VOl. 1. 1563-1570. Decei,,ber 1995 Clinical Cancer Research 1563
The abbreviations used are: MDR, multidrug resistance: SCID, severe
combined immunodeficiency.
Severe Combined Immunodeficiency (SCID) Mouse Modeling of
P-Glycoprotein Chemosensitization in Multidrug-resistant
Human Myeloma Xenografts1
William T. Bellamy,2 Abiodun Odeleye,
Elizabeth Huizenga, William S. Dalton,
Ronald S. Weinstein, and Thomas M. Grogan
Departments of Pathology [W. T. B.. A. 0., R. S. W.. T. M. G.J and
Medicine 1W. S. DI, Arizona Cancer Center, University of Arizona.
School of Medicine, Tucson. Arizona 85724, and University of
Michigan, Ann Arbor, Michigan 48105 IE. H.l
ABSTRACT
We have established a reproducible in vivo model of
human multiple myeloma in the severe combined immuno-deficiency (SCID) mouse using both the drug-sensitive
8226/S human myeboma cell line and the P-glycoprotein-expressing multidrug-resistant 8226/C1N subline. As dem-
onstrated previously, the SCID mouse is well suited as a
model for myeloma because: (a) human SCID xenografts are
readily attained; (b) human myeloma xenografts are readily
detected by their immunoglobulin secretion; and (c) differ-
ential therapy effects in drug-sensitive versus drug-resistant
cell lines are readily demonstrable by monitoring mouse
urinary human immunoglobulin output. In the current
study, we have utilized this model to evaluate the in vivo
efficacy of chemomodulators of P-glycoprotein-related mub-
tidrug resistance. In our initial experiments, doxorubicin
alone was effective in treating the 8226/S human myeloma
xenografts but had no effect on the drug-resistant 8226/C iN
xenografts, in the absence of the chemosensitizing agent
verapamil. In subsequent experiments, the combination of
verapamil and doxorubicin resulted in both a decrease in
human X light chain urinary excretion and an increase in
survival of those animals bearing the 8226/C1N tumor. The
median survival time of animals injected with 8226/C1N
cells and subsequently treated with doxorubicin was 48.6 ±
7 days, which compared to a survival of 89.6 ± 18 days in
animals receiving the 8226/S cell line and treated with doxo-
rubicin alone (P < 0.001). When verapamil was added to the
treatment regimen of those animals bearing the 8226/C1N
xenografts, there was a 179% increase in their life span (P <0.001), which corresponded with the observed decreasedlight chain in the urine. In animals receiving multiple
courses of chemotherapy, an attenuated response to vera-
Received 5/2/95: revised 7/10/95: accepted 8/2/95.
I This work was supported in part by National Cancer Institute Grants
CA57228. CA17094. CA43043. ES06694. and CA23074.
2 To whom requests for reprints should be addressed. at Department of
Pathology, College of Medicine, University of Arizona, 1501 North
Campbell Avenue, Tucson, AZ 85724. Phone: (520) 626-6032: Fax:
(520) 626-1027.
pamil and doxorubicin was observed, in a manner analogousto the clinical setting of human drug-resistant myeloma
escape from chemosensitivity. The SCID human myeloma
xenograft model thus offers a means of evaluating the in vivo
efficacy and potential toxicities of new therapeutic ap-
proaches directed against P-glycoprotein in multidrug-resis-
tant human myeloma.
INTRODUCTION
Multiple myeborna is a plasma cell malignancy which is
generally incurable in spite of a high initial response to chemo-
therapy. Relapsing disease commonly heralds increasing drug
resistance. Previous studies indicate that terminal drug-resistant
myeloma commonly expresses P-glycoprotein ( 1-5). The P-
glycoprotein is a cellular efflux pump encoded by the MDR-1
gene. and is frequently overexpressed in chernotherapy-refrac-
tory cancers in the clinic (6). One approach to modulate drug
resistance due to P-glycoprotein overexpression has involved
the use of agents known as chernosensitizers/chemomodulators
which inhibit its function. Using a munine leukemia model,
Tsuruo et (1!. (7) were the first to observe that a number of
compounds. including the calcium channel blocker venaparnil.
were able to reverse P-glycoprotein-mediated MDR1 both in
vitro and in otto. Clinical trials have shown that the use of
chemomodulators such as venapamil and cyclosponin A may be
beneficial. particularly in hematological malignancies (8-13).
Agents such as verapamib. however, have been shown to be
toxic at the concentrations required to be fully effective ( 14).
and, therefore, new chemosensitizers are sought that will be
more potent and less toxic than the initial compounds. Con-
founding the development of more potent chemosensitizers is
the observation that P-glycoprotein is expressed in normal tis-
sues including the kidney. adrenal gland. liven. and endothelial
cells lining the capillaries of the brain ( 15-17). The effects of
new chemosensitizens on these tissues is of great importance
because of the possibility of untoward toxicities as a conse-
quence of enhanced tissue uptake or altered tissue distributions
of either the chemomodulaton or the chemotherapeutic agents
( I S -2 1 ). Such interactions are best assessed through in t’it’o
models.
We have established a reproducible in viva model of hu-
man multiple myeboma in the SCID mouse using both the
drug-sensitive 8226/S human myeloma cell line and the P-
glycoprotein-expressing multidrug-resistant 8226/C I N subline
( I ). Although murine models of myeboma exist, these systems
do not specifically model human rnyeloma associated with drug
Research. on July 14, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1564 Chemosensitization in SCID Human Myeloma Xenografts
resistance. The SCID mouse, as we have previously demon-
strated, is well suited as a model for myeloma because: (a)
human SCID xenografts are readily attained; (b) tumor cells are
readily detected by their immunoglobulin secretion; and
(c) differential therapy effects in drug-sensitive versus drug-
resistant cell lines are readily demonstrable (1). The objective of
this study was to evaluate our model for its ability to assess the
in vivo efficacy and toxicities of chemosensitizers directed
against P-glycoprotein-mediated MDR using verapamil as the
prototypical chemosensitizer.
MATERIALS AND METHODS
Generation of Human Multiple Myeloma in SCID
Mice. The generation of human SCID myeloma xenografts
has been described in detail in a previous publication (1).
BALB/c/C.17 mice homozygous for the SCID defect (scid/scid)
were bred and maintained in a dedicated facility at the Arizona
Health Science Center, which is accredited by the American
Association for Accreditation of Laboratory Animal Care. The
animals were housed in microisolator cages under specific
pathogen-free conditions and were handled in a laminar flow
hood. Principles of animal care as set forth in NIH Publication
No. 85-23, revised 1985, were followed throughout. Mice were
screened at regular intervals for the presence of bacteria, Sendai
virus, mouse hepatitis virus, and Mycop!asma. All mice were
evaluated for the presence of mouse IgG by an ELISA, and
those animals with � 1 mg/liter of mouse immunoglobulin were
excluded from the studies. In brief, 5-8-week-old male and
female animals received either the P-glycoprotein-negative hu-
man 8226/S cells or the P-glycoprotein-expressing multidrug-
resistant subline 8226/C1N given i.p. Prior to injection, the
tumor cells were washed twice and resuspended in sterile PBS
(pH 7.4). In all instances, low passage number cells in logarith-
mic growth were used. Cell viability was >95% as assessed by
trypan blue dye exclusion. Both 8226 cell lines produce and
secrete is. immunoglobulin light chain, and the development of
human tumors in the recipient mice was monitored at 4-5-day
intervals using a radial immunodiffusion assay (The Binding
Site Ltd., Birmingham, England) to measure the presence of
human X light chain in the urine. The sensitivity of this assay is
�0.7 mg/liter.
Immunophenotyping. Tissue section phenotyping was
performed on formalin-fixed or snap-frozen tissues using a
standard three-stage immunohistochemistry method on an auto-
mated Ventana 320 immunostainer (Ventana Medical Systems,
Tucson, AZ; Refs. 22 and 23). Snap-frozen tissues were perme-
abilized in 4#{176}Cacetone and assayed using relevant primary
monoclonal antibodies (anti-X, anti-K, mouse antihuman; Bee-
ton Dickinson, Mountain View, CA) followed by incubation
with secondary biotin-avidin conjugates and horseradish perox-
idase labeled with diaminobenzidine tetrahydrochloride as the
detection agent. P-glycoprotein was detected using monoclonal
antibodies JSB-1 (Accurate Chemicals, Westbury, NY) and
C-494 (Centocor, Malvern, PA) using a biotin-avidin detection
method. An irrelevant isotype-matched primary antibody was
substituted as a negative control.
In Situ Hybridization. Paraffin sections were cut 4-jim
thick and heated at 60#{176}Cfor 2 h. The sections were then
deparaffinized in two changes of xylene for 12 mm each, two
changes of 100% ethanol for 5 mm each, and were taken
through a graded series of alcohols (95%, 80%, 70%) followed
by two changes of diethylpyrocarbamate H2O. Proteinase K
(100 gig/ml; Boehringer Mannheim, Indianapolis. IN) was then
placed on the sections for 60 mm at 37#{176}Cand removed by two
washes in PBS. The sections were placed through a second
series of graded alcohols (70%, 80%, 95%, 100%) and allowed
to air dry prior to hybridization. The slides were heated to 95#{176}C
and hybridization to a biotinylated human DNA probe enriched
for the repetitive A!u and Kpn sequences (Cot-l ; GIBCO-BRL,
Grand Island, NY) was carried out in 25% formarnide, 10%
dextran sulfate, 5X Denhardt’s, 4X SSC, 50 ms� sodium phos-
phate (pH 7.0), 1 mM EDTA, and 100 gig/mb salmon sperm
DNA overnight at 37#{176}C.Two stringency washes of 2.5% BSA/
0. 1 X SSC were carried out at room temperature for 1 0 mm
each. The slides were then washed in PBS and blocked with 1%
BSA in PBS for 30 mm at room temperature. For detection of
the biotinylated probe, the ExtrAvidin Alkaline Phosphatase
system was used according to the manufacturer’s protocol (Sig-
ma, St. Louis, MO).
Tumor Treatment with Doxorubicin and Verapamil.Prior to the initiation of therapy, tumor growth was confirmed
through urinary )i. measurement. Approximately 21 days after
inoculation with either 8226/S or 8226/C1N cells, treatment
with doxorubicin in the presence or absence of verapamil was
initiated. Both doxorubicin and verapamil were obtained from
Sigma Chemical Corp. and were dissolved in 0.9% saline. Drug
concentrations were adjusted so that a maximum volume of 100
gil was injected per animal.
Three doses of doxorubicin ( 1 .5 mg/kg) were administered
i_p. every 4 days. This dosing regimen was established previ-
ously as the maximum tolerated dose for doxorubicin in this
model (1). In the initial experiments, verapamib was adminis-
tered via continuous infusion using an Alzet 2001 osmotic pump
(ALZA Corp., Buena Vista, CA) implanted s.c. through a small
incision made on the dorsal surface. Pumps were implanted 24
h prior to administration of the initial dose of doxorubicin. The
infusion rate was 1.01 gil/h and resulted in a dose of 150
mg/kg/day verapamil.
In subsequent experiments, veraparnil was administered as
a bolus i.m. injection immediately prior to each dose of doxo-
rubicin. The maximally tolerated dose of verapamil by bolus
injection in our model system was determined to be 50 mg/kg
(data not shown). To minimize drug-induced toxicities, animals
were given injections of a dose of 40 mg/kg verapamil.
Following drug administration, the animals received food
and water ad !ibitum and were monitored for urinary Is. light
chain excretion as well as weight loss and other signs of toxic-
ities. A response to therapy was defined as a stabilization or a
decrease in the urinary light chain values following drug admin-
istration. All mice were necropsied at the time of tumor relapse
to document the presence of tumor.
RESULTS
Human SCID xenografts using the 8226 myeloma cell lines
were established rapidly and reproducibly under the conditions
of i.p. growth. Xenografts of both drug-sensitive and -resistant
Research. on July 14, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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CFig. I Human plasmacytoma xenognaft in a SCID mouse. A and B. xenognaft of 8226/C I N drug-resistant myeloma cells infiltrating the pancreas
of a SCID mouse. The lange blastic or anaplastic plasma cells infiltrate the pancreas. producing a mass which displaces normal pancreas. C and D,
an assay for cytoplasmic light chains (K and X. respectively) reveals a monocbonal cytoplasmic presence of X light chain with absent K light chains.
pos.
neg.
pos.
neg.
Clinical Cancer Research 1565
Table I Immunophenotype of 8226 cell lines
8226/S 8226/C1N
x Light chain pos.”
K Light chain neg.
CD-38 (Leul7) pos.CD-56 (N-CAM) neg.P-glycopnotein
JSB-l neg. pos.C-494 neg. pos.
“ p05.. positive: neg.. negative.
8226 cell lines appeared histologically as plasniacytomas (Fig.
1 ). Immunophenotyping revealed that all tumors were human X
light chain positive and human K bight chain negative (Fig. I and
Table 1 ). The human origin of the tumors was confirmed by in
situ hybridization as shown in Fig. 2. The human SCID 8226/
C 1N xenografts showed strong surface P-glycoprotein expres-
sion as evidenced by reactivity with both JSB-l and C-494.
whereas the human SCID 8226/S xenografts were negative
(Table 1).
Previous work with this model demonstrated that the Ia-
tency period from injection of tumor cells to the appearance of
human light chain in the urine was identical for mice given
injections of either the 8226/S or 8226/C I N cells ( I ). In addi-
tion, the survival times of untreated animals bearing either
tumor type were not statistically different ( 1).
When tumor-bearing SCID mice were treated with I .5
mg/kg doxorubicin administered i.p. every 4 days for three
doses, beginning 2 1 days after injection of cells. there was a
marked decrease in the level of human X light chain in the urine
of those animals bearing the P-glycoprotein-negative 8226/S
cells (Fig. 3A). In contrast, there was an increase in the X light
chain excretion of those animals inoculated with the multidrug-
resistant 8226/CIN cells under the same treatment schedule
(Fig. 3B). Thus, doxorubicin was effective in treating the 8226/S
xenografts but had no effect on 8226/C 1 N xenografts in the
absence of a chemosensitizer. In a second set of experiments.
shown in Fig. 4. we demonstrated that when veraparnil was
added to the treatment regimen. either as a continuous infusion
or as a bolus injection. there was a marked decrease in the level
of human X light chain in the urine of those animals bearing the
drug-resistant 8226/C1N cells in contrast to those without vera-
pamil. thus demonstrating a measurable chemosensitizing ef-
feet. As shown in Fig. 4A, when doxorubicin is administered in
the absence of verapamil. there is a continuous increase in the X
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p
1566 Chemosensitization in SCID Human Myeboma Xenografts
*‘
Fig. 2 I?? situ hybridization of SCID human myeloma xenograft to a DNA probe (Cot- 1 ) enriched for repetitive human Ala and Kpn sequences.
Murine tissue (left) shows no staining, while human tumor cells are readily detectable (rig/it). X 250.
light chain values, thus indicating a failure of therapy. When
doxorubicin is administered in conjunction with verapamil to
8226/C1N-beaning mice, as shown in Fig. 4B, there is a rapid
decline in the X values. The tumor is not completely eradicated
however as demonstrated by the gradual increase in It. values
following cessation of therapy. Our initial studies were based on
the observation that, to maintain adequate tissue and plasma
levels in the range demonstrated to be effective for in vitro
chemosensitization, verapamil should be administered continu-
ously in the presence of chemotherapy (24). We therefore uti-
lized a previously published protocol for continuous infusion of
verapamil using surgically implanted osmotic pumps (25). Sub-
sequent studies, using bolus injections in nontumor-beanng
SCID mice, revealed that we could achieve a mean plasma
verapamil bevel of 598 ± 75 (range, 540-710; n - 9) ng/ml
following i.m. injection of 50 mg/kg. Although bolus dosing
does not achieve the continuous serum levels which are felt to
be optimal, we were able to achieve a treatment effect similar to
that observed with the osmotic pumps. Regardless of whether
verapamil was administered as a continuous infusion or as a
series of bobus injections, we did not observe any increase in
toxicities, as assessed by increased weight loss or mortality, in
those animals receiving the combination of doxorubicin and
verapamil over those animals receiving doxorubicin alone.
When verapamil was administered alone, under conditions of
continuous infusion, we observed no treatment effect against
either tumor type (data not shown). There were no treatment
differences observed based on the sex of the animal.
With the addition of veraparnib to the doxorubicin regimen,
the reduction in the light chain values of those mice given
injections ofthe 8226/C1N tumors translated into a significantly
prolonged animal survival (Fig. 5 and Table 2). The median
survival time of animals given injections of 8226/C 1N cells and
subsequently treated with doxorubicin was 48.6 ± 7 days,
which compared to a survival of 89.6 ± 1 8 days in animals
receiving the 8226/S cell line and treated with doxorubicin alone
(P < 0.001). The addition of verapamil to the treatment regimen
did not alter the survival of animals bearing the 8226/S xe-
nografts, which was consistent with our observations that vera-
pamil did not alter the excretion of X light chain in the urine.
When verapamil was added to the treatment regimen of those
animals bearing the 8226/C1N xenografts. there was a 179%
increase in their life span (P < 0.001), which corresponded with
the observed decreased light chain in the urine. This increase in
the survival time allowed us, in a third set of experiments, to
administer multiple courses of chemotherapy to mimic the sit-
uation of escape from chemosensitization observed in human
MDR clinical trials. In Fig. 6, a group of animals bearing the
8226/C1N tumors were treated with both doxorubicin and vera-
pamil administered as bolus injections under our standard treat-
Research. on July 14, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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10
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0 10 20 60
60
50
40
30
20
0 10 20 30 40 50 60Days
0 10 20 30 40 50 60Days
Fig. 3 A and B. human X light chain excretion in the urine of 8226/S
and 8226/C1N tumor-bearing SCID niice before and after therapy with
1.5 mg/kg doxorubicin administered every 4 days. Tumor cells were
injected on day 0. and doxorubicin therapy was initiated (arrows). A.
8226/S cells: B, 8226/CIN cells (a 5 mice in each group). Symbols’,
individual mice.
ment protocol. We observed a sharp decrease in tumor burden
following the first course of treatment, which was consistent
with our previous studies. The animals were then treated with a
second course ofdoxorubicin and verapamil once the light chain
values had increased to approximately pretreatment levels. Fol-
lowing the second course of chemotherapy, an attenuated then-
apeutic response was observed.
DISCUSSION
The resistance of tumor cells to the cytotoxic effects of
chemotherapy continues to be a major obstacle to the successful
treatment of human cancers. Chemotherapy is the primary treat-
ment modality of multiple myeboma but despite improvements
in survival and quality of life, this remains an incurable disease
in part due to the development of drug resistance. The evalua-
tion of novel experimental therapeutic approaches for multiple
myeloma has been hampered by the lack of an appropriate
animal model. Although murine models of myelorna are known,
i.e. , pristane-induced BALB/c peritoneal plasmacytomas and
Fig. 4 A and B. human X light chain excretion in the urine of 8226/
Cl N tumor-bearing SCID mice before and after therapy with 1 .5 mg/kgdoxorubicin administered every 4 days in the presence or absence of the
chemosensitizer verapamil (150 mg/kg/day s.c.). Tumor cells were
injected on day 0, and doxorubicin therapy was administered (arrows).
A. X excretion in the absence of venapamil: B. X expression in the
presence of verapamil. Symbols, individual mice.
spontaneous C57BL marrow myebomas (26-29), these systems
do not specifically model human myeloma associated with drug
resistance. We have recently developed a reproducible in viva
human xenograft model in SCID mice which utilizes both the
P-glycoprotein-negative RPMI 8226 human myeloma cell line
and the P-glycoprotein-expressing multidrug-resistant 8226/
CIN subline, a derivative of the 8226/DOX4O MDR cell line
( 1 ).
The SCID xenograft model was developed to assess the
efficacy and toxicities of new chernotherapeutic agents and
chemosensitizers directed against the P-glycopnotein-rnediated
MDR phenotype and should serve to complement current in
vitro assays which are better suited to initially identify corn-
pounds effective in overcoming P-glycoprotein-mediated drug
resistance. The advantages of the SCID human myeloma xc-
nograft model have been previously stated and allow for quan-
tification of tumor burden via urinary monoclonal light chain
measurement and a reproducible, defined time frame of tumor
growth and survival allowing for assessment of outcome in a
Clinical Cancer Research 1567
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60 80Days
Treatment
8226-Sens” + Dox/sabine
8226-Sens + Dox/venapamil
8226-C1N + Dox/saline
8226-C I N + Dox/verapamil
Mean survivaltime (days) ± SE
89.6 ± 3.6
87.8 ± 6.1
48.6 ± 1.2
86.0 ± 5.63
6 Sens, definition; Dox, doxorubicin.
1568 Chemosensitization in SCID Human Myeboma Xenografts
Fig. 5 in vito activity of verapamil in combination with doxorubicin in
SCID mice bearing 8226/S or 8226/C1N xenografts. Mice were giveni_p. injections of tumor cells and treated with doxorubicin with or
without venapamib as described in ‘ ‘Materials and Methods.’ ‘ Data
presented are the results of three separate experiments (n = 21-30
animals/group). Points, surviving animals at the time on the abscissa
divided by the initial number of animals treated.
Table 2 Mean survival times of myeloma-bearing SCID mice
(‘I = 21-30/group)
speedy fashion. Measurements of serum immunogbobulin have
been demonstrated to correlate with the total tumor burden in
patients with IgG rnyeloma (30). The measurement of human
monoclonab light chain in the urine represents a noninvasive and
sensitive means of assessing human tumor burden in a murine
model. By quantitating changes in urinary light chain values at
the initiation and during chemotherapy, the efficacy of a given
treatment can be determined. One of the advantages of this
model is that tumor growth and treatment effects can be as-
sessed repeatedly by human monoclonal light chain assays
without relying solely on measurements such as the percentage
of increased life span. Treatment can be initiated at a specific
tumor burden, thus permitting a direct comparison of the effec-
tiveness of chernotherapeutic agents used at the same extent of
disease in each animal. This approach would be preferable to the
other models in which the tumor is treated at a predetermined
time interval following the inoculation of a known number of
tumor cells. In these systems, the actual tumor burden in mdi-
vidual animals may vary considerably, thus complicating direct
comparisons. Our model permits the precise quantification ofresponse and comparison of agents in mice with similar tumor
burdens. Those compounds identified as promising on the basis
of in vitro assays would then become candidates for in viva
evaluation of their ability to reverse P-glycoprotein-mediated
drug resistance.
0 20 40 60 80 100Time (Days)
Fig. 6 Human X light chain excretion in the urine of 8226/C INtumor-beaning SCID mice before and after two courses of chemotherapy
with 1.5 mg/kg doxorubicin administered every 4 days in the presence
of verapamil ( 150 mg/kg/day i.m.). Tun#{236}oncells were injected on day 0,
and doxorubicin plus verapamil was administered (arrows). Symbols.
individual mice.
Both in vitro and clinical trials have shown that chemo-
modulators such as verapamil and cyclosponin A may be ben-
eficiab in multiple rnyeborna (8, 9, 1 1 , 3 1 ). While these corn-
pounds have demonstrated a proof of concept, the clinical
results obtained to date in rnyeboma have been limited. Myelorna
patients, after an initial beneficial chemosensitizing effect, may
escape chemosensitization and experience remission. Thus, new
chernosensitizers are sought that will be more potent and less
toxic than the initial compounds in an effort to improve the
duration and number of responses. Development and testing of
candidate compounds has been hampered by the lack of a
suitable in viva model. The objective of this study was to
evaluate our SCID mouse xenograft model for its ability to
assess the in viva efficacy and toxicities of chemomodubators
directed against P-glycoprotein, using verapamil as the proto-
typical chemosensitizer. The maximum tolerated dose of doxo-
rubicin in this model was established as 1 .5 mg/kg every 4 days
x 3 ( I ) and reflects the increased sensitivity of SCID mice to
agents which induce DNA damage (32). When doxorubicin was
administered i.p. directly to the site of the tumor, we were
unable to observe a therapeutic response in those animals bear-
ing the 8226/CIN tumor in the absence of verapamil. When
veraparnil was added to the doxorubicin regimen using a second
route of administration, either s.c. or i.m., we were then able to
observe an effect on the tumor. The combination of verapamil
and doxorubicin resulted in both a decrease in light chain
excretion and an increase in survival of those animals bearing
the 8226/C I N tumor.
Thus, although the i.p. route of drug delivery was not ideal
from a clinical perspective, we were nevertheless able to dem-
onstrate a chemomodulating effect for verapamil. To avoid the
potential confounding variables associated with administering
the drug to the same site as the tumor. we have initiated studies
using the ARH-D60 human myeborna cell line, designed to
evaluate the i.v route of drug delivery. This cell line, when
injected into SCID mice, results in osteolytic lesions and may
Research. on July 14, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 1569
thus represent a more orthotopic xenograft model of myeborna
(33).
In addition to evaluating drug efficacy, the SCID xenograft
model allows investigators to examine pharmacokinetic and
pharmacodynamic parameters, which are not easily studied in
the in vitro setting, and to identify the limiting organ-specific
toxicities resulting from the use of new chernomodulators. In-
deed, we have used this model to identify increased toxicities in
the SCID mouse as a consequence of the administration of
doxorubicin and cyclosporin A (34).
Using this model, we may not be able to differentiate the
effects of inhibition of P-glycopnotein from that of altered
pharmacokinetics solely by examining changes in the urinary
light chain excretion nor by prolonged survival times. What we
will be able to do, however, is to provide information relating to
the efficacy or toxicities of a given compound under the actual
conditions of its proposed use. Several studies have demon-
strated that the use of chemomodulators directed against the
P-glycoprotein may alter the pharmacokinetics of antineoplastic
agents. resulting in decreased clearance and/or increased area
under the curve ( 18 -2 1 ). Such reports are of importance due to
the potential of increased toxicities when chemomodulators are
utilized. To assess whether the observed effects of a compound
being examined in our model are due to alterations in pharma-
cokinetics, we will utilize measurements of tissue and plasma
levels of the drugs as we have done previously (34).
Future uses of our model may also include studies of new
agents and therapeutic strategies directed against non-P-glyco-
protein-mediated MDR in myeboma. The SCID model may also
serve as a more general model of multiple rnyeboma. providing
a means to evaluate the effectiveness of various therapeutic
strategies aimed at this disease, and to elucidate additional
mechanisms of resistance by which myeborna cells may escape
from the effects of chemosensitization. Escape from chemosen-
sitization has been the clinical experience and was suggested by
the attenuated therapeutic effect of verapamil observed in ani-
mabs receiving multiple treatment courses. The current model
would seem to be ideal for studying the specifics of escape from
chemosensitization. We are currently examining this avenue of
drug resistance using the SCID xenograft model.
These observations confirm that the SCID mouse is an
excellent recipient for the human multiple myelorna cells and
may be very suitable for evaluating the efficacy and toxicities of
new chemomodulators of the P-glycoprotein. This in vivo model
should be useful not only for studying drug-resistant human
multiple myeborna but also as a more general model of multiple
myeboma, providing a means to evaluate the effectiveness of
various therapeutic strategies aimed at this disease, and to elu-
cidate the mechanisms by which rnyeboma cells may escape
from chemosensitization.
ACKNOWLEDGMENTS
We thank Yvette Frutigen, Nancy Hart. and Lynn Richter for
technical assistance.
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1995;1:1563-1570. Clin Cancer Res W T Bellamy, A Odeleye, E Huizenga, et al. human myeloma xenografts.P-glycoprotein chemosensitization in multidrug-resistant Severe combined immunodeficiency (SCID) mouse modeling of
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