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* Department of Surgery and
Department of Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA; and
Department of Microbiology and Immunology and
§ Department of Neuroscience, University of Rochester Medical Center, Rochester, New York 14642, USA
1Correspondence: Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA. E-mail: fongy{at}mskcc.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: attenuated virus • gene therapy • hepatobiliary tumors • replication • competent
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Half of those affected with colorectal cancer
will die of advanced or recurrent disease, despite aggressive treatment
(2)
. The liver is the most frequent site of distant
metastatic spread from malignancies of the colon and rectum (3
, 4)
. Patients with untreated liver metastases have a median
survival of 5 to 10 months (5
, 6)
. Although many forms of
therapy have been evaluated, only surgical resection of the affected
liver offers the possibility of cure (7)
. Two-thirds of
those who undergo successful resection, however, experience recurrence,
presumably from microscopic residual disease (8)
. For
patients with unresectable liver metastases, palliative treatment, in
the form of chemotherapy, yields clinical response in only one-third of
cases (9)
. Thus, active investigation seeks novel
therapies that may improve outcome in both resectable and unresectable
cases of this common disease entity.
Several viral-based, antineoplastic strategies are being
evaluated for treatment of colorectal cancer and liver metastases. Most
use replication-defective viruses as vectors to transfer therapeutic
genes that encode protein products such as cytokines, prodrugs, and
tumor suppressors (10
11
12
13)
. Another promising strategy
involves the use of replication-competent viruses and exploits their
natural ability to infect and lyse tumor cells (14)
. These
oncolytic viruses are genetically engineered to be less virulent to
normal tissues and more specific toward tumor cells. G207 is a
second-generation, multi-mutated, replication-competent herpes simplex
virus type-1
(HSV-1),2
which has demonstrated impressive oncolytic activity in several
neurological malignancies, while sparing normal neural tissue
(15
, 16)
.
This engineered virus, based on the wild-type HSV-1 (F-strain),
contains deletions of both copies of the 
134.5
gene, which results in attenuated neurovirulence (17
, 18)
.
Interruption of the UL39 gene restricts
replication of the G207 virus to rapidly dividing cells
(19)
. UL39 codes for ICP6, the large
subunit of ribonucleotide reductase, which is a key enzyme required for
DNA synthesis and HSV replication. Nondividing cells are deficient in
this protein, whereas rapidly dividing cancer cells express it freely
(20
, 21)
. Viral replication can only take place in the
presence of this enzyme; thus, replication of G207 is limited to
dividing cells. In addition, G207 was created with the marker gene
lacZ, which produces a histochemically identifiable protein
product. Cells infected with G207 will turn blue when exposed to X-gal
solution. Finally, G207 has two built-in safety mechanisms: expression
of herpes thymidine kinase, which renders the virus sensitive to
ganciclovir therapy; and temperature sensitivity, which halts viral
activity in the febrile host (16)
.
G207 was originally designed for treatment of neurological
malignancies, because wild-type HSV is naturally neurotropic
(16)
. Therapeutic safety and efficacy in animal model
neurological tumors encouraged us to study the effects of G207 in solid
tumors outside the central nervous system. We report the evaluation of
G207 for colorectal cancer and liver metastases in vitro and
in animal models, and demonstrate this virus to be promising for the
treatment of this disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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134.5 gene and insertion of the
Escherichia coli lacZ gene into the
UL39 sequence of the R3616 mutant
(16)
. African green monkey kidney cells (Vero cells, ATCC,
Rockville, Md.) were maintained in Dulbecco's modified Eagle's medium
with 5% fetal calf serum under standard cell culture conditions. Virus
was propagated on Vero cells at an MOI (multiplicity of infection,
number of viral particles/cell) of 0.02 at 34°C, harvested after 2
days, and subjected to freeze-thaw lysis and sonication to release G207
from the cell fraction. Cell lysates were clarified by centrifugation
(300 x g for 10 min at 4°C) and viral supernatants
were stored at -80°C. Viral titers were determined by standard
plaque assay on Vero cells.
Cell culture
Five human colorectal cancer cell lines were used in this study.
Four (C18, C29, C85, and C86) were isolated and characterized at
Memorial Sloan-Kettering Cancer Center. The fifth, HCT 8, was obtained
from the ATCC. Cells were maintained in RPMI 1640 supplemented with
10% fetal calf serum, 1% L-glutamine, 100 µg/ml penicillin, and 100
µg/ml streptomycin at 37°C in 5% CO2
humidified atmosphere and subcultured twice a week.
In vitro assay of infection and lysis of human
colorectal cancer cells
Each of the five cell lines was plated onto flat-bottom 96-well
microtiter plates (Becton Dickinson, Franklin Lakes, N.J.) at 3 x
104 cells per well. 24 h later, medium was
removed and fresh medium with the appropriate concentration of virus
was applied in a final volume of 200 µl; MOIs of 0, 0.1, 1.0, and 2.0
were evaluated. All assays were performed in triplicate.
To evaluate infection efficiency among the five cell lines, X-gal
(5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; Fisher Scientific,
Fair Lawn, N.J.) staining was performed on identically prepared serial
plates at 24, 48, and 72 h postinfection, using a previously
described technique (22)
. The percent of
lacZ-positive cells was calculated as a measure of
infection.
At 24, 72, and 120 h, wells were exposed to 0.25% trypsin; cells were counted on a hemocytometer using trypan blue exclusion to assess the ability of G207 to lyse cells from the five colorectal cancer cell lines.
Determinations of cell doubling time and S-phase fraction
Doubling time
Cells (5 x 104) were plated onto
T-25 flasks (Costar Corporation, Cambridge, Mass.) in 10 cc of medium.
Trypsinization of the monolayer and counting by hemocytometer was
performed at 24 h intervals. Data were plotted and doubling time
was calculated.
Cell cycle analysis
Cells in log-phase growth were harvested by trypsinization,
fixed in 80% ethanol, and stored at -20°C until analysis.
Subsequently, cell suspensions were digested in DNase-free RNase
(Boehringer Mannheim, Indianapolis, Ind.) for 20 min at 37°C and then
stained with propidium iodide solution (50 µg/ml, Molecular Probes,
Eugene, Oreg.). Cell cycle analysis was performed on FACScan equipped
with FACStation running CellQuest software (Becton Dickinson, San Jose,
Calif.). A forward angle, light scatter threshold trigger was used to
eliminate debris. Cell clumps were removed using analysis gates on
either fluorescence pulse width or height vs. pulse area (integral).
Data were analyzed for 1 x 104 to 2 x
104 cells per sample.
Viral growth curves
To demonstrate replication of virus in susceptible cells, viral
growth curves were established in two representative cell lines (HCT 8
and C18) as described previously (23)
. Cells (5 x
105) were plated in 6-well plates and infected
with G207 at an MOI of 0.01 (5 x 103 PFU).
Successive well contents were collected by cell scraping at 0, 12, 24,
36, and 48 h postinfection and subjected to three cycles of
freeze-thaw lysis, sonication, and centrifugation at 2000 rpm for 10
min at 4 oC. Supernatants were plated on
confluent Vero cells in 6-well plates at varying dilutions and
recovered titers were determined through standard plaque assay.
Suppression of flank tumor growth in athymic rats
All animal work was performed under guidelines approved by the
Memorial Sloan-Kettering Institutional Animal Care and Use Committee.
Athymic rats were housed in pathogen-free quarters in the animal
facility. Animals were anesthetized with intraperitoneal (i.p.)
injections of pentobarbital (50 mg/kg). Two injections of 2 x
106 tumor cells in 50 µl of culture medium were
administered to each side of the animal for a total of four tumors per
animal, and tumor growth was measured with calipers three times per
week. Estimates of tumor volumes were calculated using the formula for
a prolate spheroid, 4/3(
)ab2, with `a' as the radius
of the long axis and `b' as the radius of the short axis in
millimeters. When tumor volume reached ~50 mm3,
1 x 107 plaque-forming units (PFU) of
active G207 or heat-inactivated virus (65°C for 20 min) in 50 µl of
culture medium was injected directly into xenografts. Three cell lines
(C85, C86, and HCT8) were selected for the flank tumor model. Sixteen
tumors were evaluated for each cell line; eight were treated with
active G207 and eight with inactivated virus control. Tumor
measurements and animal weights were followed regularly. All control
animals (n=6) had to be killed at 3 wk due to ulceration of
the skin overlying the tumor. Four of the six G207-treated animals were
maintained for 8 wk to evaluate duration of response.
Treatment of hepatic metastases with regional vascular infusion of
G207
An established model of hepatic micrometastases was chosen to
assess the role of regional hepatic administration of G207. Male
Buffalo rats (National Cancer Institute, Bethesda, Md.) that receive
intrasplenic injections of 1 x 106
syngeneic Morris hepatoma McA-RH7777 cells (ATCC No. CRL 1601) will
reliably develop between 60 and 100 countable liver metastases within 3
wk of tumor challenge. Hepatoma cells were maintained in culture and
periodically implanted in flanks to ensure tumorigenicity. Infection
and lysis of hepatoma cells with G207 was confirmed in
vitro.
To evaluate the ability of G207 to suppress experimental hepatic
metastases, 17 rats underwent laparotomy, splenic tumor challenge, and
splenectomy according to the model of Lafreniere and Rosenberg
(24)
. One week later, portal vein infusions were performed
with 1 x 108 PFU of G207 (n=7)
in 0.5 ml phosphate-buffered saline (PBS) or an equal volume of PBS
alone (n=10).
Portal vein infusion technique
Rats were anesthetized with i.p. pentobarbital (50 mg/kg).
Midline laparotomy incisions were made and the anterior pyloric branch
of the portal vein was exposed. Under an operating microscope, 6–0
silk suture (USSC, Norwalk Conn.) was used to obtain proximal and
distal control of the vessel. Through a small venotomy, a polyethylene
catheter (internal diameter = 0.28 mm, Becton Dickinson) with a
beveled tip was fed into the portal vein and temporarily secured with a
microaneurysm clip (Becton Dickinson). By this technique, homogeneous
hepatic distribution was confirmed with India ink in a few animals.
Experimental animals were infused with 1 x
108 PFU of G207 in 500 µl of PBS or an equal
volume of PBS alone. Eleven days after treatment, rats were killed and
liver nodules were counted.
Determination of viral persistence and dissemination
Histochemical analysis
Tumors and organs (brain, heart, lung, liver, kidney, spleen,
testes, small bowel, and skeletal muscle) were harvested from animals
in both in vivo experiments at 1, 7, and 14 days post G207
injection. Additional flank tumors were harvested and sectioned at day
21 postinfection. Specimens were frozen in Tissue-Tek embedding medium
(Sakura Finetek, Torrance, Calif.) and sectioned by cryotome. Sections
were fixed with 1-% glutaraldehyde and evaluated for ß-galactosidase
expression by staining with X-gal solution. Slides were subsequently
counterstained with Nuclear Fast Red (Sigma, St. Louis, Mo.). Tissues
harvested from uninfected animals were used as negative controls.
Quantitative PCR analysis
To further define persistence of infection and extent of
dissemination, we performed quantitative polymerase chain reaction
(PCR) analysis on genomic DNA extracted from liver, lung, brain,
kidney, testes, and serum of Buffalo rats at day 1 (n=2) and
day 7 (n=2) postportal vein infusion of 1 x
108 PFU of G207. Additional liver and serum
samples (n=2) were analyzed at day 14. Serial femoral artery
blood samples were collected from a single rat at 0, 5, 10, 30, and 60
min after portal vein infusion of 1 x 108
PFU of G207. Serum samples were extracted with phenol/chloroform and
precipitated using yeast tRNA. Genomic DNA extraction was performed on
all tissues. Standard curves were established by doping uninfected
liver and serum with known quantities of the G207 virus prior to DNA
extraction. Real-time quantitative PCR was performed using an ABI Prism
7700 Sequence Detector (PE Biosystems), as described previously
(25
, 26)
. Sense (5'-ATGTTTCCCGTCTGGTCCAC-3'), antisense
(5'-CCCTGTCGCCTTACGTGAA-3') primers, and a dual-labeled fluorescent
TaqMan probe (5'-6FAM-CCCCGTCTCCATGTCCAGGATGG-TAMRA-3') were designed
to span the 111-bp fragment of the HSV ICP0 (immediate early gene).
Additional sense (5'-CGCCTACCACATCCAAGGAA-3'), antisense
(5'-GCTGGAATTACCGCGGCT-3') primers, and TaqMan probe
(5'-JOE-TGCTGGCACCAGCTTGCCCTC-TAMRA-3') for the 187-bp 18S rRNA coding
sequence were used in the same reaction to normalize the amount of
total DNA. Samples were subjected to 40 cycles of PCR (stage 1: 50
oC, 2 min; stage 2: 95 oC,
10 min; stage 3: 95 oC, 15 s; 60
oC, 1 min; stage 4: 25 oC,
soak). The AmpliTaq Gold nuclease cleaves a fluorescent dye (FAM or
JOE) from the nonextendable probe, liberating it from the proximity of
an associated quencher (TAMRA). Probe binding is a requisite for primer
extension. The sequence detector is coupled to a charge-coupled device
camera, which records the fluorescent emission spectra from individual
wells at 500–650 nm with each cycle. Specificity for amplified product
is conferred by both probe and primer sets, obviating the need for
Southern blot analysis of the PCR product.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). G207 demonstrated impressive infection efficiency in three of the
five cell lines tested (C85, C86, and HCT 8) and more moderate
infectivity in C18 and C29, as determined by calculating the percent of
lacZ-positive cells (Fig. 2
A). Infection correlated with cell kill. The highly
susceptible cell lines (C85, C86, and HCT8) demonstrated 76% or better
cell kill at an MOI of 0.1, 72 h after infection, whereas the
moderate responders (C18 and C29) showed 31% or less cell kill under
the same conditions (Fig. 2B
). All colorectal cancer cell
lines tested demonstrated appreciable susceptibility to G207
cytotoxicity as compared with controls, even at the lowest MOI (0.1).
In all cell lines tested, infection efficiency correlated with percent
tumor cell lysis.
|
|
Determinations of cell doubling time and S-phase fraction
In vitro doubling time and S-phase fraction were
determined for each colorectal cancer cell line. Both parameters were
determined from cell populations in log-phase growth. The two cell
lines that displayed moderate susceptibility to G207 (C18 and C29) had
doubling times of 44 and 48 h and S-phase fractions of 9.2% and
8.6%. By comparison, the three cell lines that showed strong
susceptibility to the virus (C85, C86 and HCT 8) had shorter doubling
times (19–26 h) and greater S-phase fractions (20.1%–36.6%,
Table 1
). These results demonstrate a direct correlation between the
proliferative indices of the cancer cell lines and their susceptibility
to G207 therapy in vitro (R = +0.8, linear regression
analysis of infection percentage at MOI 1.0 vs. S-phase fraction).
|
Viral growth curves
In vitro viral growth curves were performed to
demonstrate the ability of G207 to replicate in human colorectal cancer
cells. For this experiment, two cell lines were evaluated: a strong
responder to in vitro cytotoxicity (HCT 8), with a short
doubling time (19 h) and high S-phase fraction (37%), and a less
responsive line (C18) with a longer doubling time (44 h) and lower
S-phase fraction (9%). The results are depicted in Fig. 3
. At the 0 time point, ~50% of viral particles are recovered from the
initial infection in both cell lines. By 12 h, there is a modest
drop of recovered viral titers by an additional 50%, coinciding with
viral disassembly within the infected cell. Subsequently, in the
responsive line (HCT 8), the titers begin to rise at 24 h and by
48 h are increased by 1.5 logs over the number of viral particles
initially added to the well. Conversely, in the less responsive line
(C18), there is no rise in recovered titers of G207, suggesting that
this slowly dividing cell line does not foster efficient viral
replication.
|
Suppression of flank tumor growth in athymic rats
Direct injection of G207 suppressed xenograft tumor growth
significantly in all three cell lines tested as compared with controls
(Fig. 4
A, B). HCT 8 xenografts were most susceptible, with responses
in 8/8 treated tumors (4/8 partial responses and 4/8 complete
responses). C86 xenografts demonstrated similar results (4/8 partial
responses and 2/8 complete responses). C85 tumors were not completely
suppressed by G207 injection, but growth rate was reduced significantly
as compared with control.
|
As a group, G207-treated animals maintained their health as well or better than control animals. G207-treated animal gained significantly more weight than controls during the 20 day experiment (100 ± 16 g vs. 44 ± 9 g, P<0.5). All animals that developed ulceration in the skin overlying their tumors had to be killed. This occurred in 6/6 control animals and in 2/6 of the G207-treated animals by day 20 posttumor inoculation. The four G207-treated animals that showed no evidence of ulceration were evaluated for duration of response for an additional 8 wk. Tumors in these animals did not continue to grow during this period, and the animals continued to groom and gain weight appropriately.
Treatment of hepatic metastases with regional infusion of G207
Initial studies confirmed that the Morris hepatoma
McA-RH7777 cell line is sensitive to G207 in vitro (data not
shown). In the efficacy study, animals that received portal infusions
of G207 7 days after splenic tumor challenge recovered well, grooming
and gaining weight equal to the PBS-treated controls. At death (day 11
postinfusion), G207-treated livers contained far fewer nodules than
PBS-treated controls (13 ± 10 nodules vs. 80 ± 30 nodules,
P<0.05, Fig. 5
).
|
Determination of viral persistence and dissemination
Histochemical analysis
Cryosections prepared from athymic rat organs (brain, heart, lung,
liver, kidney, spleen, testes, small bowel, and skeletal muscle)
harvested at 1, 7, and 14 days post G207 flank tumor injection
demonstrated absence of ß-galactosidase expression. Conversely,
strong expression was found in all tumors that received 1 x
107 PFU via direct, single intratumoral
injection. Tumors that demonstrated only partial response to therapy
were evaluated at 21 days and found to have persistence of
lacZ positivity in a depot-like distribution. There was less
overall necrosis witnessed in these specimens, suggesting less
infectivity of the virus in neighboring cells.
In the portal vein infusion model, all organs (with the exception of
liver) were negative for ß-galactosidase activity. Nontumor liver
parenchyma demonstrated scant positive expression overall (less than 1
blue cell/LPF). This was found in half the sections studied (18/36),
with the remainder showing no blue cells in the normal parenchyma. In
animals with established gross hepatic tumor, which were then exposed
to portal vein G207, X-gal staining was impressive. Twenty-four and
48 h after exposure to G207, ~two-thirds of existing liver
nodules (~50/80) had strong expression of ß-galactosidase in
grouped arrays (Fig. 6
). About half of the nodules had a single focus, whereas the remainder
had multiple positive regions with up to five per nodule. Peripherally
located tumor-associated plaques abutted but did not cross the border
into the adjacent normal liver parenchyma.
|
Quantitative PCR analysis
Quantitative PCR analysis of tissue and serum samples was
performed to further define the presence, persistence, and extent of
dissemination of G207 in animals that were infected via portal vein
infusion. By establishing a standard curve with 1, 5, 10,
103, and 105 PFU, we first determined our limit
of reliable detection to be 10 PFU per 50 mg of tissue. Upon analysis
of the various organs (liver, lung, kidney, brain, and testes), only
liver clearly demonstrated the presence of the HSV-ICP0 DNA sequence at
any time point evaluated (Fig. 7
A). Minimal signal was observed in lung, brain, and testes,
but based on our standard curve, this expression was below the limit of
reliable detection. Furthermore, livers exposed to portal venous G207
demonstrated persistence of the viral DNA sequence at 14 days. Analysis
of serum samples showed significant recovery of viral DNA in the
peripheral arterial blood at 5 min (> 600 copies/0.2 ml) postportal
venous infusion, which decreased over the first hour and was absent
from 24 h and beyond (Fig. 7
B).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Some of the earliest work with replicating recombinant HSV vectors was
directed at development of antiherpetic vaccines, with the goal of
establishing a lasting immune response in infected hosts (27
, 28)
. As better understanding of the factors responsible for HSV
virulence (18)
(17)
and replication
(19
, 29)
developed, it became clear that these mutants
could be used to treat malignancies. Neurological tumors were first
targeted due to the natural neurotropism of herpes viruses. Selective
infection and destruction of tumors within the central nervous system,
with sparing of normal neurons, has been demonstrated by several
investigators (15
, 16
, 30
, 31)
.
More recently, several groups have tested various recombinant,
replicating HSV mutants to treat nonneurological malignancies with
success (20
, 23
, 32)
. Of note is work by Kucharczuk et al.
(23), who demonstrated safety and efficacy of HSV-1716 (a 34.5
deleted mutant) in the treatment of experimental human malignant
mesothelioma cell lines in vitro and then in an animal model
of peritoneal disease. PCR analysis was used to demonstrate that virus
was detectable in tumor, but not in normal tissues.
G207 is a second-generation, multi-mutated HSV-1 that was developed by
taking the double 34.5-deleted F-strain mutant R3616 and
inactivating the DNA sequence coding for viral ribonucleotide
reductase. This was done through an insertional mutation with the
E. coli lacZ gene (15
, 16)
. Thus,
not only is this virus attenuated in virulence, but its replication is
restricted to cells that presumably produce higher levels of endogenous
ribonucleotide reductase (i.e., rapidly dividing malignant cells) and
can be detected by assaying for production of ß-galactosidase.
Another benefit derived from using multiple mutations to create G207 is
the reduced likelihood of reversion to wild-type disease-causing parent
virus.
The precise mechanism for viral cell kill is still being deciphered. In
susceptible cell lines, characteristics of normal HSV infection such as
ballooning and formation of multinucleated giant cells are observed.
This observation and evidence of viral replication, as demonstrated
through viral growth curves, support active, productive infection with
completion of the viral lytic life cycle as one mechanism contributing
to the death of infected cells. Moreover, several authors have
supported the notion that deletion of 34.5 may promote apoptosis in
susceptible cells (17
, 18
, 33)
. The carboxyl terminus
codes for a protein product that bears homology to a highly conserved
mammalian protein known as GADD 34. This protein is up-regulated when
cells are placed under stressful conditions (e.g., serum starvation or
viral infection) and acts to preclude protein synthesis. In the absence
of this protein, infected susceptible cells sense the insult and
respond by entering the pathway to programmed cell death instead of
reevaluating and attempting to repair and persevere.
As for restricted replication, it is known that at least 45 of the 84
characterized HSV genes can be inactivated or deleted and the virus
will still replicate in cultured cells, with some altered
characteristics (34)
. Inactivation of the gene encoding
ICP6 (or viral ribonucleotide reductase) is well described. This enzyme
plays a key role in DNA synthesis in prokaryotes and eukaryotes.
Evidence exists to suggest that an ICP6-inactivated mutant herpes virus
may be able to compensate for its deficit by using cellular
ribonucleotide reductase (19
, 29
, 35
, 36)
. Thus, rapidly
dividing cells, which presumably express higher levels of
ribonucleotide reductase, may serve as more suitable hosts for G207
replication. Our laboratory is currently working on defining this
relationship in our models.
Our data indicate that G207 efficiently infects experimental human colorectal cancer cells. All five cell lines tested responded to treatment in vitro. Three of the cell lines (C85, C86, and HCT 8) had remarkable responses by 3 and 5 days postinfection, even at a low MOI of 0.1. These strong responders exhibited more rapid cell division as demonstrated by measurements of cell doubling time and S-phase fraction. The relationship between cellular proliferative rate and responsiveness to G207 was observed in the viral growth curves. G207 demonstrated active replication within the rapidly dividing HCT 8 cell line, whereas the cell line with a slower turnover (C18) did not support successful viral growth. Furthermore, HCT 8 cells had the highest S-phase fraction in vitro (36.6%) and the greatest in vivo tumor growth inhibition of the three cell lines tested in the direct intratumoral injection model, with 4/8 tumors sustaining complete responses. The observed correlation between cell line proliferation and susceptibility to G207 in vitro may serve as a clinically relevant predictor of in vivo therapeutic response.
In addition to examining direct intratumoral injections, we
investigated the efficacy of G207 as a possible agent for regional
antineoplastic therapy. Introducing the virus by selective
intravascular infusion is appealing since it allows a diffuse
distribution of virus within the tumor. The same hypervascularity that
the tumor creates to allow its own continued growth could be exploited
to increase viral delivery. HSV-1 is a large DNA virus that approaches
200 nm in diameter, raising a concern that infectious particles would
not cross the endothelium and enter tumor cells. We examined this issue
using a well-established syngeneic model of hepatic metastases, which
involves seeding the liver with tumor via portal inoculation, a path
identical to that taken by colorectal liver metastases in human beings
(3)
. Our results suggest that regional infusion with G207
may be valuable for treatment of unresectable liver malignancies or may
be useful as an adjuvant to surgical resection to reduce postoperative
local recurrence.
Although G207 is an attenuated virus with restricted replication, therapeutic use of replicating viruses still raises concerns of dissemination. In our study, we used two methods of detection. The first, X-gal staining, requires that the virus infects the cell, transfers its genetic material, and the transferred marker gene functions to produce a protein (ß-gal). We evaluated various tissues (including tumor) for the presence of the marker gene product. In the flank tumor model, only tumor demonstrated positive results. In the portal vein infusion model, high expression was demonstrated in established tumor with infrequent expression in normal hepatic parenchyma. All other organs were negative for G207 infection by this detection method.
The second method, real-time quantitative PCR, detects presence of the
herpes immediate-early gene ICP0, which plays a role in cell cycle
regulation in infected cells (34)
. Unlike X-gal staining,
this sensitive technique does not rely on the expression of viral genes
to identify infected cells. We analyzed liver tissue as our target
organ, lung tissue because it receives most of the hepatic venous blood
flow, brain because of natural herpes tropism, kidney because of its
function of filtration, and testes because this is an area with
inherent rapid cell division. We found presence of the HSV-ICP0
sequence in liver of animals that received 1 x
108 PFU of portal G207 up to 14 days and
in the serum of an animal soon after infusion of virus, but not in any
other organs examined. Moreover, no mortalities were observed, and
G207-treated rats continued to groom and gain weight appropriately at
therapeutic doses.
Primates serve as the natural host for HSV-1, which may explain selective infection of human tumor xenografts in rodents. Our hepatic metastases model addresses this issue of species tropism. Morris hepatoma McA-RH7777 is a syngeneic tumor cell line developed and passaged in Buffalo rats. In this model, G207 demonstrates impressive tumor cell selectivity. These results suggest that G207 therapy is both tumor specific and effective at suppressing tumor growth in this model.
The future of oncolytic viral therapy is evolving. Recent advances in
the literature report methods to improve potency of attenuated herpes
viruses against malignant cells. Such advances include adding ionizing
radiation as an adjuvant to enhance the virus's ability to replicate
(37)
and using attenuated, replicating viruses as helper
viruses for packaging immunostimulatory cytokine genes
(38)
. Another area worthy of investigation is cotreatment
with antiviral agents. G207 expresses HSV-thymidine kinase and is
reported to be hypersensitive to ganciclovir (16)
. Future
experimentation should investigate the added efficacy of combining the
natural oncolytic properties of G207 with this suicide gene therapeutic
strategy, bearing in mind that treatment with antiviral agents could,
in theory, inactivate the virus prematurely.
G207 kills experimental human colorectal cancers efficiently and in vitro assays may predict this agent's efficacy in vivo. This antineoplastic agent can be administered safely in experimental animals by regional infusion and direct injection and can suppress nonneural tumor growth. These results encourage pursuing future clinical studies of G207 in the treatment of human metastatic colorectal cancers.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication November 19, 1998.
Revision received March 2, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 K. Kelly, P. Brader, A. Rein, J. P. Shah, R. J. Wong, Y. Fong, and Z. Gil Attenuated multimutated herpes simplex virus-1 effectively treats prostate carcinomas with neural invasion while preserving nerve function FASEB J, June 1, 2008; 22(6): 1839 - 1848. [Abstract] [Full Text] [PDF]
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 Z. Yu, S. Li, Y.-Y. Huang, S.-F. Lin, Y. Fong, and R. J. Wong Sensitivity of Squamous Cell Carcinoma Lymph Node Metastases to Herpes Oncolytic Therapy Clin. Cancer Res., March 15, 2008; 14(6): 1897 - 1904. [Abstract] [Full Text] [PDF]
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S.-F. Lin, S. P. Gao, D. L. Price, S. Li, T.-C. Chou, P. Singh, Y.-Y. Huang, Y. Fong, and R. J. Wong Synergy of a Herpes Oncolytic Virus and Paclitaxel for Anaplastic Thyroid Cancer Clin. Cancer Res., March 1, 2008; 14(5): 1519 - 1528. [Abstract] [Full Text] [PDF]
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Z. Gil, A. Rein, P. Brader, S. Li, J. P. Shah, Y. Fong, and R. J. Wong Nerve-Sparing Therapy with Oncolytic Herpes Virus for Cancers with Neural Invasion Clin. Cancer Res., November 1, 2007; 13(21): 6479 - 6485. [Abstract] [Full Text] [PDF]
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 K. Hiraoka, T. Kimura, C. R. Logg, C.-K. Tai, K. Haga, G. W. Lawson, and N. Kasahara Therapeutic Efficacy of Replication-Competent Retrovirus Vector-Mediated Suicide Gene Therapy in a Multifocal Colorectal Cancer Metastasis Model Cancer Res., June 1, 2007; 67(11): 5345 - 5353. [Abstract] [Full Text] [PDF]
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 Y.-Y. Huang, Z. Yu, S.-F. Lin, S. Li, Y. Fong, and R. J. Wong Nectin-1 Is a Marker of Thyroid Cancer Sensitivity to Herpes Oncolytic Therapy J. Clin. Endocrinol. Metab., May 1, 2007; 92(5): 1965 - 1970. [Abstract] [Full Text] [PDF]
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D. Kuruppu, A.-L. Brownell, A. Zhu, M. Yu, X. Wang, Y. Kulu, B. C. Fuchs, H. Kawasaki, and K. K. Tanabe Positron Emission Tomography of Herpes Simplex Virus 1 Oncolysis Cancer Res., April 1, 2007; 67(7): 3295 - 3300. [Abstract] [Full Text] [PDF]
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 P. S. Adusumilli, B. M. Stiles, M.-K. Chan, T.-C. Chou, R. J. Wong, V. W. Rusch, and Y. Fong Radiation Therapy Potentiates Effective Oncolytic Viral Therapy in the Treatment of Lung Cancer Ann. Thorac. Surg., August 1, 2005; 80(2): 409 - 417. [Abstract] [Full Text] [PDF]
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Z. Yu, M.-K. Chan, P. O-charoenrat, D. P. Eisenberg, J. P. Shah, B. Singh, Y. Fong, and R. J. Wong Enhanced Nectin-1 Expression and Herpes Oncolytic Sensitivity in Highly Migratory and Invasive Carcinoma Clin. Cancer Res., July 1, 2005; 11(13): 4889 - 4897. [Abstract] [Full Text] [PDF]
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R. J. Wong, M.-K. Chan, Z. Yu, R. A. Ghossein, I. Ngai, P. S. Adusumilli, B. M. Stiles, J. P. Shah, B. Singh, and Y. Fong Angiogenesis Inhibition by an Oncolytic Herpes Virus Expressing Interleukin 12 Clin. Cancer Res., July 1, 2004; 10(13): 4509 - 4516. [Abstract] [Full Text] [PDF]
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H. Kasuya, T. M. Pawlik, J. T. Mullen, J. M. Donahue, H. Nakamura, S. Chandrasekhar, H. Kawasaki, E. Choi, and K. K. Tanabe Selectivity of an Oncolytic Herpes Simplex Virus for Cells Expressing the DF3/MUC1 Antigen Cancer Res., April 1, 2004; 64(7): 2561 - 2567. [Abstract] [Full Text] [PDF]
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R. J. Wong, M.-K. Chan, Z. Yu, T. H. Kim, A. Bhargava, B. M. Stiles, B. C. Horsburgh, J. P. Shah, R. A. Ghossein, B. Singh, et al. Effective Intravenous Therapy of Murine Pulmonary Metastases with an Oncolytic Herpes Virus Expressing Interleukin 12 Clin. Cancer Res., January 1, 2004; 10(1): 251 - 259. [Abstract] [Full Text] [PDF]
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 A. D. Cohen and N. E. Kemeny An Update on Hepatic Arterial Infusion Chemotherapy for Colorectal Cancer Oncologist, December 1, 2003; 8(6): 553 - 566. [Abstract] |
