Severe Combined Immunodeficiency (SCID) Mouse Modeling …The abbreviations used are: MDR, multidrug...

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

http://clincancerres.aacrjournals.org/

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



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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



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-



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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


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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.


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




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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