[Cancer Research 60, 3343-3347, July 1, 2000]
© 2000 American Association for Cancer Research
Inhibition of Metastatic Tumor Growth in Nude Mice by Portal Vein Infusions of Matrix-targeted Retroviral Vectors Bearing a Cytocidal Cyclin G1 Construct1
Erlinda M. Gordon2,
Peng Xuan Liu,
Zhen Hai Chen,
Ling Liu,
Michelle D. Whitley,
Conway Gee,
Susan Groshen,
David R. Hinton,
Robert W. Beart and
Frederick L. Hall
Departments of Colorectal Surgery [E. M. G., P. X. L., Z. H. C., L. L., M. D. W., R. W. B., F. L. H.], Pediatrics [E. M. G.,], and Pathology [D. R. H.], Gene Therapy Laboratories [E. M. G., P. X. L., Z. H. C., L. L., M. D. W., F. L. H.]; Norris Comprehensive Cancer Center Biostatistics Core [C. G., S. G.]; and University of Southern California School of Medicine [E. M. G., P. X. L., Z. H. C., L. L., M. D. W., C. G., S. G., R. W. B., F. L. H.], Los Angeles, California 90089
 |
ABSTRACT
|
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Tumor invasion and associated angiogenesis evoke a remodeling of
extracellular matrix components. Retroviral vectors bearing auxiliary
matrix-targeting motifs (i.e., collagen-binding
polypeptides) accumulate at sites of newly exposed collagen, thus
promoting tumor site-specific gene delivery. In this study, we assessed
the antitumor effects of serial portal vein infusions of
matrix-targeted vectors bearing a mutant cyclin G1 (dnG1) construct in
a nude mouse model of liver metastasis. The size of tumor foci was
dramatically reduced in dnG1 vector-treated mice compared with that in
control vector- or PBS-treated animals (P = 0.0002). These findings represent a definitive advance in
the development of targeted injectable vectors for metastatic cancer.
|
Introduction
|
|---|
Retroviral vectors are currently used in over 70% of human cancer
gene therapy trials: more than 3000 patients have received a gene
therapy vector in over 300 approved Phase I/II gene therapy protocols
(1)
. Thus far, it appears that retroviral vectors are
relatively safe and have minimal risk of insertional mutagenesis or
oncogenesis (2)
. For cancer gene therapy, a built-in
safety feature provided by retroviral vectors is the selective
transduction of actively dividing cells (3)
, thus sparing
normal nondividing cells. Retroviral transduction of target cells is
initiated by viral binding to cell surface receptors (4
, 5)
, followed by fusion of viral and cellular membranes
(6)
and core entry. Important advances in the production
(7
, 8)
and physiological stability (9)
of
retroviral vectors have been made in recent years. However, the
potential use of these vectors for in vivo gene delivery is
daunted by logistical problems, i.e., limited retroviral
bioavailability due to lack of tissue specificity (1
, 10)
.
Thus, the development of targeted injectable retroviral vectors for
human gene therapy has been hitherto elusive (1
, 10)
.
Current research on retroviral targeting has focused on molecular
engineering of the retroviral envelope (env) proteins
(11, 12, 13)
. In contrast to various strategies involving the
display of polypeptide ligands (11
, 14) or single-chain
antibodies (15, 16, 17)
on viral envelopes, we undertook the
seemingly counterintuitive strategy of targeting the
ECM3
itself, i.e., targeting ECM proteins exposed in
pathophysiological lesions to concentrate vectors in the ECM in the
vicinity of target cells (18
, 19)
. For transduction of
human cells, an optimized targeting construct was engineered to display
a matrix-targeting motif on the NH2-terminal
region of the amphotropic 4070A envelope (20)
. Resultant
single enveloped vectors (which would be readily amenable to
large-scale clinical vector production) exhibited both ECM targeting
and WT infectivity. We subsequently demonstrated successful
transduction of human cancer cells in vivo by portal vein
infusions of the matrix-targeted vector but not the nontargeted vector
in a nude mouse model of liver metastasis (20)
. In the
present study, we report dramatic reductions in the sizes of metastatic
tumor foci in nude mice by portal vein infusions (via an indwelling
catheter) of matrix-targeted retroviral vectors bearing a cytocidal
mutant cell cycle control gene.
|
Materials and Methods
|
|---|
Cells, Cell Culture Conditions, Plasmids and Vectors Bearing
Marker, and Cell Cycle Control Genes.
NIH3T3, 293T, and human pancreatic cancer MiaPaca2 cells were supplied
by American Type Culture Collection. NIH3T3 and 293T cells were
maintained in DMEM supplemented with 10% fetal bovine serum (D10;
Biowhittaker). The pcgp plasmids containing the viral gag
pol genes and a retroviral vector, pcnBg, expressing a
nucleus-targeted ß-galactosidase construct were kindly provided
by Drs. Paula Cannon and Ling Li, respectively (University of Southern
California Gene Therapy Laboratories, Los Angeles, CA). The plasmid
containing VSVG env protein was kindly provided by Dr.
Theodore Friedmann (University of California, San Diego, CA). A
truncated (amino acids 41–249) cyclin G1 construct (dnG1) was cloned
into the retroviral expression vector pREX.
Production of Matrix-targeted Retroviral Vectors Bearing Mutant
Cyclin G1 Constructs.
High-titer vectors were generated using a transient three- or
four-plasmid cotransfection system (7)
in which the
packaging components gag-pol and a chimeric murine leukemia
virus-based env bearing a vWF-derived
collagen-binding (matrix-targeting) motif expressed from the
cytomegalovirus promoter were placed on separate plasmids, each of
which contained the SV40 origin of replication. The vectors expressed
without WT env were named Bv1 or Hs2 (Bv, bovine
vWF-derived; Hs, human vWF-derived; LF or 1, linkers
derived from natural vWF sequences; LS or 2, standard linkers). To
further increase viral titer, a fusogenic VSVG env protein
(21)
was coexpressed with Bv1 or Hs2 env
proteins in a four-plasmid cotransfection protocol.
Viral titers in murine NIH3T3 cells were determined as described
previously, based on expression of the ß-galactosidase or neomycin
phosphotransferase resistance (neor) gene
(22)
. Viral titer was expressed as the number of
G418-resistant cfu/ml and ranged from
106-108 cfu/ml, depending
on the nature and amount of plasmid DNA used in the transfection
protocol.
In Vitro Efficacy Studies.
To assess the cytocidal/cytostatic effects of the dnG1 vector, the
transduced cells were evaluated for their proliferative potential by
counting the number of viable cells in each culture at serial intervals
(up to 4 days) after transduction without G418 selection. Western
analysis of endogenous and mutant cyclin G1 protein expression was
performed as described previously (22)
.
In vivo efficacy studies were conducted in compliance with a
protocol approved by the University of Southern California Institution
Animal Care and Use Committee. To evaluate the efficiency of targeted
gene delivery based on the antitumor effects of dnG1 vector treatment
in vivo, a model of liver metastasis simulating the route of
dissemination of human colon cancer was established in nude mice.
Briefly, 7 x 105 tumor cells were
infused slowly into the portal vein via an indwelling catheter that was
kept in place for 14 days. Intracatheter infusions of either a low- or
high-dose dnG1 vector (titers, 3 x 106 or 9 x 108 cfu/ml at 200 µl/day), a high-dose control
vector bearing a ß-galactosidase gene (titer, 8.5 x 108 cfu/ml), or PBS (pH 7.4) were begun 3 days
later and continued daily for a total of 9 days. The mice were
sacrificed by cervical dislocation 1 day after completion of treatment.
Histological and Morphometric Analysis.
The liver lobes were excised, fixed in 10% formalin, labeled (A,
right and caudate lobes; B, left lobe; C, median lobe), processed
separately, and embedded in paraffin blocks. The antitumor efficacy of
dnG1 vector treatment was assessed as follows: H&E-stained tissue
sections were examined by light microscopy; and the SAs of
representative liver sections and tumor foci from lobes A, B, and C
were measured by morphometric analysis using an Optimas image analysis
system. Evaluation of retroviral safety included assessment of the
integrity of the liver architecture and examination for the presence of
hepatocellular swelling or necrosis, inflammatory infiltrates,
cholestasis, and/or thrombosis. Tissue sections were also immunostained
to assess the presence of ß-galactosidase transgene
(20)
, cytokeratin, apoptosis, PAS, vimentin, and
CD68.
Statistical Analysis.
For the in vivo efficacy study, four treatment groups were
compared: (a) low-dose Bv1/dnG1 (titer, 3 x 106 cfu/ml); (b) high-dose
Hs2/VSVG/dnG1 (titer, 9.5 x 108
cfu/ml); (c) high-dose control vector bearing a
ß-galactosidase (nBg) gene (titer, 8.5 x 108 cfu/ml); and (d) PBS control.
Initially, 12 mice were studied; 4 were treated with a high-dose dnG1
vector, 4 were treated with a high-dose control vector, and 4 were
treated with PBS. Subsequently, four additional mice were treated with
a low-dose dnG1 vector. The response variables (total SA of the liver,
total SA of the tumor, tumor SA:liver SA ratio, and mean SA of tumor
foci) were log-transformed before formal analysis. A repeated measures
analysis with lobe as the repeated measures factor was used to
determine whether or not the treatment had an effect on each of the
response variables. Pairwise comparisons were also performed for the
outcome variables with overall F test Ps of <0.05
between groups.
|
Results and Discussion
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In 1994, our laboratory cloned a human G-type cyclin
(CYCG1), which was markedly overexpressed in a subset of
osteosarcoma cells (23)
. Since then, the retroviral
transfer of an antisense cyclin G1 construct has been repeatedly shown
to induce down-regulation of cyclin G1 expression and apoptosis of
target cells, inhibit proliferation of numerous cancer cells in
vitro (22
, 24
, 25)
, and, subsequently, arrest tumor
xenograft growth in vivo (24
, 25) . Recently, a
mutant human cyclin G1 (dnG1) was created with a deletion in the cyclin
box, a conserved region among cyclins that helps determine cyclin-Cdk
association that induces Cdk
activation.4
Initially shown to exhibit antiproliferative properties in vascular
smooth muscle cells, dnG1 may act to inhibit the function of WT cyclin
G1 or form inactive complexes with target Cdk molecules. Hence, the
performance of a series of cytocidal mutant cyclin G1 constructs were
tested in vitro to determine the optimal construct for
additional in vivo studies.
A human undifferentiated cancer cell line of pancreatic origin was
selected as the prototype of a metastatic gastrointestinal cancer.
Retroviral transduction efficiency in these cancer cells was excellent,
ranging from 26–85%, depending on the multiplicity of infection
(multiplicity of infection = 4 and 250,
respectively; Fig. 1A
). For selection of an optimal therapeutic gene, cell
proliferation studies were conducted in transduced cells using vectors
bearing various cyclin G1 constructs. Fig. 1B
shows the
cytocidal/cytostatic effects of mutant and antisense cyclin G1
retroviral vectors in transduced cancer cells. Under standard
conditions, the dnG1 vector consistently exhibited the greatest
antiproliferative effect, concomitant with the appearance of
immunoreactive cyclin G1 at the region of
Mr 20,000, representing the
dnG1 protein (Fig. 1C)
. The expression of the dnG1 protein
in cancer cells results in unscheduled cell death; hence, the
low-level expression of dnG1 protein in cell lysates from transduced
unselected cell cultures. Based on the increased cytocidal activity of
the dnG1 vector (Fig. 1B)
in transduced MiaPaca2 cancer
cells, the dnG1 vector was used in subsequent in vivo
efficacy studies.
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Fig. 1. A, transduction efficiency of
matrix-targeted retroviral vectors in MiaPaca2 cells.
ß-galactosidase-expressing cells are shown with blue-stained
nuclei. B, cytostatic effects of matrix-targeted
retroviral vectors bearing mutant cyclin G1 constructs in MiaPaca
cancer cells. The number of cells/well (plotted on the vertical axis)
is expressed as a function of time (days after transduction), which is
plotted on the horizontal axis. D10, control medium;
Null, vector bearing only the neor gene;
AS 587 and AS 693, vectors bearing
antisense cyclin G1 constructs; dnG1, dnG1 vector
bearing a deletion in the NH2 terminus of human cyclin G1.
C, Western analysis of human cyclin G1 protein
expression in dnG1 vector-transduced versus null
vector-transduced cancer cells without G418 selection. Immunoreactive
dnG1 (cyclin G1 DN41) is detected as a distinct band in the region of
Mr 20,000 (Lane 3), whereas
the endogenous cyclin G1 protein is seen as an intensely stained band
in the region of Mr  30,000 (Lanes
1–4).
|
|
Considering the overall mass of the liver, the physiological dilution
factors involved, and the practical limitations of vector volume
(10% of blood volume), the efficiency of matrix-targeted gene
delivery by portal vein infusions was remarkably high, ranging from
1–3% (20)
to 
50% in focal areas (Fig. 2)
. Under these conditions, repeated injections of a high-dose
matrix-targeted retroviral vector bearing a ß-galactosidase marker
gene were required to detect an appreciable gene transfer in the tumor
foci. As shown in Fig. 2
, enhanced transduction of metastatic tumor
cells (50%) was observed not only within established tumor nodules
as described previously (20)
but also at points of tumor
exit from the circulation (Fig. 2, A–C
).
Moreover, significant transduction (50%) of tumor-associated stromal
cells and endothelial cells was observed in some tumor foci undergoing
active angiogenesis (Fig. 2, D–F
). No evidence
of transduction of neighboring hepatocytes was apparent, indicating the
selective retroviral transduction of actively dividing tumor cells.
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Fig. 2. X-Gal immunostaining for detection of the
ß-galactosidase transgene in metastatic tumor nodules.
A, H&E- and X-Gal-stained tissue section of liver from a
ß-galactosidase vector-treated mouse model of liver metastasis
(x40). B, X-Gal-stained tissue section of
A counterstained with nuclear fast red stain (x40).
C, higher magnification of B (x200).
ß-Galactosidase-expressing tumor cells (cells with
blue-stained nuclei) near a disrupted hepatic venule
(v) are indicated by arrows.
D, H&E-stained tissue section of liver from a
ß-galactosidase vector-treated mouse model of liver metastasis (x40)
showing the tumor nodule undergoing active angiogenesis.
E, X-Gal-stained liver sections counterstained with
nuclear fast red stain (x40). F, higher magnification
of E (x200). ß-Galactosidase-expressing tumor stromal
and endothelial cells are indicated by arrows.
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Histological and immunohistochemical evaluation of metastatic tumor
foci from mice treated with either PBS or the low-dose dnG1 vector was
performed and evaluated by morphometric analysis with an Optimas
imaging system. Histological examination of liver sections from control
animals revealed substantial tumor foci with attendant areas of
angiogenesis and stroma formation (Fig. 3, A and C)
; the epithelial components stained
positive for cytokeratin, and the associated tumor stromal/endothelial
cells stained positive for vimentin and FLK receptor (data not
shown). This heterogenous population of tumor cells, stromal
cells, and endothelial cells constituted the tumor nodule. In contrast,
the mean size of tumor foci in the low-dose dnG1-treated animals was
significantly reduced compared with that in PBS controls (Fig. 3, B and D
, arrows;
P = 0.001), simultaneously revealing a focal
increase in the density of apoptotic nuclei (Fig. 3, F and H
, arrows) compared with that in the PBS-treated
control group (Fig. 3E)
. Furthermore, infiltration by
PAS+, CD68+, and hemosiderin-laden macrophages (Fig. 3D
, arrow) was observed in the residual tumor
foci of dnG1 vector-treated animals, suggesting active clearance of
degenerating tumor cells and tumor debris by the hepatic
reticuloendothelial system.
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Fig. 3. H&E-stained tissue sections of liver reveal the tumor
foci in the PBS-treated control group (A and
C; x40 and x100) and the dnG1 vector-treated animals
(B and D; x40 and x100). Apoptosis in
the tumor foci of the PBS-treated control group (E;
x100) and the dnG1 vector-treated animals (F and
H, x100 and x200; arrows) is depicted
as reddish-brown immunostaining in an ApopTagPlus
peroxidase in situ apoptosis assay. G,
negative staining control without the terminal
deoxynucleotidyltransferase enzyme; t, tumor foci;
H, hepatocytes in liver parenchyma.
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Morphometric analysis of tumor foci confirmed that the targeting
strategy for therapeutic gene delivery was effective in that portal
vein infusions (via indwelling catheter) of high-dose matrix-targeted
dnG1 vectors induced dramatic reductions in the sizes of tumor foci
when compared with the PBS- and control vector-treated animals based on
all response variables (P < 0.0002; Tables 1
2
). In pairwise comparisons for the three outcome variables, a
dose-dependent tumor response to dnG1 vector treatment was apparent,
and additional studies are currently underway to better determine tumor
responsiveness to various vector doses in terms of tumor shrinkage
versus complete disappearance of the tumor foci and to
predict the minimum effective vector dose that could achieve the
desired tumor response in Phase I/II gene therapy trials. Importantly,
no evidence of hepatocellular damage, necrosis, thrombosis, or
cholestasis was detected in tissue sections from dnG1 vector-treated
animals, indicating that the matrix-targeted dnG1 vector (cumulative
dose,106-109 cfu) may have
a wide margin of safety. Whereas our previous studies showed that the
level of retroviral transduction of tumor nodules in vivo
was only 1–3% (20)
, the efficiency of gene delivery was
dramatically enhanced by increasing the number of vector infusions
(Fig. 2)
. A heterogeneous population of cells within the tumor nodule
was transduced, with the greatest number of transduced tumor cells and
tumor stromal and endothelial cells at sites of tumor invasion and in
areas of active angiogenesis. An appealing concept is that tumor
stromal cells and associated endothelial cells were transduced by the
dnG1 vector simultaneously with the tumor cells, resulting in
disproportionate tumoricidal effects caused by a decrease in vascular
supply and/or growth factor stimuli. We have shown previously that
blockade of cyclin G1 function using an antisense cyclin G1
(22)
or a dnG1 vector (data not shown) induces apoptosis
in 35% of transduced cancer cell cultures. The simultaneous
increase in the incidence of apoptotic nuclei in the tumor foci of dnG1
vector-treated mice indicates that dnG1 inhibits tumor growth by
evoking apoptotic mechanisms.
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Table 1 Inhibition of liver metastatic tumors after portal vein infusions of
matrix-targeted retroviral vectors bearing a mutant cyclin G1 construct
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In conclusion, we conducted in vivo efficacy and safety
studies in a unique nude mouse model of liver metastasis and
established the proofs of principle that (a) therapeutic
gene delivery can be achieved by repeated portal vein infusions (via an
indwelling catheter) of matrix-targeted retroviral vectors bearing a
cytocidal mutant cyclin G1 construct, as evidenced by statistically
significant reductions in the sizes of tumor foci in dnG1
vector-treated mice compared with those of tumor foci in control
animals, and (b) matrix-targeted dnG1 vectors may be
systemically administered with a wide margin of safety, as indicated by
the absence of associated hepatocyte necrosis, thrombosis, or
cholestasis. Taken together, these findings represent a definitive
advance toward the development of targeted injectable gene therapy
vectors for metastatic cancer.
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ACKNOWLEDGMENTS
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We thank Drs. Fan Xu and Liqiong Liu for technical assistance in
cloning of the dnG1 vector and the retroviral vector targeting
constructs.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by a grant from the Whittier
Family Foundation (Pasadena, CA) and by National Cancer Institute/NIH
Grant P30CA14089. 
2 To whom requests for reprints should be
addressed, at Gene Therapy Laboratories, 1975 Zonal Avenue, KAM300, Los
Angeles, CA 90089-9025. Phone: (323) 865-0609; Fax: (323) 442-3618;
E-mail: emgordon{at}genome2.hsc.usc.edu
3 The abbreviations used are: ECM, extracellular
matrix; VSVG, vesicular stomatitis virus G; WT, wild-type; vWF, von
Willebrand factor; cfu, colony-forming unit(s); SA, surface area; Cdk,
cyclin-dependent kinase.
4 Unpublished data.
Received 12/13/99.
Accepted 5/17/00.
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ABSTRACT
Introduction
