Establishment and Serial Quantification of Intrahepatic ...€¦ · surface antigen (HBsAg), the...

Vol. 2, 695-706, April 1996 Clinical Cancer Research 695

Establishment and Serial Quantification of Intrahepatic Xenografts

of Human Hepatocellular Carcinoma in Severe Combined

Immunodeficiency Mice, and Development of Therapeutic

Strategies to Overcome Multidrug Resistance1

Cynthia R. Levei11e-Webster� and Irwin A. Arias

Department of Physiology, Tufts University School of Medicine,

Boston, Massachusetts 021 1 1

ABSTRACT

A murine model in which to study multiple drug resis-tance in human hepatocellular carcinoma was developed.

PRF/PLC/5 hepatoma cells (Alex 0) and an induced multi-drug resistant clone (Alex 0.5) were injected intrasplenically

into severe combined immunodeficiency mice. In 70% of

injected mice, hepatoma cells engrafted in the liver and grew

as intrahepatic metastasis. Since Alex cells contain an inte-

grated hepatitis B virus genome and secrete hepatitis B

surface antigen (HBsAg), the serum HBsAg concentration in

tumor-bearing mice was used to quantitate tumor burden.

Tumor wet weight determined at necropsy was directlyproportional to the serum HBsAg concentration.

In Alex 0 cells, IC5�,s for doxorubicin, vinblastine, andcis-platinum were 0.35 riM, 0.029 ELM, and 3.70 ELM, respec-

tively. Alex 0.5 cells were 25-, 14-, and 1.4-fold more resis-

tant to doxorubicin, vinblastine, and cis-platinum, respec-

tively. Immunoblotting of Alex 0 cell membranes with an

anti-P-glycoprotein antibody (C219) revealed small amounts

of P-glycoprotein, whereas Alex 0.5 membranes overex-pressed the protein. Concurrent exposure to verapamil (10

pM) sensitized both cell lines to the cytotoxic action of

vinblastine and doxorubicin but had no effect on the cyto-

toxicity of cis-platinum. Mice bearing intrahepatic xe-

nografts derived from Alex 0 and 0.5 cells had no responseto treatment with i.v. vinblastine or doxorubicin, as was

anticipated from in vitro drug testing. Addition of verapamilto vinblastine treatment did not improve the success of in

vivo chemotherapy. Immunotherapy with a human anti-P-

glycoprotein antibody (MRK16) suppressed the in vivo

growth of tumors derived from both cell lines. The effect wasmost pronounced in mice bearing Alex 0.5 tumors. Immu-

noblotting of tumors which initially responded to MRK16therapy, but subsequently relapsed, revealed a marked de-

crease in P-glycoprotein expression when compared to re-

sults in tumors that were untreated or treated with vinblas-

tine or control antibody. In summary, we have developed an

intrahepatic tumor xenograft model of human hepatocellu-

lar carcinoma in mice that permits noninvasive serial quan-

tification of tumor burden by determination of serum HB-

sAg levels and demonstrated a positive response to

immunotherapy with anti-P-glycoprotein antibodies.

INTRODUCTION

HCC,3 a prevalent tumor worldwide, is often intrinsically

resistant to multiple chemotherapeutic agents (1). When re-

sponse to chemotherapy does occur, it is often quickly followed

by acquired drug resistance and clinical relapse (1).

Little is known concerning factors responsible for multiple

chemotherapy drug resistance (MDR) in HCC. One proposed

mechanism involves overexpression of P-glycoprotein, a plasma

membrane protein, which functions as an ATP-dependent drug

efflux pump (for review, see Refs. 2 and 3). In cancer cell lines,

P-glycoprotein is responsible for efflux of a variety of structur-

ally and functionally unrelated chemotherapeutic drugs includ-

ing anthracyclines, Vinca alkaloids, epipodophyllotoxins, and

taxenes.

Several lines of evidence suggest a role for P-glycoprotein

in drug resistance in HCC. P-glycoprotein is found on the apical

biliary canalicular membrane in normal liver (4) where it me-

diates AlT-dependent chemotherapeutic drug transport (5).

Overexpression of MDR1, the human P-glycoprotein gene, has

been demonstrated in drug-resistant human hepatoma cell lines

and clinical cases of human HCC (6-8). Increased P-glycopro-

tein expression accompanies transformation of hepatocytes in

rodent models of hepatocarcinogenesis (10, 1 1 ) and can be

induced in rodent cell lines by exposure to cytotoxic agents (12).

The extent to which P-glycoprotein contributes to intrinsic

or acquired MDR in HCC has important clinical implications,

since several therapeutic strategies to circumvent P-glycopro-

tein-mediated MDR are under investigation. Many drugs, in-

cluding verapamil, cyclosporine, quinidine, and progesterone,

inhibit P-glycoprotein-mediated drug efflux in cancer cell lines

(2, 3, 13). These so-called chemosensitizing agents correct drug

accumulation defects and restore drug sensitivity in MDR cell

lines. In mice harboring tumor xenografts from MDR cancer cell

lines, concurrent administration of chemotherapy agents with

chemosensitizing drugs restored drug sensitivity and was asso-

ciated with tumor regression (13). Clinical trials to evaluate the

Received 8/22/95; revised 12/I 1/95; accepted 12/21/95.

1 This work was supported by NIH Grant R37DK35652.

2 To whom requests for reprints should be addressed, at Tufts UniversitySchool of Veterinary Medicine, 200 Westboro Road, North Grafton,MA 01536. Phone: (508) 839-7950; Fax: (508) 839-7922.

3 The abbreviations used are: HCC, hepatocellular carcinoma: MDR.multidrug resistance; SCID, severe combined immunodeficiency; �50% inhibitory concentration; ADCC, antibody-dependent cell-medi-

ated cytolysis.

Research. on December 24, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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696 Multidrug Resistance in a Murine Hepatoma Model

efficacy of these chemosensitizers in reversing MDR in human

malignancies have shown modest success in hematological ma-

lignancies, but results with solid tumors, such as colon and

breast cancer, have been disappointing (14). A small clinical

trial evaluating the effect of verapamil combined with doxoru-

bicin therapy in unresectable HCC failed to show any beneficial

effect (15).

Monoclonal antibodies against external epitopes of P-gly-

coprotein also modulate the growth of cancer cells which over-

express P-glycoprotein (16-24). When two of these antibodies

(MRKI6 and HYB-24l) were administered in combination with

cytotoxic agents, drug sensitivity was restored in mice bearing

human tumor xenografts derived from MDR cancer cell lines

( I 8, 21 , 24). In addition, MRK16 given alone was capable of

modulating the growth of murine tumor xenografts ( I 7, 20).

A major limitation in the design of strategies to circumvent

P-glycoprotein-mediated drug resistance in solid tumors, such as

HCC, is lack of an appropriate animal model in which to study

the phenomenon. Present murine models involve growth of

human tumor xenografts as ascitic or subcutaneous tumors

which limit extrapolation of results to solid tumors. Intraperito-

neal ascitic tumor models, which are often treated by i.p. drug

injections, fail to consider the efficiency of vascular drug dcliv-

cry to the tumor site and the need for drug penetration through

multiple cell layers, which are critical determinants of success-

ful clinical therapy of solid tumors. In ascitic models, it is

impossible to verify whether tumor exists on initiation of treat-

ment or to quantitate tumor burden during therapy. Ascitic

tumor models rely on prolongation of survival as the end point

with which to judge efficacy. In mice bearing subcutaneous

tumor xenografts, tumor size can be directly measured using

calipers and response to therapeutic intervention monitored

quantitatively. However, when some cell lines are injected sc,

resultant tumors grow as well-encapsulated, poorly vascularized

masses so that early tissue necrosis and hemorrhage may com-

plicate evaluation of tumor volume. Both ascitic and subcuta-

neous models fail to consider the rapidly emerging concept that

the microenvironment in which tumor cells grow can pro-

foundly influence their response to cytotoxic therapy (25-27).

Such limitations may explain, in part, the discrepancy between

successes reported with in vito chemosensitization protocols in

murine models and failure of these protocols in clinical trials

with solid tumors.

A recently described murine human myeloma tumor model

in SCID mice overcomes one of the limitations of an ascitic

tumor model in that it permits serial quantification of tumor

engraftment (28). Mice injected i.p. with myeloma cells devel-

oped peritoneal tumor implants and metastasis. Because the

myeloma cells secrete immunoglobulin light chain, urinary light

chain concentrations progressively increased in injected mice,

presumably reflecting an increasing tumor burden, although

tumor mass was not measured.

We have developed a murine xenograft model of human

HCC in which the role of P-glycoprotein in intrinsic and ac-

quired drug resistance can be investigated. We sought a model

in which human HCC grows within the hepatic parenchyma and

contains a quantifiable marker permitting serial noninvasive

monitoring of tumor burden. Several investigators demonstrated

the feasibility of establishing intrahepatic metastasis of human

carcinoma cell lines after intrasplenic injection in immunodefi-

cient mice (29-31). Therefore, we injected the human HCC cell

line PRFIPLC/5 (32) intrasplenically into SCID mice. This cell

line contains an integrated hepatitis B viral genome and selec-

tively secretes viral surface antigen (HBsAg), but not infectious

virion or other viral protein (33). In tissue culture, secretion of

HBsAg into the medium is proportional to cell mass (34). Mice

bearing subcutaneous tumors from PRFIPLC/5 cells contain

circulating levels of HBsAg which correlated positively with

tumor size (34, 35). We exploited the secretion of HBsAg by

PRF/PLC/5 cells to monitor the growth of HCC xenografts

introduced into SCID mice by intrasplenic injection.

Intrasplenic injection of PRFIPLC/5 cells into SCID mice

resulted in reproducible engraftment of tumor cells within the

hepatic parenchyma. Secretion of HBsAg by tumor cells en-

abled noninvasive and serial quantification of tumor burden. We

explored several therapeutic strategies to treat tumors derived

from the parental HCC cell line and an MDR clone which was

induced to overexpress P-glycoprotein.

MATERIALS AND METHODS

Animals. Four- to 6-week-old male and female C.B-l7

SCID mice were obtained from Charles River Laboratories, Inc.

(Wilmington, MA). All mice were maintained under sterile

conditions. Serum samples (20 p.1) were obtained from mice at

4 weeks of age for determination of immunoglobulin levels to

detect significant breakthrough of the SCID phenotype (36).

Animals with IgG or 1gM levels greater than 5 jig/mI or 2

p.g/ml, respectively, were excluded from the study.

Drugs. Doxorubicin HC1 (Adriamycin) was obtained

from Adria Laboratories (Columbus, OH). Vinblastine sulfate

was obtained from Lyphomed (Deerfield, IL). R-verapamil was

a gift from BASF Bioresearch Corporation (Worcester, MA).

cis-Platinum, racemic verapamil, and the mouse myeloma pro-

tein UPC 10 were obtained from Sigma (St. Louis, MO).

MRK16, a murine monoclonal antibody to human P-glycopro-

tein, was generously supplied by Professor Takashi Tsuruo

(University of Tokyo, Tokyo, Japan). C2l9, a polyclonal anti-

body to hamster P-glycoprotein, was purchased from Centocor

(Malvern, PA). [3H]Daunorubicin was obtained from New En-

gland Nuclear (Boston, MA).

Cell Lines. The human HCC cell line PLCIPRF/5 was

obtained from Professor David Shafritz (Albert Einstein College

of Medicine, Bronx, NY). The parental cell line was designated

Alex 0. A MDR clone was developed by sequential selection in

increasing concentrations of doxorubicin. This clone, Alex 0.5,

has stable resistance to growth in 0.5 p.g/ml doxorubicin, over-

expresses P-glycoprotein, and exhibits a MDR phenotype (37,

38). Alex cells were cultured in Eagle’s MEM supplemented

with 2 mM L-glutamine, 0.01 msi nonessential amino acids, 10%

FCS, penicillin (100 units/ml), and streptomycin (0.1 mg/mI).

Alex 0.5 cells were grown in doxorubicin (0.5 p.g/ml) until 2

weeks before experiments were conducted.

CH’C5, Chinese hamster ovary cells which overexpress

P-glycoprotein, were obtained from Professor Victor Ling (On-

tario Cancer Institute, Toronto, Ontario, Canada) and main-

tamed in a-MEM supplemented with 10% FCS, 2 mM glu-

tamine, penicillin (100 units/mI), and streptomycin (0.1 mg/mI).



Clinical Cancer Research 697

All cell lines were maintained in a humidified environment

at 37#{176}Cin an atmosphere of 5% CO2/95% air.

In Vitro Cytotoxicity Studies. Alex 0 and 0.5 cells were

plated at 1 and 1.5 X l0� cells/35-mm dish, respectively. Cells

were allowed to attach for 24 h, after which fresh media con-

taming cytotoxic drug and/or reversing agent were added. After

incubation for 72 h, cells were harvested using a 0.05% trypsin/

0.002% EDTA solution prepared in 0.9% NaCl. Viable cell

counts were by trypan blue dye exclusion using a hemocytom-

eter. IC50 was calculated.

Drug Accumulation and Retention. Alex 0 and 0.5

cells were plated in 35-mm dishes at 4 X l0� cells/dish and

incubated overnight under standard tissue culture conditions.

Accumulation and efflux of [3H]daunorubicin (1 p.M) were

determined (39). In brief, 1 p.M daunorubicin containing a tracer

amount of [3H]daunorubicin was added to fresh media with or

without addition of 10 p.M verapamil. Dishes were incubated

under tissue culture conditions and at 0, 15, 30, and 60 mm after

drug addition, were quickly washed in ice-cold PBS, and cells

were removed by treatment with trypsin. The cell suspension

was diluted with 1 ml cold PBS, and cells were counted in a

hemocytometer. The tubes were spun at 10,000 rpm at 4#{176}Cin a

microfuge, and the pellet was lysed in PBS containing 0.05%

SDS. Radioactivity in the lysate was determined by liquid

scintillation counting. For efflux studies, cells were plated as

described above and then loaded with 1 p.M daunorubicin in

glucose-free medium containing 10 msi sodium azide and 10%

dialyzed FCS. After loading for I h under tissue culture condi-

tions, the cells were washed with PBS, and regular culture

media were added with or without 10 p.M verapamil. The

amount of daunorubicin remaining in the cells was determined

at 0, 5, 15, 30, and 60 mm as described above.

Tumor Induction. Subconfluent cultures of Alex 0 or

0.5 cells were harvested from monolayer culture after

trypsinization. Cells were collected by centrifugation, washed

twice with PBS, and resuspended to a final concentration of

12 X 106 cells/ml in PBS.

Six- to 8-week-old male and female SCID mice were

anesthetized with Avertin (0.024 mg/kg of a 1 .2% tribromoeth-

anol solution i.p.), placed in left lateral recumbancy, and the

right paracostal area aseptically prepared for surgery. The spleen

was exteriorized through a right paracostal incision and 0.15-

0.2 ml of the cell suspension corresponding to 2 X 106 cells was

injected subcapsularly into the spleen. After allowing 2 mm for

movement of cells out of the spleen, splenectomy was per-

formed. The splenic pedicle was ligated with 6-0 polyglactin

suture (Vicryl; Ethicon, Somerville, NJ), and the abdomen was

closed with stainless steel skin stables. Each experimental group

consisted of three to four mice given injections on the same day.

Tumor Detection. After tumor cell injection, mice were

bled (100-200 p.1) from the tail vein. Serum was analyzed for

HBsAg by RIA (Ausria II; Abbott Laboratories). A HBsAg

standard curve from 2.5 to 20 ng/ml was constructed using the

positive serum control included in the kit. When necessary,

mouse serum samples were diluted to fall within this standard

curve. HBsAg titers were expressed in ng/ml.

Representative mice with positive HBsAg titers varying

from 1 to 2500 ng/ml were euthanized and necropsied. Grossly

visible liver tumors were dissected free from normal liver tissue,

and tumor wet weight was obtained. Neoplastic tissue and

remaining normal liver were fixed in formalin, processed for

histopathology, stained with H&E, and evaluated using light

microscopy by a veterinary pathologist. Histological evaluation

verified tumor in the excised tissue and failed to detect addi-

tional evidence of tumor in the remaining liver.

Drug Treatment Protocols. Mice with positive HBsAg

titers for 2 or more consecutive weeks were entered into che-

motherapy or immunotherapy treatment protocols. For chemo-

therapy trials, mice were treated with single i.v injections of

doxorubicin (6-8 mg/kg) or 3 weekly iv. injections of vinblas-

tine (5 mg/kg) with or without concurrent i.p. administration of

racemic verapamil (25 mg/kg) or R-verapamil ( 100 mg/kg). The

vinblastine dose was selected on the basis of pharmacokinetic

data in mice, which showed that this dose results in serum

vinblastine concentrations (40) within the range associated with

in vitro cytotoxicity in Alex 0 cells. In addition, this schedule of

Vinca alkaloid administration has been successfully employed

in published studies on MDR reversal in solid tumor models (18,

2 1 , 24). The verapamil doses utilized were the maximum toler-

ated doses in our mice. In several mice, osmotic pumps (Model

lOO7D; ALZET, Palo Alto, CA) were implanted i.p. under

Avertin anesthesia as previously described (4 1 ). Pumps were

preloaded with 100 p.1 of a 125 mg/ml solution of R-verapamil

dissolved in 0.9% saline and operated at 0. 1 p.1/h to deliver a

continuous i.p infusion. Mice with i.p pumps received a single

i.v injection of vinblastine (5 mg/kg) 4-6 h after pump place-

ment. Immunotherapy with MRKI6 or UPC 10 was adminis-

tered at 500 �ig i.v. weekly for 3 weeks.

During therapy, serum HBsAg titers were monitored

weekly. Partial response was defined as stabilization of or

decrease in HBsAg titer. Complete response to therapy was

defined as disappearance of HBsAg titer at two or more assay

points. Treatment cures were documented by gross and histo-

logical absence of tumor at necropsy. All mice that underwent

treatment were necropsied at the time of relapse, the presence of

gross hepatic tumors was recorded, and tumor wet weight was

obtained. Representative samples of Alex 0 and 0.5 tumors from

animals in each treatment group were frozen at -80#{176}C for

subsequent analysis of P-glycoprotein expression.

Immunoblotting. Plasma membrane preparations were

made from subconfluent cultures of Alex 0, Alex 0.5, and

CHrC5 cells and from mouse tumor xenograft tissue obtained at

necropsy. Tumor samples were thawed and minced, whereas

tissue culture cells were scraped into hypotonic lysis buffer ( 10

mM Tris-HC1, 10 m� MgCl2, 1.5 msi NaC1, 1 nmi dithiotreitol,

pH 7.4) containing 2 p.M pepstatin, 2 p.g/ml leupeptin. and I mr�i

phenylmethylsulfonyl fluoride. The cells remained on ice for 10

mm and then were homogenized with I S strokes of a Wheaton

A dounce homogenizer. Cell homogenates were spun at 400 X

g for 10 mm at 4#{176}Cto remove nuclei. The resultant supernatant

was further spun at 22,000 X g for 30 mm at 4#{176}C.The resultant

membrane-rich pellet was resuspended in hypotonic lysis buffer

in 50% glycerol and stored at -70#{176}C. Protein concentrations

were determined according to the method of Lowry et al. (42)

using BSA as a standard.

Membrane protein preparations were subjected to electro-

phoresis on 8% SDS-PAGE gels. Gels were electrophoretically

transferred to nitrocellulose overnight at 30 V at 4#{176}C.Blots were




Table I In vitro chemosensiti vity patterns in Alex cells

Chemotherapy

IC50 (p.MY�

Alex 0 Alex 0 Alex 0.5 Alex 0.5

agent +vrp -vrp +vrp p388b

Doxorubicin

Vinblastine

035d.e

(0.079)0029d.e

(0.006)

0.077

(0.006)

0.0015(0.0002)

861d

(2.49)

041d

(0.010)

0.98(0.69)

(0.038)(0.006)

0.02

0.003

cis-Platinum 3.70(0.329)

3.23(0.421)

5.22(0.925)

5.52(0.876)

0.075

�aIC�50 represents the concentration of drug necessary to cause 50% inhibition of cell growth. Values are means +/-SE from three independent

experiments.S From Refs. 40 and 41.C vrp, verapamil; values in the presence (+vrp) and absence (-vrp) of 10 p.M verapamil.d Significanfly different ( p < 0.01) than IC50 in the presence of verapamil.e Siginficantly different ( p < 0.05) than IC50 in Alex 0.5 cells.

blocked for I h in 10% nonfat dry milk dissolved in PBS

containing 0.05% Tween 20 and incubated overnight at 4#{176}C

with 100 ng/ml C2l9 prepared in 5% nonfat milk dissolved in

PBS. Blots were washed in PBS and reacted with goat anti-

mouse horseradish peroxidase-conjugated IgG (1:3000) dis-

solved in 5% nonfat milk for 1 h at room temperature. After

washing in PBS, blots were developed with an enhanced chemi-

luminescence kit (ECL; Amersham Life Science, Amersham,

United Kingdom) using Kodak X-OMAT/AR film.

Statistical Analysis. All values are expressed as mean ±

SE. The significance of differences between groups and linear

regressions were analyzed using Student’s t test or Wilcoxon’s

signed rank list as appropriate. The x2 test was used to evaluate

in vivo drug efficacy.

RESULTS

In Vitro Testing. In vitro, the parental cell line Alex 0

exhibited low levels of resistance to doxorubicin and vinblastine

(Table 1) when compared to the well-characterized chemosen-

sitive murine leukemia cell line P388 (43, 44). Alex 0.5 cells

were 25 times more resistant to doxorubicin and 14 times more

resistant to vinblastine than were Alex 0 cells. This level of drug

resistance was maintained even when Alex 0.5 cells were grown

in the absence of doxorubicin for up to 45 days (data not

shown). In vitro resistance to doxorubicin and vinblastine in

Alex 0 and 0.5 cells was decreased by exposure to 10 p.m

verapamil (Table 1). In both cell lines, verapamil reversed

vinblastine resistance better than it reversed doxorubicin resis-

tance. Verapamil concentrations as low as 2.5 p.M showed

similar chemosensitizing ability in both cell lines. An equipotent

chemosensitizing effect was seen with the racemic mixture of

verapamil or the R-isomer (data not shown). When compared to

P388 cells, both hepatoma cell lines showed in vitro resistance

to cis-platinum, which was not altered by the addition of vera-

pamil (Table 1).

Fig. IA shows the effect of verapamil on the accumulation

of daunorubicin in Alex 0 and 0.5 cells. Alex 0 cells had a time

dependent increase in daunorubicin accumulation, and the ad-

dition of verapamil increased accumulation at all time points

(P < 0.01 at t = 60 and 90 mm). Alex 0.5 cells accumulated

appreciable amounts of daunorubicin only in the presence of

verapamil (P < 0.05 at all times points). The inability of Alex

0.5 cells to accumulate daunorubicin in the absence of verapamil

was associated with enhanced drug efflux as reflected in the

rapid loss of preloaded daunorubicin (Fig. 1B). Addition of

verapamil significantly increased the retention of daunorubicin

in Alex 0.5 cells at all time points (P < 0.01). Alex 0 cells

retained greater amounts of daunorubicin than did Alex 0.5 cells

(P < 0.001 at all time points) and manifested improved reten-

tion upon addition of verapamil. These results are consistent

with the expression of P-glycoprotein-mediated drug resistance

in both cell lines, albeit at different levels. Since verapamil was

not capable of completely reversing either increased daunoru-

bicin efflux or in vitro doxorubicin cytotoxicity, it is likely that

alternative methods of anthracycline drug resistance exist in

Alex 0.5 cells. This would not be an unexpected finding in a cell

line that was selected for drug resistance by growth in doxoru-

bicin.

Expression of P-Glycoprotein in Alex Cells. Immuno-

blotting of crude membrane fractions prepared from Alex 0,

Alex 0.5, and CH�C5 was performed with C2l9, an anti-P-

glycoprotein antibody (Fig. 2). Low-level P-glycoprotein cx-

pression was detected in Alex 0 cells consistent with previous

studies (6); Alex 0.5 cells had substantially greater amounts of

P-glycoprotein, but less than that seen in the P-glycoprotein

overexpressing cell line CHrC5. Expression of P-glycoprotein

was maintained in Alex 0.5 cells even when the cells were

grown in doxorubicin-free media for 45 days (data not shown).

Secretion of HBsAg. Both Alex cell lines secreted HB-

sAg into the culture medium in direct proportion to cell mass

(data not shown; Ref. 34).

Induction of Tumor Xenografts. More than 1 80 mice

were entered into the study over a 3-year period. Mice received

1 .5-2.0 x 106 cells by intrasplenic injection. When larger cell

inocula were attempted, death from portal thromboembolism

occurred. Even at these cell doses, occasional thrombotic com-

plications developed, especially with the Alex 0.5 cells. These

episodes were minimized by passing cells through a 30-gauge

needle to break up cell aggregates just before injection into the

spleen. This treatment had no effect on cell viability as assessed

by trypan blue exclusion.

Serum samples from mice given injections were analyzed



A U AlexO�-c�-�-- Alex 0 + verapamil

S Alex 0.5-0--- Alex 0.5 + verapamil

BU AlexO

-0-U-- AIexO+verapamil. Alex 0.5

-�0--- Alex 0.5 + verapamil

0 10020 40 60 80 0 20 40 60

Time (minutes) Time (minutes)

80

Fig. 1 Time-dependent accumulation (A) or retention (B) of daunorubicin in Alex 0 or Alex 0.5 cells in the presence and absence of 10 p.M

verapamil. Daunorubicin accumulation was measured as described in ‘ ‘Materials and Methods’ ‘ at various time points following the addition of 1 p.M

daunorubicin. Daunorubicin retention was measured at the times indicated after loading Alex cells in the presence of I p.M daunorubicin for 60 mm

and then transferring the cells to drug-free media. Values are the means of three independent experiments: bars, SE. For some time points. the SEbars are not visible due to the small variability.

kDa

200->�

116-

80-

Fig. 2 Immunoblots of crude membrane preparations (50 p.g) fromAlex 0, Alex 0.5, and CH’CS cells with the anti-P-glycoprotein antibody

C2l9. Lanes I and 2, Alex 0; Lane 3, CH’C5; Lwze 4, Alex 0.5. Left,

molecular weight markers. Arrowhead, position of P-glycoprotein.

weekly for HBsAg. In initial experiments to determine the

association between serum HBsAg and tumor weight, mice with

positive titers ranging from I to 2500 ng/ml were sacrificed. All


C0

�u)

0 ,_,

4w0

.-

�0.C

2 3 4 mice with positive titers had grossly visible liver tumors atnecropsy. Single or multiple hepatic tumors grew as distinct

white to tan nodules within the hepatic parenchyma. Extrahe-patic tumor sites were rare and included the root of the mesen-

tery (n = 10), the incision site (ii = 2), and the lung (n = 1).

Microscopic examination of tumor sections prepared from Alex

0 or 0.5 tumors revealed features of a moderately well-differ-

entiated HCC identical to those previously reported in subcuta-

neous xenografts derived from the parental cell line (29, 30).

Mice that remained HBsAg negative never showed gross or

histological evidence of tumor formation.

Tumors consistently developed after intrasplenic injection

of Alex 0 or 0.5 cells. Successful engraftment occurred in 73%

(89/I 12) and 71% (50/70) of mice given injections of Alex 0

and 0.5 cells, respectively. The latent period from injection to

appearance of HBsAg titers was longer in mice receiving injec-

tions of Alex 0.5 cells (39.9 days; range, 28-63 days) than in

mice that received Alex 0 cells (30. 1 days; range, 14-56 days).

The differences in latency may reflect both a lower inoculating

dose of Alex 0.5 due to these cells enhanced tendency to clump

at the high concentrations and the slightly slower in vitro growth

rate noted in this drug resistance cell line (data not shown).

Serial determination of serum HBsAg titers in mice receiv-

ing injections revealed progressive increases over a 4-8-week

interval (Fig. 3). Fig. 4 shows the statistically significant posi-

tive correlation between the serum HBsAg titer and tumor wet

weight when wet weight was less than 200 mg. Correlation of

the HBsAg titer with tumor weight decreased as tumor mass

increased above 200 mg. This disparity was reflected in finding

necrosis and hemorrhage in larger tumors, which had been

included in the measurement of total tumor weight. Mice ap-

peared to be clinically normal when HBsAg titers were less than

200 ng/ml. Once titers increased over 200 ng/mI, mice started

showing mild weight loss. As titers continued to rise, mice

exhibited more profound weight loss, lethargy, and an unthrifty

coat and were humanely sacrificed in compliance with the

UKCCR guidelines.



=‘ 400

E

�300

4

l� 200I

E

Cl)

.

A

EC)C

C)

4

I

B

EC)C

C)

4

mI

Days Post Injection50 0

200

150

100

50

100 200

Tumor Wet Weight (mg)

Fig. 4 Correlation between mouse serum HBsAg titer and the wetweight of intrahepatic tumor xenografts derived from Alex 0 or 0.5 cellsdetermined at the time of necropsy. The correlation coefficient was0.976, and the linear association was significant at P < 0.001


�o 2O� 80

Days Post Injection

Fig. 3 Progressive increase in serum HBsAg titer as monitored by lilAin SCID mice bearing tumors derived from Alex 0 (A) or Alex 0.5tumors (B). The four lines represent the results of serial determination ofHBsAg titers in four animals given injections on the same day of eitherAlex 0 or 0.5 cells.

As monitored by HBsAg titer, tumors derived from Alex 0

or 0.5 cells grew at comparable rates once engrafted, although

some variation among mice within experimental groups receiv-

ing injections on the same day was noted (Fig. 3)

Immunotherapy of HCC Xenografts. Table 2 summa-

rizes the results of immunotherapy with MRK16. Fourteen mice

bearing Alex 0-derived tumors (HBsAg titers, 1.5-128 ng/ml;

median, 23 ng/ml) and seven mice bearing Alex 0.5 tumors

(HBsAg titers, 12-144 ng/ml; median, 14 ng/ml) were treated

with MRK16. All mice bearing tumors derived from Alex 0.5

cells responded to therapy. Two were cured (HBsAg titers, 1.5

and 15.8 ng/ml) and three had complete responses (HBsAg

titers, 4.4, 14, and 12.3 ng/ml), but relapsed at 57, 79, and 119

days after treatment (Fig. SA). Two mice with the highest

HBsAg titers on initiation of therapy had sustained partial

responses (38 and 42 days), but relapsed when therapy ended

(Fig. SB). Response to MRK16 in mice bearing tumors from

Alex 0 cells was less consistent. Six mice (HBsAg titers, 5.8-

128 ng/ml; median, 19 ng/ml) had no response (Fig. SC). Six

mice (HBsAg titers, 9.8-104 ng/ml; median, 25 ng/ml) had

partial responses, lasting up to 68 days (Fig. SD). Two mice with

the lowest titers (HBsAg titers, 3.2 and 1.5 ng/ml) had a com-

plete response and were cured.

Ten mice (HBsAg titers, 4.1-118 ng/ml; median, 23 ng/ml)

were treated with an isotype-matched control antibody, UPC 10

(Table 3). Two mice with Alex 0-derived tumors had stabiliza-

tion of titers for the first week, but titers rose sharply thereafter.

In the remaining eight mice, there was no response to treatment

with UPC 10.

Statistical analysis comparing animals treated with UPC 10

or MRK16 that had no response to treatment with the number of

animals that responded (partial responders plus complete re-

sponders) revealed a significant increase in the number of re-

sponders in MRK16-treated animals (P < 0.05 for Alex 0

tumors and P < 0.001 for Alex 0.5 tumors).

Chemotherapy of HCC Xenografts. Initial chemother-

apy protocols used doxorubicin at a previously published toler-

able dose in mice (8 mg/kg i.v.). Consistent with the finding that

SCID mice have a decreased ability to repair DNA breaks (28,

36), doxorubicin, at this dose, caused significant morbidity and

mortality. When the dose was reduced to 6 mg/kg i.v., mice

tolerated a single dose of doxorubicin. Nine mice bearing tu-

mors (HbsAg titers, 1-209 ng/ml; median, 22 ng/ml) from either

cell line were treated with this dose of doxorubicin (Table 3).

Two had a transient partial response (7-10 days). The remaining

seven mice had no response.

Because of the SCID mouse’s unique susceptibility to

doxorubicin and in vitro studies with vinblastine, which sug-

gested that the Alex cell lines were more sensitive to this drug

than to doxorubicin, vinblastine was used in subsequent drug

treatment protocols. Therapy with vinblastine at S mg/kg i.v.

weekly for 3 weeks was ineffective in treating mice bearing

Alex 0 (HBsAg titers, 5.5-64 ng/ml; median, 55 ng/ml) or Alex

0.5 tumors (HBsAg titers, 4.1-68 ng/ml; median, 14.1 ng/ml).

Short-term partial responses represented by stabilization of HB-

sAg titers were seen in both groups (Table 3). Mice tolerated

vinblastine chemotherapy with only weight loss noted.

Chemosensitization with Verapamil. Reversal ofP-glycoprotein-mediated drug resistance was attempted by corn-

bining vinblastine chemotherapy with verapamil (Table 4). In

the first protocol, mice were given three weekly injections of

either racemic verapamil (25 mg/kg) or R-verapamil (100 mg/

kg) as a single i.p. bolus at the time of vinblastine administra-

tion. These doses of verapamil represented the maximum toler-



BA

EC)C

C)

4U)

I

C

EC)C

C)4U)

I

D

0 10 20 30 40

Days Post Treatment0 50 100 150

Days Post Treatment


T able 2 Results of imm unotherapy with MR K16 in SCID mi cc bearing human HCC xenografts

Tumor

Therapeutic response

Complete CompleteTreatment” origin None Partial with relapse with cure

MRK16 AlexO

Alex 0.5

6/14 6/14

(14-68)”

2/7

(38-42)3n

(57-1 19)

2/14

2/7

UPC 10 AlexO 3/5 2/5(7)

AlexO.5 4/4

a Antibodies were given at 500 p.g iv. weekly for 3 weeks.b Duration of response in days.

Fig. 5 Response in SCID mice bearing in-trahepatic xenografts derived from Alexcells to immunotherapy with anti-P-glyco-protein antibody MRK16. Sequential serumHBsAg titers in mice bearing tumors de-rived from Alex 0.5 (A and B) or 0 (C andD) cells treated with 500 p.g MRK16 iv.weekly for 3 weeks. The first injection ofMRK16 in each mouse was administered attime 0, and the subsequent two doses wereadministered during the succeeding 2weeks. A, five mice with Alex 0.5 tumorsthat had complete responses. B, two micewith Alex 0.5 tumors that had partial re-sponses. C, five mice with Alex 0 tumorsthat had no response. D, seven mice withAlex 0 tumors that had partial responses.Note the variation in the X- and Y-axes inthe four graphs.

ated doses in our mice. In these studies, increased efficacy of

vinblastine chemotherapy was not apparent (HBsAg titers,

9.1-68 ng/ml; median, 17 ng/ml). Previous studies suggested

that to maximize chemosensitization, verapamil should be ad-

ministered before and continuously throughout chemotherapy

(41 , 45) to ensure serum and tissue verapamil concentrations in

the range capable of in vitro chernosensitization. Since previous

studies demonstrated that i.p. administration of 75 mg/kg race-

mic verapamil resulted in transient appearance of drug levels

which produce in vitro chemosensitization, it was possible that

our single i.p. bolus injections of verapamil were adequate. We,

therefore, used a previously published protocol (41) to infuse

R-verapamil continuously using an i.p. osmotic pump at 150

mg/kg/day, which maintains verapamil concentrations of 10 p.M

in plasma and 100 p.M in liver (41). One course of therapy

consisting of verapamil infusion with vinblastine chemotherapy

04003002001000 20 40 60 8 0

did not significantly improve response to therapy (Table 4). One

mouse with an Alex 0 tumor (HBsAg titer, 52 ng/ml) had a

complete response for 2 weeks, but the other six mice (HBsAg

titers, 8.4-100 ng/ml; median, 24 ng/ml) with Alex 0 or 0.5

tumors had responses no better than those seen when vinblastine

was administered alone. Since this chernosensitization protocol

was associated with increased morbidity in the mice as noted by

increased weight loss, enhanced immobilization, and the occa-

sional development of diarrhea, weekly treatments were not

possible in all animals. In two mice, an additional cycle of

treatment was administered, but did not improve response.

P-Glycoprotein Expression in Hepatic Xenografts.Immunoblotting for P-glycoprotein expression in tumor samples

from untreated mice and mice subjected to various therapeutic

protocols was performed (Fig. 6). When gels were overloaded

with 300 jig membrane protein derived from untreated Alex 0




Table 3 Chemotherapy in SCID mice bearing human HCCxenografts

Treatment Tumor origin

erapeutic response

None Partial Complete

Doxorubicin” Alex 0

Alex0.5

5/6

3/4

1/6

(10)”

1/4

(7)

Vinblastine’ Alex 0

AlexO.5

3/9

1/5

6/9

(7-21)

4/5

(7-23)

‘, Doxorubicin was given at 6-8 mg/kg iv. once.

S Duration of response in days.( Vinblastine was given at 5 mg/kg iv. weekly for 3 weeks.

tumors (ii = 4), low levels of P-glycoprotein were detected.

Untreated Alex 0.5 tumors (n 4) expressed large amounts of

P-glycoprotein easily visualized with only 50 jig crude mem-

brane protein. Representative immunoblots of untreated Alex 0

and Alex 0.5 tumors are shown in Fig. 6, Lane 6 and Lane 5,

respectively. Tumors from Alex 0 or 0.5 cells treated with

vinblastine or UPC 10 control antibody, in which no response to

therapy was noted, had P-glycoprotein levels similar to those

seen in untreated tumors. However, in mice that had a complete

or partial response to MRK16 immunotherapy followed by

relapse, relapsed tumor tissue contained considerably less P-

glycoprotein than was observed in untreated tumors. In an Alex

0.5 tumor-bearing mouse that had a complete response to

MRK16, P-glycoprotein was detected only after six times as

much protein was loaded onto the gels. Nonspecific staining in

Lanes 7 and 8 (Fig. 6), verified by reactivity with non-P-

glycoprotein antibody, was associated with protein overloading

of the gel.

DISCUSSION

Intrasplenic injection of Alex cells into SCID mice repro-

ducibly produced intrahepatic metastasis. This tumor xenograft

model offers several advantages over other murine tumor mod-

els. First, the malignant hepatoma cells grow as solid tumors

within a microenvironment similar to that seen clinically. 5ev-

eral studies have suggested that neighboring cells and extracel-

lular matrix components profoundly effect tumor cell phenotype

(25-27). Second, tumor engraftment can be reliably monitored.

Alex cells, which contain an integrated hepatitis B virus ge-

nome, secrete large amounts of HBsAg when growing as hepatic

xenografts. HBsAg accumulates in the mouse’s circulation and

progressively increases over time. Tumor burden, as determined

by wet weight, was proportional to serum HBsAg titer. Regres-

sion of tumors was accompanied by a decrease in HBsAg titers.

HBsAg titers provide a noninvasive method for serially quanti-

tating tumor burden at initiation of therapy and during chemo-

therapy. HBsAg is an ideal circulating marker since it is rapidly

secreted by tumor cells, is nonpathogenic, has a short half-life,

and can be quantified by a highly sensitive reproducible RIA

(46). Finally, the fact that the parental cell line Alex 0 is also

intrinsically MDR represents an important component of this

physiologically relevant HCC tumor model. Since the cellular

Table 4 Use of verapamil to chemosensitize human HCCxenografts

Tumor

Th erapeutic response

CompleteTreatment origin None Partial with relapse

Vinblastine/verapamil bolus” Alex 0

Alex 0.5

4/6

2/4

2/6(7-26)”

2/4

(7-23)

Vinblastine/R-verapamil Alex 0 4/6 2/6bolus’ (7-28)

VinblastinefR-verapamil Alex 0 4/6 1/6 1/6infusion”

Alex 0.5 4/6

(7)2/6

(7-26)

(14)

(I Vinblastine was given at 5 mg/kg iv. and verapam il at 25 mg/kg

i.p.h Duration of response in days.C Vinblastine was given at 5 mg/kg iv. and R-verapamil at 100

mg/kg i.p.(I Vinblastine was given at 5 mg/kg iv. and R-verapamil was

infused via an i.p. pump at 150 mg/kg/day.

basis for acquired and intrinsic drug resistance may differ,

development of a HCC model in which modulation of both

forms of resistance can be evaluated is of considerable impor-

tance.

We exploited this murine model to examine MDR in HCC.

Clinically, HCC is often resistant to chemotherapy, but identi-

fication of important mediators of drug resistance has received

little attention. In vitro both Alex cell lines express P-glyco-

protein-mediated MDR, as demonstrated by the presence of

P-glycoprotein on immunoblots and the finding of in vitro drug

resistance to P-glycoprotein substrates that is associated with

verapamil-reversible defects in drug accumulation and efflux.

The intrinsically drug-resistant Alex 0 cells have a lower level

of P-glycoprotein-mediated MDR than did Alex 0.5 cells, which

were selected for acquired MDR. Chemoresistance was also

seen in vito because hepatic tumors derived from Alex 0 or 0.5

cells were resistant to treatment with doxorubicin and vinblas-

tine.

in vitro studies using verapamil as a chemosensitizer in

combination with vinblastine suggested that tumors from Alex 0

cells should be rendered sensitive to vinblastine therapy by

coadministration of verapamil. In cell culture, the IC5() for

vinblastine in the presence of verapamil in Alex 0 cells was

similar to that in cell lines which are sensitive to vinblastine

when grown as ascitic tumors in mice (43). However, when

grown as intrahepatic xenografts, Alex 0 tumor cells were

unaffected when verapamil was administered as a bolus injec-

tion i.p or a continuous i.p infusion with vinblastine.

The discrepancy between in vivo and in vitro responses

may result from several mechanisms. Since tissue and blood

levels of verapamil were not measured, verapamil concentra-

tions in tumor tissue may not have been high enough to inhibit

P-glycoprotein. In other studies in mice, the i.p verapamil infu-

sion protocol used in this study maintains hepatic verapamil

concentrations at 100 p.M, which exceeds concentrations needed

to inhibit P-glycoprotein in cell culture (41 ). Even if verapamil



>


4 Unpublished observations.

kDa

200-

116-

23456789I I I I I I � I

�, .� -

Fig. 6 Immunoblotting of crude membrane preparations (50 or 300 p.g protein) from intrahepatic xenografts derived from Alex 0 (Lanes 6-9) orAlex 0.5 (Lanes 2-5) cells with the anti-P-glycoprotein antibody C2l9. Lane 1, 50 p.g of control membrane preparation prepared from Alex 0.5 cellsgrown in culture. Lane 2, Alex 0.5 tumor (300 p.g) treated with MRK16, complete response with relapse. Lane 3, Alex 0.5 tumor (300 jig) treatedwith MRKI6, complete response with relapse. Lane 4, Alex 0.5 tumor (300 p.g) treated with MRKI6, partial response. Lane 5, Alex 0.5 tumor (50p.g), untreated. Lane 6, Alex 0 tumor (300 p.g), untreated. Lanes 7 and 8, Alex 0 tumors (300 p.g) treated with MRKI6, partial responses. Lane 9,

Alex 0 tumor (300 p.g), untreated. Lane 1 is overexposed to enable visualization of designated bands in all samples. Nonspecific staining in Lanes

7 and 8 is due to overloading of the gel. Arrowhead, position of P-glycoprotein.

concentrations this high were obtained in the HCC xenografts,

tumor concentrations of chemotherapy agents may have not

have been altered. In mice bearing subcutaneous xenografts of

human MDR rhabdomyosarcoma, treatment with vincristine

and verapamil did not increase tumor vincristine concentration

when compared to results in control mice treated with chemo-

therapy alone (41). In vivo drug resistance might also develop

through mechanisms which require interaction of tumor cells

with host tissue (25, 26). Mouse mammary tumor carcinoma

cells made drug resistant in vivo by repeated administration of

alkylating agents to tumor-bearing mice exhibited a drug-resis-

tant phenotype only when growing in vivo and not when tested

for drug sensitivity in monolayer cultures (25). When these cells

were grown as as three-dimensional spheroids in vitro, they

again expressed drug resistance (26). The basis for in vivo drug

resistance is unknown, but may involve release of substances

which alter tumor drug uptake or interactions of tumor cells with

host cells and components of the extracellular matrix. Other

pharmacokinetic parameters, such as lack of adequate drug

penetration through multiple cell layers in solid tumor xc-

nografts or changes in vasculature dynamics, pH, or oxygen

tension in deep layers of tumor tissue, may have been important

components of the in vivo drug resistance we observed.

Drug resistance mechanisms other than P-glycoprotein

may be operative in Alex cells. The presence of additional

mechanisms of drug resistance in Alex cells is suggested by the

fact that the cells were resistant not only to vinblastine and

doxorubicin but also to cis-platinum, which is not a substrate for

P-glycoprotein. In addition, verapamil was unable to totally

reverse the daunorubicin accumulation defect or the in vitro

cyotoxocity of doxorubicin or vinblastine in Alex 0.5 cells.

Several mechanisms of chemotherapy drug resistance have been

proposed (for review, see Ref. 47), including increased gluta-

thione or glutathione S-transferases, decreased topoisomerase II

activity, or increased expression of the MRP gene. In addition,

changes in intracellular pH, which promote sequestration of

chemotherapeutic agents in cytoplasmic compartments or inter-

fere with their binding to cellular targets, may also contribute to

drug resistance (47). Preliminary results in our laboratory show

that the Alex cells have increased glutathione S-transferase and

glutathione levels.4

Immunotherapy with MRK16 modulated growth of HCC

xenografts. MRK16 is a monoclonal antibody against a drug-

resistant human myelogenous leukemia cell line (15). Since

MRK16 modulates the uptake of anthracyclines and Vinca al-

kaloids in MDR cell lines (16, 18), it has been used as a

chemosensitizing agent in mice bearing MDR human xc-

nografts. In mice with intraperitoneal tumors from human co-

lonic cancer cells, i.p. administration of MRK16 with doxoru-

bicin or vinblastine enhanced survival (18). Administration of

MRK1 6 alone had no effect. In mice bearing subcutaneous

xenografts from a MDR human ovarian cancer cell line or a

human colorectal carcinoma cell line, i.v. administration of

MDR 16 alone prevented tumor appearance when given at the

same time as cell injection and inhibited growth of tumors when

treatment was started after tumors were palpable (17, 20). No

effects of MRK1 6 immunotherapy were seen when tumors from

parental cell lines, which did not express P-glycoprotein, were

treated. In our studies, i.v. administration of MRK16 decreased

tumor burden and, in some cases, was curative.

Several tumor-related factors could have influenced mono-

clonal antibody accumulation in tumor xenografts and, thereby,

affected efficacy of immunotherapy (48). Our results are con-

sistent with previous observations that the amount of antigen

expressed on the surface of the tumor and the size of the tumor

are important determinants of the success of monoclonal anti-




body immunotherapy (48, 49). The best response to MRK16

therapy occurred in Alex 0.5 tumors which overexpressed

P-glycoprotein. Presumably, the greater the amount of mono-

clonal antibody bound, the greater the tumor killing activity. We

also observed better responses to MRK16 when tumor burdens

were small (<20 mg). This finding may be associated with poor

penetration of MRKI6 into cells deep within large tumors due to

decreased vascular supply or permeability in deep tissue. Inter-

action of MRK16 with P-glycoprotein on surface tumor cells

may also retard antibody entrance into the tumor. Large tumors

may also have a heterogeneous distribution of P-glycoprotein

such that some tumor cells lose expression of the protein and no

longer bind MRK16. Our results support suggestions that im-

munotherapy may be most effective in solid tumors for control-

ling residual primary or metastatic tumor following surgery,

radiation, or chemotherapy (48, 49).

It should be noted that MRKI6 does not recognize native

mouse P-glycoprotein. If used to treat human tumors, the anti-

body would recognize and bind to naturally occurring P-glyco-

protein present on several normal tissues. This binding would

undoubtedly have implications in the design of dosage sched-

ules as well as have an impact on the development of potential

side effects.

Several mice initially responded to MRK16 immunother-

apy, but relapsed. By immunoblotting, these relapsed tumors

had markedly decreased P-glycoprotein expression when com-

pared to untreated tumors or tumors treated with vinblastine or

control antibody. If pretreatment tumor cell populations are

heterogeneous in P-glycoprotein expression, MRK16 may bind

to and mediate cell death only in P-glycoprotein-positive cells,

leaving behind a population of P-glycoprotein-negative tumor

cells which subsequently proliferate. Hence, tumor cells remain-

ing after MRK16 therapy may be more sensitive to the cytotoxic

actions of chemotherapy agents. In two Phase I clinical chemo-

sensitization trials, patients with P-glycoprotein-positive hema-

tological malignancies treated with chemotherapy in combina-

tion with the MDR-reversing agent cyclosporine had decreased

expression of P-glycoprotein in biopsy samples posttreatment

(50, 5 1). It would be of interest to determine in vivo chemosen-

sitivity of Alex tumor cells which remain after immunotherapy

to determine whether they respond to chemotherapy immedi-

ately following a course of MRKI6 or at the time of clinical

relapse.

The mechanism whereby MRK16 modulates growth of

murine MDR xenografts is unknown. MRKI6 alone has no

effect on the growth of MDR tumor cells in culture (16-18).

Complement-dependent cytotoxicity has been documented in a

MRK16-treated MDR human ovarian carcinoma cell line in

culture ( I 7), although we were unable to document this in vitro

with the Alex cell lines.4 Alternatively, ADCC may be respon-

sible for antitumor activity. ADCC effector cells may be lym-

phocytes, monocyte/macrophages, natural killer cells, or neu-

trophils. MRK16 induced ADCC in a human ovarian cell line

when mouse spleen cells were used as effector cells (17).

MRK I 6 also promoted human monocyte and lymphocyte-me-

diated tumor killing in ovarian carcinoma and myelogenous

leukemia cell lines (52). Since SCID mice lack functional T and

B lymphocytes, these cell populations are unlikely effector cells

in our study. Natural killer cells may be the effector cells in

hepatic immunotherapy (31, 53, 54). These cells are important

in the response to immunotherapy in colonic and gastric carci-

nomas growing as intrahepatic metastasis in immunodeficient

mice (31, 53). When murine lymphocyte populations were de-

pleted of natural killer cells, the cytotoxic action of human

mouse chimeric MRK16 antibody was significantly diminished

(54).

Preliminary studies suggest that magnetic resonance imag-

ing of tumor xenogratfts with a new asialoglycoprotein receptor-

targeted magnetic resonance imaging contrast agent. AG-USPIO

(Advanced Magnetics, Inc.; Ref. 55), may provide an additional

mechanism for monitoring tumor burden. Alexander cells lack

surface expression of the hepatic asialoglycoprotein receptor

(56), which is responsible for the removal of desialyated glyco-

proteins and is abundant on normal hepatocytes (57). AG-

USPIO is an ultrasmall superparamagnetic iron oxide particle

coated with arabinogalactan, a ligand for the asialoglycoprotein

receptor (55). During magnetic resonance imaging of mice

bearing tumor xenografts, normal mouse hepatocytes internal-

ized the agent with a resultant decrease in signal intensity while

tumor xenografts failed to take up the contrast agent and main-

tam their signal intensity (58). This imaging sharply demarcated

normal and neoplastic hepatic tissue, permitting visualization of

very small tumors ( 1 mm) and accurate computer-generated

reconstructions to determine tumor volume. Tumor volume de-

termined by this method appears to be proportional to the serum

HBsAg titer.4

We developed a physiologically relevant intrahepatic xc-

nograft model of human HCC in SCID mice in which tumor

burden can be noninvasively and serially quantitated by deter-

mination of a soluble, secreted tumor marker. The relative

contribution of drug resistance mechanisms, as defined by in-

vestigations in cell culture, to clinical drug resistance in vito can

be evaluated in this murine model. Since HCC xenografts grow

within the hepatic parenchyma, mechanisms of in s’ivo drug

resistance which may require tumor cell interactions with the

extracellular matrix can also be evaluated. Our initial investiga-

tions of P-glycoprotein-mediated drug resistance using this

model suggest that treatment modalities which target drug de-

livery to cells expressing P-glycoprotein, such as immunother-

apy with anti-P-glycoprotein antibodies, may be a more effec-

tive means of overcoming P-glycoprotein-mediated MDR in

solid tumors than are protocols employing the combined use of

chemosensitizing agents along with chemotherapy drugs.

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1996;2:695-706. Clin Cancer Res C R Leveille-Webster and I A Arias therapeutic strategies to overcome multidrug resistance.combined immunodeficiency mice, and development ofxenografts of human hepatocellular carcinoma in severe Establishment and serial quantification of intrahepatic

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