells were washed to remove bound
IL-6. They were cultured for 5 hours in culture medium alone and washed
again. They were then incubated in 96-well flat-bottomed microplates
for 5 days with either culture medium alone or with a mixture of a pair
of the anti-gp130 MoAbs (B-S12 + B-P8 or B1 + I2) or IL-6 at a
concentration of 10,000 XG or 5,000 B9 cells per well. Tritiated
thymidine (0.5 µCi, 25 Ci/mmol/L; CEA, Saclay, France) was added for
the last 8 hours of culture to measure tritiated thymidine
incorporation.
Measurement of blood murine and human Ig concentration.
Veinous blood was collected by retro-orbital puncture and Ig
concentrations were determined by a standard double-antibody enzyme-linked immunoabsorbent assay (ELISA). To determine the concentration of murine Ig, microtiter plates (Maxisorb; Nunc, Rosklide, Denmark) were coated with rabbit antibodies to mouse Ig
cross-absorbed to human and bovine Ig (DAKO Z0259; Dakopatts A/S,
Glostrup, Denmark; 200 ng/100 µL in phosphate-buffered saline [PBS]) at 4°C overnight. The protein specific binding sites were saturated by 2 hours of incubation in PBS and 5% of bovine serum albumin (BSA; A-6793; Sigma). Serial dilutions of mouse serum samples
were incubated for 2 hours at room temperature, and, after three
washes, peroxidase-conjugated rabbit antibodies to mouse Ig (DAKO
P0260) were used as a second antibody. Purified mouse IgG (I5381;
Sigma) were used to get a standard curve. After additional washes,
ortho-phenylene diamine (OPD P-6912; Sigma) in sodium acetate buffer
(pH 5) was added as substrate solution and the optical density was
determined using a Titertek Multiskan PLUS (ICN, Meckenheim, Germany).
Human Ig concentration was measured by the same method as the one used
for mouse Ig determination, except that plates were coated with an
affinity-purified goat antihuman IgG antiserum with minimal
cross-reaction to mouse serum proteins (Jackson Immunoresearch Lab,
West Grove, PA) and human Ig were detected by the same antiserum conjugated with peroxidase (Jackson Immunoresearch Laboratories). Home-purified human IgG were used to get the standard curve.
Measurement of circulating human soluble gp130 and IL-6R.
Circulating human soluble IL-6R (sIL-6R) and gp130 (sgp130) were
measured using asymmetric ELISA, as already published.18,20 The M182 and biotinylated M91 anti-IL-6R MoAbs and the A1 and biotinylated D2 anti-gp130 MoAbs were used to coat the immunoplates and
show the bound material, respectively. The epitopes recognized by A1
and D2 anti-gp130 MoAbs are different from those recognized by the
B-S12, B-P8, B1, and I2 MoAbs.20
Flow cytometry.
Cells (5 × 105) were incubated with 1 µg of murine
MoAb in 100 µL of PBS, 30% human AB serum, and 0.01% sodium azide
for 45 minutes at 4°C. Isotype-matched Ig was used as a control
(human or murine IgG). After two washes, cells were stained with either FITC-conjugated or PE-conjugated (Fab)2 fragments of
goat antibodies to mouse IgG. Flow cytometry was performed with a
FACScan apparatus (Becton Dickinson, Palo Alto, CA).
SCID mice.
SCID/SCID/Bg/Bg (CB-17/IcrHsd-scid-bg) were purchased from Harlan
(Gannat, France) and bred in our sterile animal facility. Mice with a blood Ig concentration greater than 10 µg/mL were eliminated (leaky mice). Human myeloma cells (20 to 50 × 106) were washed and resuspended in 150 µL of RPMI 1640 medium without additives to be grafted in the mice. In the majority of
the experiments, they were mixed with basement membrane matrix
(Matrigel; 40234A; Becton Dickinson, Bedford, MA) using precooled
pipettes to avoid gelling. The matrigel stock solution was frozen in
150 µL aliquots and stored at  20°C. An aliquot of 150 µL
was thawed immediately before being mixed with 150 µL of cell
suspension (50 × 106 cells) and the mixture was
surgically implanted into the peritoneum. An autopsy examination was
performed on each animal and aliquots of excised tissues were placed in
4% buffered formalin for immunohistological studies.
Immunohistological studies.
Tissues were embedded in paraffin and sections were stained with
haematoxylin and eosin for histological examination. Additional sections were used for immunochemical staining for cytoplasmic human
or Ig light chains and syndecan-1. Sections were incubated with
murine antihuman or Ig light chains MoAbs or the MI15 anti-syndecan-1 MoAb. They were then incubated with biotin-conjugated goat antimouse antibodies followed by streptavidin-horseradish peroxidase. The staining reaction was performed for 10 minutes with
3,3-diamino-benzidine-tetrahydrochloride in PBS. Tissues of control
SCID mice were not stained with MoAbs to human or Ig light
chains or to human syndecan-1.
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RESULTS |
The agonist antihuman gp130 MoAbs do not recognize or activate murine
gp130.
The survival and growth of XG myeloma cell lines was dependent on
addition of IL-6; after starvation of IL-6, XG cells ceased to
proliferate and progressively died through apoptosis.23,24 In agreement with our previous results,15 the B-S12 and
B-P8 MoAbs to human gp130 supported the long-term growth of the
IL-6-dependent human myeloma XG-1 cell line and labeled membrane gp130
(Fig 1A through C). We have previously
shown that an optimal proliferation was obtained when the antibodies
were added together at a 1:1 ratio.15 The two antibodies
failed to label the murine hybridoma B9 cells or to support their
long-term growth, unlike human IL-6 (Fig 1B through D). As shown in
Fig 2A, the two antibodies induced an
optimal survival and proliferation of XG-1 cells at concentrations ranging from 1 µg (0.5 µg each) to 100 µg (50 µg each). Control murine IgG1 had no effect (Fig 2A). As shown previously,23
an optimal survival and proliferation was obtained with 1 ng/mL of IL-6. Similar results were obtained with the other IL-6-dependent human myeloma cell lines (results not shown). The B1 and I2 anti-gp130 MoAbs that recognized different epitopes than the B-S12 and B-P8 MoAbs20 also supported the growth of IL-6-dependent
myeloma cell lines but not that of B9 cells.16 Optimal
proliferation of myeloma cell lines was obtained when these MoAbs were
used at concentrations similar to those of B-S12 and B-P8 MoAbs, except that concentrations greater than 40 µg/mL became slightly inhibitory (Fig 2B).
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| Fig 1.
The agonist antihuman gp130 MoAb does not recognize or
activate murine gp130. Human XG-1 (A) and murine B9 hybridoma (B) cells were extensively washed and cultured at 5 × 104
cells/mL (XG-1) or 3 × 104 cells/mL (B9) with 10 µg/mL
of control murine IgG1 (control) or with 3 ng/mL (XG-1) or 20 pg/mL
(B9) of IL-6 or with 10 µg/mL of a mixture of B-S12 and B-P8 (5 µg
each) antihuman gp130 IL-6 transducer MoAbs. Every 5 days, the cells
were counted and cultures were diluted at the initial cell
concentration with culture medium containing fresh cytokine or the
initial concentration of anti-gp130 MoAb. Results are the cumulative
numbers of cells generated in the cultures. XG-1 (C) or B9 (D) cells
were labeled with biotinylated B-S12 or B-P8 MoAb or control
biotinylated murine IgG1 and then with FITC-conjugated avidin. Results
are the fluorescence profiles obtained with the different antibodies
analyzed with a FACSCAN apparatus.
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| Fig 2.
Proliferation response of XG cells in the presence of
agonist anti-gp130 MoAbs. XG-1 or XG-2 myeloma cells were extensively washed and cultured for 5 days with various concentrations of a mixture
(1:1) of agonist anti-gp130 MoAbs or of control murine IgG1 or of IL-6.
At the end of the culture, the proliferation was assayed by tritiated
thymidine incorporation. XG-1 cells were cultured with B-S12 + B-P8
antibodies and XG-2 cells with B1 + I2 antibodies. Results are the
mean ± SD tritiated thymidine incorporation determined on sextuplate
culture wells. For some points, the SD was too small to be visible on
the graph.
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Protocol of injection of the anti-gp130 MoAb.
When 100 µg of the B-S12 + B-P8 mixture were injected
intraperitoneally (IP), the concentration of the circulating MoAbs in the blood picked up to 12 µg/mL and progressively decreased, with a
mean half-life of 14.5 days (Fig 3A). These
data suggest a retention of the MoAbs in the peritoneum and a
progressive release into the circulation (Fig 3A). The B1 and I2 MoAbs
behave similarly, with a half-life of 17.1 days (Fig 3B). According to
these data, the following protocol was used to achieve a mean blood
concentration of 5 to 10 µg/mL of the pair of anti gp130 MoAbs
according to the calculations we had previously developed for the
diffusion of MoAb in humans25: IP injection of an initial
dose of 100 µg (50 µg of each MoAb) and IP injection of 50 µg (25 µg of each MoAb) every fortnight. This is an easy way of delivering a
human gp130-activating signal without any toxicity in the SCID mice.
For more than 1 year, 20 mice were injected IP every fortnight with
these antibodies without any obvious toxicity or mortality (results not
shown).
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| Fig 3.
Half-life of the anti-gp130 MoAbs in SCID mice. One
hundred micrograms of a mixture of B-S12 and B-P8 (A) or B1 and I2 (B) anti-gp130 MoAbs (50 µg each) was injected IP in 4 SCID mice. Another
4 mice were injected with physiological saline. At days 1, 4, 8, 12, 16, 20, or 24 after injection, blood was collected and the
concentration of circulating murine Ig was determined by ELISA. Results
are the mean values ± SD obtained in the 4 mice. The concentration of
murine Ig in mice injected with saline was less than 10 µg/mL.
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Growth of IL-6-dependent human myeloma cell lines in SCID mice.
Having determined a protocol of an anti-gp130 MoAb injection to provide
optimal concentration of circulating MoAb in SCID mice, we looked for a
site where tumor cells could be grafted. XG-1 myeloma cells were
injected in SCID mice either intravenously (IV), in the spleen (IS), or
IP. In some experiments, myeloma cells were imbedded in matrigel before
being implanted IP, as this gel has been shown to favor
angiogenesis.26 We have previously shown that matrigel
supported human myeloma cell growth in the presence of IL-6 in vitro
(results not shown). Tumors and/or syndecan 1+ and
HLA-class I+ human cells were looked for in bone marrow,
spleen, blood, or liver 200 days after tumoral cell implantation. As
outlined in Table 1, no tumoral
infiltration or tumors were detected in the IV and IS groups. Tumor
nodules were detected only when myeloma cells were imbedded in matrigel
and implanted IP surgically. Tumors were detected 4 to 5 weeks after an
XG-1 cell graft at the site of matrigel implantation. These tumors
further developed along the peritoneal epithelium as a solid tumor.
Mice died with a massive tumoral invasion of the peritoneum (tumor
weight of about 5 g) 4 to 6 weeks after tumor detection. No myeloma
cells were detected in the blood and bone marrow. Immunohistological
examination showed infiltration of spleen, liver, and pancreas by
tumoral plasmablasts that produced cytoplasmic human Ig light
chains.
The tumors were put into suspension, and fluorescence-activated cell
sorting (FACS) analysis indicated that they were human myeloma cells expressing human HLA class I antigen, human syndecan-1, but no B-cell antigens (Fig 4). In
addition, tumoral cells had the phenotype of the original XG-1 cells:
HLA-DR, CD28+, and
CD80.23 They weakly expressed human
gp130 (Fig 4). They had the cytology of plasmablasts and expressed
cytoplasmic human Ig light chains. When cultured in vitro, tumoral
cells failed to proliferate without addition of agonist anti-gp130
MoAbs or of IL-6 (Fig 5A). In a group of 10 mice grafted with XG-1 cells in matrigel, we measured sequentially the
blood concentration of human Ig, sIL-6R, and sgp130 as markers of tumor
cell mass. As shown in Fig 6, circulating
sIL-6R was the earliest tumoral marker to be detected before the tumors
were palpable. The blood concentration of human Ig or sgp130 increased
later, 2 and 4 weeks after tumor detection, when the tumor size was
already very large (Fig 6). As shown in Table 1, XG-1 cells ended up
forming tumors in mice that did not receive anti-gp130 antibodies.
However, these tumors were detected 20 weeks after the XG-1-cell graft,
16 weeks later than tumors arising in mice injected with anti-gp130
MoAbs. These tumors were composed of human myeloma cells (HLA-class
I+, syndecan-1+, CD28+,
CD19; results not shown) that were able to survive
without IL-6 for several weeks in vitro (Fig 5B). These findings were
not surprising, because we had previously found that such autonomously
growing clones could be obtained in vitro from this IL-6-dependent
XG-1 cell line by long-term culture in vitro at a high
density.27
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| Fig 4.
Phenotype of XG-1 tumors in SCID mice. The tumors growing
in SCID mice grafted with XG-1 cells and injected with agonist
anti-gp130 MoAbs were harvested and put in suspension. Viable cells
were recovered by centrifugation on a Ficoll-hypaque density medium and
cells were labeled with MoAbs to various antigens or control murine
antibodies. Fluorescence was analyzed with a FACSCAN apparatus. The
dashed line represents the fluorescence profile with a control antibody
and the solid line is with MoAbs to specific antigens. The percentages
of cells labeled with the different MoAbs are indicated in the
panels.
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| Fig 5.
Growth of XG-1 tumor cells in vitro. Tumors growing in
SCID mice grafted with XG-1 cells and injected (A) or not (B) with agonist anti-gp130 MoAbs were put in suspension and viable cells were
cultured at a concentration of 2.5 × 105 cells/mL in
culture medium supplemented with 10 µg/mL of control murine IgG1
(control), 3 ng/mL of IL-6 (IL-6), or 10 µg/mL of a mixture of the
B-S12 and B-P8 anti-gp130 MoAbs (5 µg each; anti-gp130 antibodies).
Every 5 days, cells were counted and diluted at the initial cell
concentration with fresh culture medium and the initial concentration
of cytokine or antibodies. Results are the mean cumulative numbers of
cells determined on six different culture wells.
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| Fig 6.
Blood levels of human Ig, soluble IL-6R, and soluble
gp130 in mice grafted with XG-1 cells. Ten mice received IP 100 µg of B-S12 + B-P8 MoAbs (50 µg each) 2 days before graft of 50 × 106 XG-1 cells in matrigel. They then received IP 50 µg
of B-S12 + B-P8 MoAbs (25 µg each) every fortnight. Tumors were
detected by palpation at the site of inoculation in all mice 4 to 5 weeks after the graft. Blood was collected every 2 weeks and assayed for human Ig, human sIL-6R, and human sgp130. The limit of sensitivity of the ELISA was 1 µg/mL for human Ig, 20 ng/mL for human sIL-6R, and
5 ng/mL for human sgp130. Results are the mean ± SD of the determinations. For some points, the error bars were too small to be
visible on the graphs.
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We next investigated whether tumoral cell growth could be obtained with
other IL-6-dependent human cell lines in the presence or absence of
agonist anti-gp130 MoAbs. XG-2 cells imbedded or not with matrigel
formed tumors in vivo with a mean appearance time of 11 weeks (range, 9 to 14 weeks). No tumors were detected without anti-gp130 MoAb
injections. These tumors had the characteristic phenotype of XG-2
myeloma cells (syndecan 1+, HLA-class I+,
CD28+, CD40+++),23 expressed
cytoplasmic chains, and had the morphology of malignant
plasmablasts. Similar to XG-1 tumors, XG-2 tumors grew as solid tumors
along the peritoneal epithelium. Unlike XG-1 tumors, a massive tumoral
infiltration into the bone marrow was found at autopsy (60% of
syndecan-1+ tumor cells in the bone marrow). Tumoral
infiltration of spleen, liver, and pancreas were also found.
Circulating human Ig were detected before clinical tumor detection
(Fig 7). They were 12-fold higher than
those observed in mice bearing XG-1 tumors in agreement with a larger
in vitro Ig production by XG-2 cells compared with XG-1 cells. The
tumor uptake was also associated with the presence of circulating sIL-6
and sgp130 (Fig 7). When cultured in vitro, the tumors grew in an
agonist-gp130-dependent fashion similar to parental cells
(Fig 8).
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| Fig 7.
Concentration of blood murine and human Ig in SCID mice
receiving XG-2 cells and anti-gp130 MoAbs. Three mice were injected IP
with 50 × 106 XG-2 cells and B1 +I2 anti-gp130 MoAbs.
Peripheral blood was harvested 2, 4, 6, 8, 10, and 14 weeks after
XG-2-cell inoculation and the concentrations of human Ig, human sIL-6R,
and human sgp130 were determined by ELISA. The limit of sensitivity of
the ELISA was 1 µg/mL for human Ig, 20 ng/mL for human sIL-6R, and 5 ng/mL for human sgp130. Results are the mean ± SD of the
determinations. For some points, the error bars were too small to be
visible on the graphs.
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| Fig 8.
Growth of XG-2 tumors in vitro. Tumors growing in SCID
mice injected with XG-2 cells and agonist anti-gp130 MoAbs were put in
suspension and the viable cells recovered by centrifugation on Ficoll
hypaque. These cells were cultured at a concentration of 2.5 × 105 cells/mL in culture medium supplemented with 10 µg/mL
of control murine IgG1 (control), 3 ng/mL of IL-6 (IL-6), or 10 µg/mL
of a mixture of the B1 and I2 anti-gp130 MoAbs (5 µg each; anti-gp130 antibodies). Every 5 days, cells were counted and diluted at the initial cell concentration with fresh culture medium and the initial concentration of cytokine or antibodies. Results are the mean cumulative numbers of cells determined on six different culture wells.
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Graft of patients' myeloma cells.
Myeloma cells from 5 patients with MM were purified, imbedded with
matrigel and agonist anti-gp130 MoAb, and implanted in vivo in SCID
mice pretreated with agonist anti-gp130 and further injected every
fortnight with these anti-gp130 antibodies.
A tumor was obtained in a SCID mouse transplanted with the myeloma
cells from a patient with plasma cell leukemia. The growth pattern of
this solid tumor was similar to those reported for the XG-1
IL-6-dependent myeloma cell line. In particular, no human CD45+ cells could be detected in bone marrow or blood.
Cells from this SCID tumor were human CD45+ cells that
expressed no CD3 or CD19 antigens but expressed cytoplasmic Ig
chain, syndecan-1, CD38, and CD56, the same as the donor patient's
myeloma cells (Fig 9). They also expressed
HLA class I but failed to express HLA class II antigens (Fig 9).
Cytological examination indicated that these cells were plasmablastic
cells. At the time of tumor detection, a large concentration of
circulating human Ig was detected in the blood of the mice (150 µg/mL; Table 2). When cultured in vitro,
tumoral cells could grow only in the presence of agonist anti-gp130
antibodies or IL-6 (Fig 10). This cell
line was termed XG-13.
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| Fig 9.
Phenotype of tumor cells developing in SCID mice grafted
with patient's myeloma cells. The tumors growing in SCID mice grafted with a patient's myeloma cells and injected with agonist anti-gp130 MoAbs were harvested and put in suspension. Viable cells were recovered
by centrifugation on a Ficoll-hypaque density medium and cells were
labeled with MoAbs to various antigens or control murine antibodies.
Fluorescence was analyzed with a FACSCAN apparatus. The dashed line
represents the fluorescence profile with a control antibody and the
solid line is with MoAbs to specific antigens. The percentages of cells
labeled with the different MoAbs are indicated in the panels.
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| Fig 10.
In vitro growth of tumor cells harvested from SCID mice
grafted with a patient's myeloma cells. The tumor growing in SCID mice
grafted with myeloma cells from a patient with MM were put in
suspension. Cells were cultured at a concentration of 5 × 105 cells/mL in culture medium supplemented with 10 µg/mL
of control murine IgG1 (control), 3 ng/mL of IL-6 (IL-6), or 10 µg/mL
of a mixture of the B-S12 and B-P8 anti-gp130 MoAbs (5 µg each;
anti-gp130 antibodies). Every 5 days, cells were counted and diluted at
the cell concentration with fresh culture medium and the initial
concentration of cytokine or antibodies. Results are the mean
cumulative numbers of cells determined on six different culture
wells.
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Circulating human Ig were detected in the plasma of mice grafted with
tumoral cells from the 4 patients with MM. The concentration ranged
from 8.1 to 11.9 µg/mL (Table 2). SIL-6R was detected in the mice
grafted with myeloma cells from 2 patients, unlike sgp130 (Table 2). No
solid tumors were detected 360 days after transplantation. No human
syndecan-1 cells or CD45 cells were detected in the bone marrow, blood,
spleen, and liver. Immunohistological examination showed the presence
of human myeloma cells in the mesenteric adipose tissue. These data
indicated that a minority of myeloma cells survived in these mice.
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DISCUSSION |
We describe here a model that, in SCID mice, makes it possible to grow
human myeloma cells whose survival and growth is dependent on gp130
transducer activation in vitro. This SCID model has several advantages.
First, we use agonist antihuman gp130 MoAbs that have a long half-life
(2 weeks) in vivo and do not activate murine gp130. Then, we avoid a
daily injection of human IL-6 that has a short half-life in vivo (20 to
60 minutes)10,11 and is likely to have all the toxicities
reported for IL-6 in vivo as human IL-6 binds to murine
IL-6R.12-14 Another advantage is that we have to target
only the gp130 transducer and avoid a cascade of activation of IL-6R
and then of gp130 transducer by IL-6/IL-6R complexes.28 This is likely to be important for in vivo models, because large levels
of circulating agonist IL-6R circulate in the human (ie, 200 ng/mL in
patients with MM) and may replace the need for membrane IL-6R.29,30 In the early phases after tumoral implantation, it is possibly very important to provide an optimal gp130 activation to
prevent tumor cells from dying, as is the case with gp130 antibodies. It is noteworthy that one group has reported the possibility of growing
in vitro human myeloma cells with a combination of IL-6 and sIL-6R but
not with IL-6 alone, which is probably due to a too weak membrane IL-6R
expression on myeloma cells.31
By using this optimal strategy to provide human gp130 transducer
activation in SCID mice, we have been able to grow IL-6-dependent human myeloma cell lines and freshly explanted myeloma cells from 1 patient with extramedullary proliferation. In addition, we have observed the persistence of circulating human Ig for more than 1 year
with freshly explanted myeloma cells from patients with medullary
involvement. For one cell line (XG-2), tumors were obtained only in
mice injected with anti-gp130 MoAbs, whereas for the XG-1 cell line,
tumors were obtained both with and without anti-gp130 MoAbs. However,
tumors without anti-gp130 MoAbs occurred 20 weeks after tumoral cell
inoculation, 16 weeks later than in the presence of anti-gp130 MoAbs.
This again emphasized than an optimal gp130 transducer activation in
the early days after tumor cell injection is likely to be important to
promote an optimal tumoral cell survival. The occurrence of XG-1
tumoral growth without anti-gp130 MoAbs in SCID mice was not
surprising, because this cell line produced a weak amount of autocrine
IL-6, and autonomously growing subclones can be obtained in vitro by
culturing cells at a high cell density or with myeloma cell survival
factors such as interferon- .24,27
The tumors developed as a solid tumor along the peritoneal membrane and
were enveloped in peritoneal epithelial cells. Tumors were fully
vascularized. In the late stages of tumoral invasion, tumoral
infiltration of the liver, pancreas, and spleen were found. Tumor
infiltration of bone marrow was found for the XG-2, unlike the XG-1
myeloma cells, suggesting that the murine bone marrow environment is a
suitable one for certain populations of human myeloma cells. Tumor
cells retained all the phenotypical characteristics of the inoculated
myeloma cells and produced human Ig, sIL-6R, and sgp130 that circulated
in the mouse blood. This made it possible to monitor tumoral cell mass
and growth by assaying the concentration of circulating human sIL-6R
and human Ig.
The tumors growing in SCID mice also retained the dependence on gp130
activation to survive and grow in vitro, as did the inoculated myeloma
cells. These findings again emphasize the usefulness of the agonist
antibodies to provide a continous human gp130 activation in SCID mice.
The tumor uptake was increased when myeloma cells were imbedded in
matrigel together with agonist anti-gp130 antibodies before being
implanted. In particular, no XG-1 tumor occurred spontaneously in mice
injected with XG-1 cells not imbedded in matrigel. The improvement of
tumor uptake through using matrigel is in agreement with previous
reports showing that matrigel favored angiogenesis,26 thus,
the feeding of myeloma cells by various blood nutrients, in particular
by the circulating agonist anti-gp130 antibodies.
The fact that we have been able to get tumors from IL-6-dependent cell
lines or from freshly explanted myeloma cells from a patient with
extramedullary proliferation, unlike those with medullary involvement,
is in agreement with in vitro data and like the pattern of disease
growth in patients. In vitro, by using IL-6 or these agonist
antibodies, we, and others, have been able to obtain IL-6-dependent
cell lines from myeloma cells from patients with extramedullary
proliferation only.7,23 Myeloma cells from patients with
medullary involvement survive for several weeks with the agonist
anti-gp130 antibodies but are blocked in the G1 phase of the cell cycle
(unpublished results). For 2 patients, we obtained a
survival lasting for several months without cell proliferation. For
these patients' cells, activation of the gp130 transducer was a
necessary signal to promote myeloma cell survival but not sufficient to
promote G1 to S phase transition and cell cycling (unpublished
results). When inoculated in SCID mice with agonist
anti-gp130 antibodies, these myeloma cells could also survive for more
than 1 year, as assayed by the detection of circulating human Ig, and
they did not proliferate. In patients with medullary involvement, the
situation is likely to be very similar, because only a minor fraction
of myeloma cells proliferate in vivo.32 In these patients
with medullary involvement, beside gp130 transducer activation, an
additional signal might be delivered by stromal cells that make it
possible for a minor fraction to enter the cell cycle. One of these
signals might be linked to the FGF/FGR3 signalling. Indeed, t(4;14)
translocations involving the FGR3 gene have been shown to occur in
myeloma cell lines and in freshly explanted cells from some
patients.33 In addition, FGR3 mutations similar to those
found in dwarnish syndrome and leading to constitutive activation of
the receptor have been shown in some cell lines.34 The
recent demonstration that fetal bone favors the homing of myeloma cell
lines in SCID mice may help to identify this cosignal.5
In patients with extramedullary proliferation, activation of gp130
transducer induces G1 to S phase transition,35,36
suggesting that genetic disregulations contribute to this transition.
In these patients, most of the abnormalities have been linked to gene
coding for proteins regulating the apoptosis and the G1 to S phase
transition: hypermethylation of P16 gene,37 deletion of
Rb,38,39 mutations in P53 gene,40,41
translocation involving the cyclin D1 gene,42,43 or the
MUM1/IRF4 gene.44
This SCID mouse model of MM should be interesting as an assay for
various therapeutical strategies, including immunotherapies and
chemical agents. Because it takes into account the gp130 dependence on
myeloma cell survival and proliferation,24 inhibitors of the gp130 transduction pathway could be assayed. For example, tyrphostin AG490 that blocks JAK 2 activation was recently shown to
inhibit the growth of human leukemic cells in SCID mice without affecting mouse viability.45 We are now testing to find out whether these inhibitors may block JAK2 activation in myeloma cells as
well as their in vitro survival and growth. If positive results are
obtained, it would be useful to look for an inhibition of the
gp130-dependent myeloma growth in SCID mice. However, because the
agonist activity of gp130 antibodies is not affected by anti-IL-6 or
IL-6R antibodies, one limitation of this model is that inhibitors of
IL-6, IL-6 production, or IL-6R cannot be assayed, contrary to recently
published models.6 In addition, the overproduction of IL-6
in MM is associated with accompanying symptoms related to IL-6
toxicities46-49 that cannot be observed in the present SCID
model due to the lack of biological activity of anti-gp130 antibodies
on murine cells.
In conclusion, the SCID model we present here is a unique model making
it possible to obtain gp130-dependent survival and growth of myeloma
cells from cell lines or from patients with active disease. This model
should be useful to study various agents able to prevent tumor survival
and growth in vivo.
| |
FOOTNOTES |
Submitted November 19, 1997;
accepted February 11, 1998.
Supported by grants from ARC (Paris, France), LFNC (Paris, France), and
DRC (CHU Montpellier, France).
Address reprint requests to Bernard Klein, PhD, INSERM U475, 99 Rue
Puech Villa, 34197 Montpellier Cedex 5, France; e-mail: klein{at}montp.inserm.fr.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract