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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1697-1706
Ibandronate Reduces Osteolytic Lesions but not Tumor Burden in a
Murine Model of Myeloma Bone Disease
By
Sarah L. Dallas,
I. Ross Garrett,
Babatunde O. Oyajobi,
Mark R. Dallas,
Brendan F. Boyce,
Frieder Bauss,
Jiri Radl, and
Gregory R. Mundy
From the Department of Medicine (Division of Endocrinology and
Metabolism), and the Department of Pathology, University of Texas
Health Science Center at San Antonio, TX; the Department of Preclinical
Research and Development, Bone Metabolism, Boehringer Mannheim,
Germany; and TNO Institute for Prevention and Health, Leiden, the
Netherlands.
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ABSTRACT |
We determined the effects of the potent bisphosphonate ibandronate
in a murine model of human myeloma bone disease. In this model, bone
lesions typical of the human disease develop in mice following
inoculation of myeloma cells via the tail vein. Treatment with
ibandronate (4 µg per mouse per day) significantly reduced the
occurrence of osteolytic bone lesions in myeloma-bearing mice. However,
ibandronate did not prevent the mice from developing hindlimb paralysis
and did not produce a detectable effect on survival. There was no
significant effect of ibandronate on total myeloma cell burden, as
assessed by morphometric measurements of myeloma cells in the bone
marrow, liver, and spleen, or by measurement of serum IgG2b levels.
These results support clinical findings that bisphosphonates may be
useful for the treatment of myeloma-associated bone destruction, but
suggest that other therapies are also required to reduce tumor growth.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PATIENTS WITH multiple myeloma frequently
have extensive bone involvement, which is manifested as osteolytic
lesions. These osteolytic bone lesions are responsible for some of the most distressing symptoms of the disease, such as intractable bone
pain, increased susceptibility to fractures, hypercalcemia, and nerve
compression syndromes such as spinal cord compression. An agent that
could prevent or reduce the osteolysis associated with multiple myeloma
would be of major therapeutic benefit for patients with this disease.
Recent studies have shown that specific treatment of the bone disease
associated with myeloma using inhibitors of bone resorption can be
beneficial to the patient (for reviews see1-4). Berenson et al5 showed that in patients treated with the
bisphosphonate pamidronate, there was a decrease in the number of
skeletal-related events including the need for analgesics, courses of
radiation therapy, episodes of hypercalcemia, and pathologic fractures. Similar data were published by Lahtinen et al,6 Laakso et
al,7 and McCloskey et al8 using clodronate.
These findings have led to the Federal Drug Administration (FDA)
recently releasing pamidronate for the treatment of not just
hypercalcemic patients but also nonhypercalcemic patients with
osteolytic bone disease due to myeloma.
An important question is whether treatment with bisphosphonates will
influence tumor burden, as shown by Sasaki et al using experimental
models of human breast cancer cell metastasis.9 In these
studies, bisphosphonates were found to reduce tumor burden, specifically in the skeleton, with no effect on the growth of breast
cancer metastases in soft tissues. This suggested that the breast
cancer cells may be dependent on factors released during bone
resorption for their growth in the bone microenvironment. It is not
known currently whether myeloma cells are similarly dependent on the
products of bone resorption for their growth in the bone marrow cavity
and whether inhibitors of bone resorption, such as bisphosphonates,
will inhibit tumor growth. Shipman et al10 and Aparicio et
al11 have recently reported that some bisphosphonates
induce apoptosis of myeloma cells in vitro. However, it is not yet
clear whether bisphosphonates have a beneficial effect on survival in
multiple myeloma patients. McCloskey et al8 reported no
significant improvement in survival in myeloma patients treated with
oral clodronate. Similarly, Brincker et al12 reported no
improvement in survival of myeloma patients treated with oral
pamidronate. However, this may have been due to poor uptake of
pamidronate given orally. In the studies of Berenson et
al,5,13 pamidronate treatment, administered by intravenous
infusion, did not improve overall survival in myeloma patients.
However, in a subgroup of patients on salvage therapy, a significant
improvement in survival was observed. Unfortunately, the issues raised
by these studies are difficult to answer definitively in patients
because (1) bisphosphonates are rarely administered in the absence of
other treatments such as chemotherapy and radiation; (2) there are many
confounding variables in all patients with advanced malignant disease,
including other non-bone-related complications; (3) it is difficult to
assess responses to specific therapies in patients with advanced bone
disease; and (4) patient studies often take many years to complete.
One practical way of overcoming some of these problems is with the use
of an appropriate animal model of myeloma bone disease. Such an animal
model is the 5T murine model of myeloma in which myelomas arise
spontaneously in an inbred substrain of C57 black mice (C57BL/KaLwRij
substrain).14-16 The myelomas can be propagated from mouse
to mouse in this inbred strain by marrow transfer. Several of the 5T
myeloma lines closely mimic myeloma disease in humans, with monoclonal
gammopathy, marrow replacement, focal osteolytic bone lesions, hindlimb
paralysis, and occasionally hypercalcemia. Cell lines have been
established from this myeloma model, which also mimic the human
disease.17,18
In the present study, we used the 5T murine myeloma model to examine
the effects of a potent bisphosphonate on myeloma-associated bone
destruction. We found that ibandronate, an amino
bisphosphonate,19 markedly inhibited myeloma-associated
bone resorption in this model. Ibandronate has potential advantages
over pamidronate because it can be used orally and is more potent.
Treatment with ibandronate (4 µg per mouse per day) significantly
reduced the development of osteolytic lesions in myeloma-bearing mice.
Ibandronate was not effective in preventing animals from developing
hindlimb paralysis and did not prolong survival of myeloma-bearing
animals. Consistent with its lack of effect on survival, ibandronate
did not reduce the total tumor burden, as assessed by serum IgG2b
levels, or the tumor burden in the bone marrow, liver, and spleen.
These results suggest that ibandronate may be a useful adjunctive
therapy for the treatment of myeloma to specifically inhibit the
increased bone resorption that typically occurs in this disease.
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MATERIALS AND METHODS |
Cell Culture
Unless stated otherwise, all tissue culture reagents were obtained from
Life Technologies Inc (Gaithersburg, MD) or JRH Biosciences (Kansas
City, MO).
5TGM1 myeloma cells.
5TGM1 myeloma cells were derived from a myeloma, designated 5T33, which
arose spontaneously in an aged C57BL/KaLwRij mouse.14 The
characterization of these cells is described below. These cells were
propagated by marrow transfer in the inbred C57BL/KaLwRij strain of
mice and reliably produced disease exhibiting many of the features of
human myeloma. In our hands, the 5TGM1 variant of 5T33 grows more
avidly and causes more bone destruction in vivo. Additionally, this
variant grows well in culture without supplementation with IL-6 or
stromal cell-conditioned media. For preparation of myeloma cells for
inoculation, marrow was flushed from femurs and tibias of 5TGM1
myeloma-bearing mice that exhibited increased serum IgG2b monoclonal
protein using a syringe and 27-gauge needle. The marrow cells were then
centrifuged and resuspended in 20 mL Iscove's modified Dulbecco's
media (IMDM) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L
L-glutamine (LG), 100 U/mL penicillin-streptomycin (P/S). After
overnight culture in a 90-mm Petri dish, nonadherent cells were
recovered and replated in 75-cm2 tissue culture flasks in
20 mL IMDM supplemented with 10% FBS, 2 mmol/L LG, and 100 U/mL PS.
These nonadherent cells were expanded in culture for 7 days and then
prepared for injection into the tail vein of recipient mice as
described below.
5T33 myeloma cell line.
We have also recently established a clonal myeloma cell line (5T33)
that can be maintained in long-term culture and that, when injected
into C57BL/KaLwRij mice, produces myeloma disease exhibiting most of
the features of human myeloma as described above. The isolation and
characterization of this IgG2b-producing clonal cell line has been
described previously.18
Preparation of myeloma cells for intravenous injection.
All cultures for inoculation were harvested at subconfluency and re-fed
with fresh culture medium 24 hours before use. Cells were centrifuged,
washed twice in 50 mL phosphate-buffered saline (PBS), and then
resuspended at 5 × 106 cells per mL of PBS. Two hundred
microliters of this cell suspension (ie, 106 cells) was
inoculated into experimental mice via tail vein injection using a
27-gauge needle. Control animals received injections of PBS alone.
Characterization of 5TGM1 Myeloma Cells
5TGM1 myeloma cells were characterized by histological examination of
affected bones and soft tissues in both the founder animal from which
the myeloma variant was isolated and in mice inoculated with 5TGM1
myeloma cells. Besides routine histology, cytocentrifuge preparations
of cells isolated from the bone marrow of 5TGM1 myeloma-bearing mice
were also stained by the May-Grünwald-Giemsa method for
examination. Radiographs of the myeloma-bearing animals were taken to
confirm the presence of osteolytic lesions in the founder animal and in
mice inoculated with 5TGM1 myeloma. Serum IgG2b levels were measured by
ELISA as described below and were found to be elevated to >30 mg/mL
in the founder animal and to increase to similar levels after 4 weeks
in mice inoculated with 5TGM1 myeloma cells. The increase in serum
IgG2b in 5TGM1 myeloma-bearing mice was confirmed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western
blotting. Details of these methods are described below.
Animals
Animal studies were conducted using 8- to 10-week-old female
C57BL/KaLwRij mice in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals. Whole blood samples
were collected at baseline and then weekly after tumor inoculation by
retroorbital puncture under methoxyflurane inhaled anesthesia. Body
weights of animals were also determined at baseline and weekly thereafter.
Ibandronate Administration
Ibandronate, a potent bisphosphonate,19 was kindly donated
by Boehringer Mannheim (Mannheim, Germany). Ibandronate was diluted in
sterile PBS at a concentration of 20 µg/mL for injection into animals. Two hundred microliters of this solution was administered daily into experimental animals by subcutaneous injection (the dosage
used, expressed as free acid equivalents of the monosodium salt
monohydrate, was 4 µg per mouse per day). Control animals received
injections of vehicle (PBS).
The dose of ibandronate used (0.16 mg/kg) was at the highest end of the
range of doses tested in various animal models of normal and pathologic
bone resorption, including: ovariectomized rats and dogs,
thyroparathyroidectomized rats with and without treatment with
parathyroid hormone-related protein, and rats bearing the Walker 256 carcinoma.20 In these experiments the doses used ranged
between 0.001 to 0.1 mg/kg. The dose used in the present study (0.16 mg/kg) is approximately 100-fold higher than the doses required to
inhibit bone resorption in these animal models. This dose was selected
based on the above studies and also based on our previous results
showing this dose to be effective in reducing both osteolytic lesions
and tumor burden in bone using a murine model of breast cancer
metastasis to bone.21
Analytical Methods
Measurement of serum IgG2b levels.
Serum IgG2b levels were measured using a specific two-site ELISA. The
capture antibody (rat anti-mouse IgG2b; Zymed Laboratories Inc, San
Francisco, CA) was coated onto EIA/RIA plates (Costar, Cambridge, MA)
overnight at 4°C at a concentration of 2 µg/mL in PBS. The plates
were washed three times in PBS + 0.05% tween 20 and blocked in PBS + 5% bovine serum albumin (BSA) for 3 hours at room temperature. The
plates were then incubated for 1 hour at 37°C with IgG2b standard
(mouse IgG2b kappa; Cappel Research Products, West Chester, PA) or
serum samples which had been diluted in PBS + 0.5% BSA. The plates
were washed six times in PBS + 0.05% tween 20 and then incubated for 1 hour at 37°C with detection antibody (peroxidase-conjugated rat
anti-mouse IgG antibody; Biodesign International, Kennebunk, ME), used
at a 1:5000 dilution in PBS + 0.5% BSA. The plates were washed 10 times in PBS + 0.05% tween 20, and then O-phenylenediamine tablets
were used as the reaction substrate according to manufacturer's
instructions (Sigma Chemical Co, St Louis, MO). The reaction was
stopped by addition of 3 mol/L HCl (25 µL/100 µL reaction volume).
The absorbance at 492 nm was read on an EIA plate reader. IgG2b
concentrations were calculated by linear regression using the Immunofit
EIA/RIA program, version 2.00 (Beckman Instruments Inc). This ELISA has
a linear detection range of approximately 1 to 100 ng/mL.
Quantitation of osteolytic bone lesions from radiographs.
After death, whole animal radiographs were obtained as described
previously22 using a Faxitron radiographic inspection unit (Field Emission Corporation Inc, McMinnville, OR). Similar x-rays were
also taken of the limbs, spine, and calvaria after dissection of the
tissues, removal of the skin, and fixation in 10% buffered formalin.
Quantitation of lesions visible on radiographs was performed by
computerized image analysis as described previously.22
Using this technique, lesions as small as 0.1 mm2 can be
visualized as radiolucent areas in the bones. The number and area of
these lesions was quantified by an individual who was without knowledge
of the experimental protocols.
Histology and morphometric analysis.
After fixation in 10% buffered formalin, skeletal tissues were
decalcified in 14% EDTA and embedded in paraffin by standard techniques as described previously.9,23 Soft tissues were fixed in 10% buffered formalin and embedded in paraffin without prior
decalcification. Nonconsecutive sections were cut longitudinally through the sagittal plane of the lumbar vertebrae and through blocks
of liver and spleen of each animal for histomorphometric analysis using
a standard microtome. The sections were then placed on
poly-L-lysine-coated glass slides and stained with hematoxylin and
eosin.9,23
Histomorphometric analysis of vertebral trabecular bone volume (bone
volume/tissue volume; BV/TV, %) and the percentage of vertebral bone
marrow replaced by tumor was performed stereologically in two
representative nonconsecutive sections of lumbar vertebrae L2 through
L7 using point counting and a Zeiss Integrationsplatte II eyepiece
graticule along with an Olympus BHS microscope and a ×20
magnification objective lens. Measurements were made in a minimum of
three vertebrae to obtain a mean value for each animal. Trabecular bone
volume was measured in two fields (0.314 mm2) in the
center of each vertebral body at a standard location (0.056 mm) from
each growth plate. The percentage of marrow replaced by tumor was
measured in five fields (0.078 mm2) chosen randomly
within the marrow cavity of each vertebra. The mean thickness of the
cortices of lumbar vertebra was measured in two nonconsecutive
longitudinal sections of lumbar vertebrae L2 through L7 using an
Olympus BX40 microscope fitted with a drawing tube, together with the
Osteomeasure computerized histomorphometry system (Osteometrics,
Atlanta, GA). A total of 20 individual measurements of cortical bone
thickness was made in each vertebra on each section using a ×10
objective lens. The percentage of liver and spleen replaced by tumor
was measured in 10 randomly chosen fields (0.168 mm2) in
each of two nonconsecutive sections from each organ using the
Osteomeasure histomorphometry system and a ×20 objective lens.
SDS-PAGE and Western blotting.
For Western blotting analysis, 0.25 µL serum samples from control and
tumor-bearing animals were separated on duplicate 7% SDS-PAGE gels
under nonreducing conditions. Coomassie blue staining was performed by
standard techniques. For Western blotting analysis, proteins were
transferred onto a nitrocellulose membrane and immunoblotting was
performed as described previously.24 The primary antibody was a rat anti-mouse IgG2b (Zymed Laboratories Inc, San Francisco, CA)
used at 2 µg/mL in TBS + 1% BSA and the secondary antibody was an
HRP-conjugated donkey anti-rat (Jackson Immunoresearch, Westgrove, PA)
used at a 1:2,500 dilution in 5% skimmed milk. Immunostained proteins
were detected using an enhanced chemiluminescence (ECL) kit according
to manufacturer's instructions (Amersham International PLC,
Arlington Heights, IL).
In Vitro Growth and Cell Viability Assays
To examine the effects of bisphosphonates on in vitro growth and
viability of 5TGM1 myeloma cells a dye exclusion assay was used. 5TGM1
myeloma cells were plated in 24 well plates at 105 cells
per milliliter in IMDM supplemented with 5% FBS in the presence or
absence of 1 to 100 µmol/L ibandronate or risedronate (kindly donated
by Rhone-Poulenc-Rorer, Collegeville, PA). After incubation for 24 to
72 hours, trypan blue was added to 0.04% and the plates incubated at
37°C for 30 minutes. The total number of cells and the number of
trypan blue-positive cells was then counted using a Neubauer chamber
under brightfield illumination. Percentage viability was calculated by
subtracting the number of trypan blue-positive (ie, nonviable) cells
from the total cell number and expressing this figure as a percentage
of the total cell number.
Statistical Analysis
Student's t-test was used for comparisons made between two
groups of data. In experiments where comparisons were made between more
than two treatment groups, analysis of variance was used followed by
the Student Newman-Keuls method of multiple comparisons. Statistical
differences in the survival rate of the animals and the number of
animals developing hindlimb paralysis were analyzed by the generalized
Wilcoxon test.25 In all cases, data were accepted as
significantly different with a probability of .05 or less.
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RESULTS |
Characterization of 5TGM1 Myeloma Cells
Histological analysis of affected bones from the founder animal from
which the 5TGM1 myeloma variant was isolated revealed myeloma cells
that had almost completely replaced the normal marrow and had caused
bone destruction. An example of this is shown in Fig
1A, which shows a lumbar vertebra from the
founder animal. Note the loss of cortical bone (arrowheads), and the
reduced amounts of trabecular bone at the endochondral growth plates.
Myeloma cells (MY) can also be seen to have invaded the surrounding
soft tissues. The spinal cord (SP) is surrounded by myeloma cells, which has resulted in spinal cord compression (the animal was paraplegic at the time of death). A similar pattern of tumor growth occurred after 4 weeks in mice that were inoculated with
106 5TGM1 myeloma cells (for examples refer to Fig 5).
The myeloma-bearing mice also showed osteolytic lesions that
were visible on radiographs (see Fig 2).
Giemsa staining of cytocentrifuge preparations of 5TGM1 cells isolated
from the marrow of myeloma-bearing mice revealed a typical myeloma
morphology (see Fig 1B). ELISA measurements revealed that the serum
IgG2b levels were elevated approximately 10- to 50-fold in
myeloma-bearing animals compared with non-tumor-bearing controls (for
examples refer to Fig 6). SDS-PAGE and Western blotting analysis
confirmed increased serum monoclonal protein in myeloma-bearing animals, which co-migrated with a purified IgG2b standard and was
recognized by an anti-IgG2b antibody by Western blotting (see Fig 1C
and D).
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| Fig 1.
Characterization of 5TGM1 myeloma cells. (A) Hematoxylin
and eosin (H&E) stained section of a lumbar vertebra from the 5TGM1
founder animal, showing myeloma cells (MY), which have almost
completely replaced the normal bone marrow and are associated with
osteolysis. Note that trabecular bone is absent from the growth plate
shown at left and greatly reduced in the growth plate shown at right.
Arrowheads indicate areas where cortical bone has been destroyed,
allowing tumor cells to invade the surrounding tissue. This animal was
paraplegic at the time of death and in this section, the spinal cord
(SP) can be seen to be surrounded by myeloma cells, which has resulted
in spinal compression (bar = 200 µm). (B) Giemsa stained
cytocentrifuge preparation of cultured 5TGM1 myeloma cells, exhibiting
a characteristic myeloma morphology (bar = 100 µm). (C) Coomassie
blue-stained SDS-PAGE analysis of serum from control mice (lanes 1, 2),
5TGM1 myeloma-bearing mice (lanes 3, 4), and the 5TGM1 founder animal
(lane 5), lane 6 shows 1 µg of IgG2b standard for comparison. The
arrow indicates the IgG2b band. (D) Identical gel to C, which was
analyzed by Western blotting using antibodies against mouse IgG2b.
Lanes 1 through 6 are the same as for C.
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| Fig 2.
Radiographs showing the effect of ibandronate (4 µg per
mouse per day for 28 days) on osteolytic lesions (arrows) in the lumbar
vertebrae of 5TGM1 myeloma-bearing mice. (A) Non-tumor-bearing control
treated with PBS. (B) Non-tumor-bearing control treated with
ibandronate. (C) 5TGM1 myeloma-bearing mouse treated with PBS. (D)
5TGM1 myeloma-bearing mouse treated with ibandronate. Note the large
number of lesions visible in myeloma-bearing mice treated with PBS (C),
which are prevented by treatment with ibandronate (D). Note also the
reduction in height of the lumbar vertebrae in myeloma-bearing mice
treated with PBS (C) as compared with controls (A), (B), and
myeloma-bearing mice treated with ibandronate (D). There are seven
vertebrae visible in the field in (C), as compared with six in (A),
(B), and (D).
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Effects of Ibandronate Treatment in 5TGM1 Myeloma-Bearing Mice
A number of different experimental protocols were designed using the
5TGM1 model of multiple myeloma to assess the effects of ibandronate on
myeloma bone disease. Initial experiments were designed to determine
the effect of continuous (prophylactic) treatment with ibandronate on
5TGM1 myeloma bone disease and also to determine the effects of
ibandronate in non-tumor-bearing control animals. Ibandronate (4 µg
per mouse per day) was administered at the time of tumor inoculation
and daily thereafter for the duration of the experiment. Four
experimental groups (n = 6) were used: group A received myeloma cells
and daily injections of ibandronate, group B received myeloma cells and
daily injections of vehicle (PBS), groups C and D did not receive
myeloma cells, but received daily injections of ibandronate and vehicle
(PBS), respectively. The experiment was terminated when the first
animal developed hindlimb paralysis in either of the tumor-bearing groups.
Mice injected with 5TGM1 myeloma cells showed osteolytic bone lesions
by 4 weeks after tumor inoculation and experiments were terminated on
day 28. Lesions in the vertebrae were readily detectable in 5TGM1
myeloma-bearing animals that were treated with PBS (representative radiographs are shown in Fig 2). In contrast, 5TGM1 myeloma-bearing animals that were treated with daily ibandronate showed a dramatic reduction in the number of osteolytic lesions visible in their vertebrae. Results from quantitation by computerized image analysis of
the osteolytic lesions in the vertebrae of 5TGM1 myeloma-bearing mice
with and without ibandronate treatment are shown in Fig
3A and B. Ibandronate (4 µg per mouse per
day) significantly reduced both the number and area of vertebral
osteolytic lesions in 5TGM1 myeloma-bearing mice. Moreover, a
significant reduction in the mean height of the lumbar vertebrae was
noted in 5TGM1 myeloma-bearing mice compared with non-tumor-bearing
controls, presumably due to crush fractures (see Figs 2 and 3C). This
reduction in height was less severe in myeloma-bearing mice that were
treated with ibandronate compared with myeloma-bearing mice treated
with PBS. Myeloma-induced osteolytic lesions in the long bones were
also significantly reduced by ibandronate treatment (see Fig 4A and B).
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| Fig 3.
Histograms showing (A) the number and (B) the area of
osteolytic lesions in the lumbar vertebrae of 5TGM1 myeloma-bearing and
control mice with and without ibandronate treatment (4 µg per mouse
per day for 28 days). Lesions were quantified by computerized image
analysis from radiographs taken at sacrifice. Data are mean ± SEM
(n = 6). *, Significant reduction compared with myeloma + PBS.
(C) Histogram showing the effect of ibandronate treatment (4 µg per
mouse per day for 28 days) on the mean height of the lumbar vertebrae
in 5TGM1 myeloma-bearing and control mice. The height of each lumbar
vertebra was measured by image analysis from radiographs taken at
sacrifice and the mean vertebral height calculated for each animal.
Data are mean ± SEM (n = 6). +, Significant decrease compared
with PBS treated non-tumor-bearing control. *, Significant increase
compared with myeloma + PBS.
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| Fig 4.
Histograms showing (A) the number and (B) the area of
osteolytic lesions in the long bones of 5TGM1 myeloma-bearing and
control mice with and without ibandronate treatment. Lesions were
quantified by computerized image analysis from radiographs taken at
death. Data are mean ± SEM (n = 6). *, Significant reduction
compared with myeloma + PBS.
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Figure 5 shows representative histological
sections of lumbar vertebrae from control and myeloma-bearing mice
treated with PBS or ibandronate. In myeloma-bearing mice treated with
PBS (Fig 5C) a reduction in trabecular bone as well as occasional
thinning and destruction of cortical bone was observed when compared
with non-tumor-bearing control mice (Fig 5A and B). This loss of bone was prevented by treatment with ibandronate in myeloma-bearing animals
(Fig 5D). Histomorphometric analysis confirmed that mean values for
trabecular bone volume in the vertebrae of 5TGM1 myeloma-bearing mice
treated with PBS were significantly lower than those in
non-tumor-bearing controls (see Table 1).
This reduction in bone volume was prevented by treatment with
ibandronate. Mean values for trabecular bone volume in the vertebrae of
both control and tumor-bearing animals treated with ibandronate were
higher than those in the PBS-treated controls, but these differences
failed to reach statistical significance.
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| Fig 5.
H & E stained sections of lumbar vertebrae from control
and 5TGM1 myeloma-bearing mice treated with PBS or ibandronate (4 µg
per mouse per day for 28 days). (A) Non-tumor-bearing control treated
with PBS, (B) non-tumor-bearing control treated with ibandronate,
(C) 5TGM1 myeloma-bearing mouse treated with PBS, (D) 5TGM1
myeloma-bearing mouse treated with ibandronate. The spinal cord (SP) is
indicated in each figure. Note that in the non-tumor-bearing animals,
(A) and (B), the marrow cavity is filled with normal marrow (BM). In
contrast in myeloma-bearing animals (C) and (D), the normal bone marrow
has been replaced by myeloma cells (MY), which have also invaded the
surrounding tissues. In the myeloma-bearing animal treated with PBS
(C), there is a clear loss of trabecular bone and also some loss of
cortical bone. This bone loss is prevented by ibandronate treatment
(D). Bar = 200 µm.
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Table 1.
Histomorphometric Analysis of Lumbar Vertebrae From
Control and 5TGM1 Myeloma-Bearing Mice With or Without Ibandronate
Treatment
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In myeloma-bearing mice treated with PBS, cortical bone loss was
observed only in vertebrae that had a relatively high degree of marrow
replacement by tumor. Histomorphometric analysis of cortical bone
thickness in myeloma-bearing mice was therefore restricted to vertebrae
in which the marrow replacement by tumor was shown to be greater than
70% (see Table 1). The mean cortical bone thickness was significantly
lower in myeloma-bearing mice treated with PBS compared with
myeloma-bearing and non-tumor-bearing mice treated with ibandronate,
consistent with the anti-resorptive effects of this drug. Although
myeloma-bearing animals treated with PBS showed a lower mean cortical
bone thickness compared with non-tumor-bearing controls treated with
PBS, this difference did not reach statistical significance.
In contrast to its effect on osteolytic lesions and trabecular bone
volume, ibandronate treatment did not significantly affect the
percentage of bone marrow replaced by tumor cells (see Table 1). The
liver and spleen are the two major sites other than bone where myeloma
growth occurs in the 5T33 and 5TGM1 myeloma models. Ibandronate
treatment had no significant effect on the 5TGM1 tumor volume in the
liver and spleen, as reflected in the organ weights at death and by
histomorphometric analysis (see Table 2).
Ibandronate also did not significantly reduce the total tumor burden,
as assessed by serum IgG2b concentrations (see Fig 6).
Body weights of the animals were similar in all groups throughout the
experiment and showed a modest reduction in tumor-bearing animals
between days 21 and 28. However, this reduction was not significantly
different from control non-tumor-bearing animals (data not shown).
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| Fig 6.
Graph showing the effect of ibandronate treatment (4 µg
per mouse per day) on serum IgG2b concentrations in 5TGM1
myeloma-bearing and control mice. Serum IgG2b levels were measured by
ELISA. Data are mean ± SEM (n = 6). *, Significantly different
from control + PBS. No significant differences were observed between
myeloma-bearing animals treated with PBS or with ibandronate.
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Effect of Ibandronate on Hindlimb Paralysis and Survival in 5TGM1
Myeloma-Bearing Animals
Further experiments were designed to determine whether ibandronate
treatment of 5TGM1 myeloma-bearing animals had any beneficial effect on
their survival or the onset of hindlimb paralysis, which occurs
frequently in the murine 5T myeloma model. Two experimental groups were
used (n = 10). Group A received myeloma cells and daily injections of
vehicle (PBS) beginning at the time of tumor inoculation and continuing
until the death of each animal. Group B received daily injections of
ibandronate (4 µg per mouse per day) beginning at the time of tumor
inoculation and continuing until the death of each animal.
Treatment with ibandronate did not significantly reduce the occurrence
of hindlimb paralysis or prolong survival in 5TGM1 myeloma-bearing mice
(see Fig 7). At necropsy, both PBS and
ibandronate-treated mice showed enlargement of the liver and spleen,
due to growth of the myeloma in these sites. It is therefore assumed
that the animals died from the consequences of large tumor burdens in
these organs because of the large amount of circulating monoclonal
protein and/or opportunistic infections or from some other
illness due to the immunocompromization resulting from replacement of
marrow with myeloma cells.
View larger version (16K):
[in this window]
[in a new window]
| Fig 7.
Graphs showing the effect of ibandronate treatment (4 µg per mouse per day) on (A) hindlimb paralysis and (B) survival in
5TGM1 myeloma-bearing mice. Significant differences were not observed
between myeloma-bearing mice treated with PBS or with ibandronate.
|
|
Effect of Ibandronate Treatment in 5T33 Myeloma-Bearing Mice
The effects of continuous (prophylactic) treatment with ibandronate
were also assessed in another murine model of myeloma, which uses the
5T33 clonal myeloma cell line. Ibandronate (4 µg per mouse per day)
was administered at the time of tumor inoculation and thereafter
administered daily for the duration of the experiment. Two experimental
groups (n = 6) were used: group A received myeloma cells and daily
injections of ibandronate, group B received myeloma cells and daily
injections of vehicle (PBS). The experiment was terminated when the
first animal developed hindlimb paralysis in either of the
tumor-bearing groups.
Mice injected with the clonal 5T33 myeloma cell line showed osteolytic
bone lesions similar to the 5TGM1 myeloma, by 10 weeks after tumor
inoculation. These lesions occurred predominantly in the long bones.
Figure 8 shows results from quantitation by image
analysis of the area and number of osteolytic lesions seen on
radiographs of the long bones of 5T33 myeloma-bearing mice at death.
Treatment of myeloma-bearing mice with ibandronate (4 µg per mouse
per day) significantly reduced both the number and area of osteolytic
lesions. Similar to the results obtained with the 5TGM1 myeloma,
ibandronate treatment did not reduce the total tumor burden, as
assessed by the serum IgG2b concentrations and the liver and spleen
weights (data not shown). Body weights of the animals were similar in
all groups (data not shown).
View larger version (15K):
[in this window]
[in a new window]
| Fig 8.
Histograms showing (A) the number and (B) the area of
osteolytic lesions in the long bones of 5T33 myeloma-bearing mice with
and without ibandronate treatment. Lesions were quantified by
computerized image analysis from radiographs taken at death. Data are
mean ± SEM (n = 6). *, Significant reduction compared with
myeloma + PBS.
|
|
Effect of Bisphosphonate Treatment on 5TGM1 Cell Growth In Vitro
The above data suggested that ibandronate inhibited bone destruction
without reducing tumor burden in the 5TGM1 mouse myeloma model,
however, bisphosphonates have recently been reported to induce
apoptosis in myeloma cell lines in vitro.10,11 To further investigate the lack of effect of ibandronate on tumor burden in the
5TGM1 in vivo model of myeloma, we examined its effects on in vitro
growth and apoptosis of the 5TGM1 myeloma cell line. The bisphosphonate
risedronate was also tested for comparison, as it is a non-amino
bisphosphonate with similar potency.26 In the present
study, using a dose of 4 µg ibandronate per mouse, we estimate the
peak serum concentration of ibandronate to be approximately 5 µmol/L
(assuming a blood volume of approximately 2 mL for a 20-g mouse).
Initial experiments to examine the effects of bisphosphonates on growth
of 5TGM1 cells in vitro were therefore performed using a dose range of
1 to 5 µmol/L ibandronate or risedronate. Using this dose range, no
significant effect was seen with ibandronate or risedronate on the
total number of myeloma cells or on the percentage of viable cells in
the cultures, even in prolonged cultures of up to 72 hours (data not
shown). An extended dose range of 10 to 100 µmol/L ibandronate was
then tested (see Table 3). Treatment for 72 hours with
10 µmol/L ibandronate produced a slight but significant reduction in
the total number of myeloma cells with no significant effect on the
percentage of viable cells. Treatment with 50 µmol/L caused a modest
reduction in both cell number and cell viability, and a more dramatic
effect was seen with 100 µmol/L, where there was an almost complete
loss of cell viability.
| |
DISCUSSION |
We have used the murine 5T in vivo model of myeloma to study the
effects of the bisphosphonate ibandronate, which is a powerful inhibitor of osteoclastic bone resorption, on the osteolytic bone disease associated with this cancer. The advantage of this in vivo
animal model is that the effects of the bisphosphonate can be
quantified in the complete absence of other cancer therapies, which
often compound the results of studies in human
patients.5-8,12,13 We have found that ibandronate markedly
inhibits myeloma-associated bone destruction without reducing the total
tumor burden in this animal model, using two different myeloma cell lines.
In contrast to the data reported here, we have previously shown that
ibandronate and other bisphosphonates reduce tumor burden to the
skeleton in models of osteolysis induced by breast cancer cells.9,21,27 In those studies, although the
bisphosphonates had no beneficial effect on growth of breast cancer
metastases in soft tissue organs, they dramatically reduced tumor
burden in the bone and bone marrow. We hypothesized that factors
released by the breast cancer cells stimulate osteoclastic resorption. The actively resorbing osteoclasts then in turn release factors such as
transforming growth factor beta (TGF ) and insulin-like growth
factors (IGFs), which change the local bone microenvironment so that it
is more favorable for growth of the breast cancer cells. This may lead
to a vicious cycle between growth of the breast cancer cells and
pathologic bone resorption.4 Myeloma cells, in contrast to
breast cancer cells, are derived from hematopoietic cell lineages,
which normally reside in the bone marrow. Thus, the marrow
microenvironment may already be favorable for growth of the myeloma
cells independent of local rates of bone resorption, possibly by the
production by marrow stroma of cytokines such as
interleukin-6.28-30 This may explain why myeloma cells,
unlike breast cancer cells, appear to be able to grow in the marrow
cavity and completely replace the normal marrow cells even when
osteoclastic activity is inhibited by bisphosphonates such as
ibandronate. Thus myeloma cells may be less dependent than breast
cancer cells on factors released by resorbing osteoclasts for their
growth in the bone marrow cavity.
Our data showing that bisphosphonates reduce skeletal lesions in the 5T
model of myeloma bone disease confirms and extends an earlier study
using pamidronate.31 In this study, pamidronate was
effective in reducing osteolysis in mice bearing the 5T2 myeloma line.
Similar to the present study, no effect of pamidronate on the total
tumor burden or tumor burden to the skeleton was observed. However, the
effect of pamidronate on tumor burden in non-bone sites was not
assessed. In the present study, ibandronate treatment was effective in
reducing myeloma-associated osteolytic lesions when administered at the
time of tumor inoculation and continually for the duration of the
experiment. Future studies are required using therapeutic regimens in
which treatment is initiated after establishment of the myeloma or is
given in conjunction with other anti-cancer therapies. This may more
closely model the clinical situation, in which treatment is initiated
after clinical diagnosis of multiple myeloma, at which stage the
patient frequently already has extensive bone involvement. Our results
provide support for recent clinical studies using bisphosphonates,
which report that they are effective in reducing the occurrence of
skeletal-related events (ie, pathologic fractures, radiation therapy to
bone, surgery to bone, spinal cord compression), reducing progression
of osteolytic lesions on x-ray, reducing pain scores, and improving
quality of life assessments in myeloma patients.5,7,13
In the present study ibandronate treatment was not effective in
preventing the development of hindlimb paralysis in tumor-bearing mice.
Hindlimb paralysis is a frequent complication of disease in 5T
myeloma-bearing mice and probably occurs due to spinal cord compression
by myeloma cells. However, at present it is not clear whether these
cells gain access to the spinal cord by escaping from the vertebral
marrow cavity through resorption-induced cavities or by direct invasion
of the spinal canal through pre-existing vascular channels independent
of bone resorption.
Although ibandronate had beneficial effects on the osteolytic component
of the myeloma disease, it did not prolong survival of 5TGM1
myeloma-bearing mice. Consistent with this observation, ibandronate did
not reduce the total tumor burden, as assessed by serum IgG2b levels,
or the tumor burden in the bone marrow cavities, liver, and spleen.
However, the natural history of the disease in these mice runs a rapid
course over a few weeks, and thus effects on survival are not as easy
to discern as they may be in humans, where the median survival is
several years. Animals probably died of other complications of the
myeloma disease, such as liver involvement, renal failure,
and/or infections due to compromised immunity. Interestingly,
in a follow-up to the study of Radl et al,31 Croese et al
reported a significant prolongation of survival in 5T2 myeloma-bearing
mice treated with pamidronate, although no significant effect on tumor
growth was observed.32 The beneficial effect of pamidronate
in this model might be due to differences in the modes of action of
different bisphosphonates or to the longer times (months as opposed to
weeks) required for the 5T2 myeloma disease to run its course.
Our results support those of a number of human clinical trials in which
the effects of bisphosphonates on survival have been reported. Lahtinen
et al,6 Laakso et al,7 and McCloskey et
al8 showed no improvement in survival in myeloma patients treated with clodronate. Similar results were reported by
Brincker et al12 using oral pamidronate.
In a randomized double-blind, placebo-controlled study of intravenous
pamidronate treatment for multiple myeloma, no significant effect of
pamidronate on overall patient survival was observed; however, in a
subgroup of patients who entered the trial receiving salvage
chemotherapy, a significant improvement in survival was
observed.5,13
Recent in vitro studies have raised the intriguing possibility that
bisphosphonates may exhibit anti-tumor activity by directly stimulating
apoptosis in myeloma cells.10,11 However, in these studies
the concentrations of bisphosphonates used were generally over 10 µmol/L, even for the most potent bisphosphonate presently available, Zoledronate (Novaritis, Basel,
Switzerland).11 These doses are high relative to the
peak serum concentrations achieved in patients undergoing treatment
with bisphosphonates. Thus, it remains to be determined whether
sufficiently high levels of bisphosphonates could readily be attained
in patients for this anti-tumor activity to manifest. We estimate that
the peak serum concentrations of ibandronate achieved in the
present study were approximately 5 µmol/L. This concentration of
ibandronate did not affect the growth or viability of 5TGM1 cells in
vitro, and cytotoxic effects were seen only at doses of 50 µmol/L and
higher. This cytotoxic effect is probably nonspecific, as a similar
loss of viability was observed in normal mouse bone marrow cultures and
in a murine osteoblast cell line, 2T3, using similar concentrations of
ibandronate (data not shown). In contrast, the doses of bisphosphonates
effective in inducing apoptosis in osteoclasts are several orders of
magnitude lower,33 suggesting a specific effect of
bisphosphonates on osteoclast apoptosis. Although we report no
significant effect of ibandronate on growth and viability of 5TGM1
myeloma cells in vivo or in vitro, except at high doses that produced
nonspecific effects on other cell types, the possibility remains that
bisphosphonates with different chemical structures may have different
effects on tumor cell growth and apoptosis. Thus, the cytotoxic effects of bisphosphonates on myeloma cells may be limited to particular bisphosphonates with specific structural features. Future studies are
clearly warranted to address this important question. The 5T mouse
myeloma model may be an ideal system in which to compare the effects of
bisphosphonates with different chemical side groups for their effects
on both tumor-associated bone resorption and on growth and apoptosis of
the tumor cells.
In summary, our results suggest that although not useful as a direct
therapy to reduce tumor burden in myeloma patients, ibandronate may be
extremely useful as an adjunctive therapy for the treatment of the
osteolytic component of myeloma disease. Since it is this bone
destructive component that causes the most distressing and painful
symptoms for the patient, the use of bisphosphonates such as
ibandronate in myeloma disease may improve dramatically the quality of
life of the patient. However, other treatments, such as chemotherapy,
radiotherapy, and marrow transplantation should continue to be the main
therapies directed at preventing growth of the myeloma cells.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge the technical assistance provided by Arlene
Farias and secretarial assistance provided by Nancy Garrett.
| |
FOOTNOTES |
Submitted May 8, 1998; accepted October 22, 1998.
Supported by Grant No. P01-CA40035 (from the National Institutes of
Health) and by Boehringer Mannheim, GmbH, D-68305 Mannheim, Germany.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Sarah L. Dallas, PhD, Department of
Medicine/Endocrinology, University of Texas Health Science Center, San
Antonio, TX 78284-7877; e-mail: dallas{at}uthscsa.edu.
| |
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K. C. Anderson, R. A. Kyle, W. S. Dalton, T. Landowski, K. Shain, R. Jove, L. Hazlehurst, and J. Berenson
Multiple Myeloma: New Insights and Therapeutic Approaches
Hematology,
January 1, 2000;
2000(1):
147 - 165.
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Abstract
Introduction