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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
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).
Research. on December 24, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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
Research. on December 24, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
698 Multidrug Resistance in a Murine Hepatoma Model
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
Research. on December 24, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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
Clinical Cancer Research 699
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.
Research. on December 24, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
=‘ 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
700 Multidrug Resistance in a Murine Hepatoma Model
�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-
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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
Clinical Cancer Research 701
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
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702 Multidrug Resistance in a Murine Hepatoma Model
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
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>
Clinical Cancer Research 703
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-
Research. on December 24, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
704 Multidrug Resistance in a Murine Hepatoma Model
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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