Beruflich Dokumente
Kultur Dokumente
doi:10.1016/S1525-0016(03)00236-3
To whom correspondence and reprint requests should be addressed at the Department of Microbiology, University of Pennsylvania School of Medicine,
319a Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6067. Fax: (215) 898-3849. E-mail: nfraser@mail.med.upenn.edu.
The HSV-1 1716 mutant virus and similar oncolytic herpesviruses deficient in the 34.5 neurovirulence gene are able to reduce the growth of tumors in mice. Here we demonstrate that HSV-1
1716 therapy moderately reduced the growth of tumors of the highly malignant, spontaneously
metastasizing 4T1 mouse mammary carcinoma model. This moderate effect on 4T1 tumor growth
was likely due to poor replication kinetics of HSV-1 1716 in 4T1 cells. Interestingly, HSV-1 therapy
of the primary tumor increased the survival time of mice. Coincident with this increase was a
reduction in metastases as determined by quantification of the number of metastatic cells in the
lungs. HSV-1 therapy of the primary tumor was also able to reduce the establishment of a second
challenge of 4T1 tumors. Moreover, infiltrates of both CD4 and CD8 T cells were detected in
HSV-1 1716-treated tumors. An important role for the T cell infiltrates was confirmed when HSV-1
therapy did not reduce the growth of 4T1 tumors in SCID mice. Collectively, these results
demonstrate that an HSV-dependent anti-tumor immune response is required for the reduction
in primary 4T1 tumor growth and for the reduction in the establishment of metastases in this
tumor model.
Key Words: HSV-1, gene therapy, 4T1 mammary tumor, metastasis, immune response
INTRODUCTION
Herpes simplex viruses with mutations in the 34.5 neurovirulence gene-encoding infected cell protein (ICP34.5)
do not replicate in terminally differentiated cells, such as
cells of the central nervous system, but can replicate in
dividing cells, such as cancer cells [13]. ICP34.5 functions to inactivate the dsRNA-induced PKR pathway, to
prevent host protein synthesis shutoff, by complexing
with protein phosphatase 1, which results in the dephosphorylation of eIF-2 [4]. The selective replication of
34.5-mutant viruses has been exploited to design replicating HSV-1 vectors for cancer therapy. HSV-1 therapy
can increase the survival of mice harboring established
brain tumors and reduce the growth (sometimes completely) of established subcutaneous tumors in mice after
intraneoplastic injection. Herpes vectors like G207,
R3616, and 1716 have been shown to be effective in therapy
of human tumors in immune-deficient mouse models, such
as colon, ovarian, glioma, breast, prostate, and melanoma,
as well as in therapy of immune-competent mouse models of glioma, colorectal, and melanoma [59 and re-
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FIG. 1. Replication of HSV-1 17 and HSV-1 1716 in 4T1 and Vero cells. 4T1 and Vero cells were infected at a m.o.i. of 0.1 for 1 h with (A) HSV-1 17 or (B)
HSV-1 1716 as described under Materials and Methods. The zero time point was taken after washing of the cell monolayers immediately following the 1-h
infection. Time points were taken in duplicate and were titered on Vero cells in triplicate. The mean pfu at each time point is presented in log scale standard
error.
RESULTS
HSV-1 1716 Replicates Poorly in 4T1 Mammary
Tumor Cells
Although Balb/c mice are susceptible to HSV-1 infection,
not all murine cell lines derived from susceptible mice can
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FIG. 2. HSV-1 1716 therapy of 4T1 mammary tumors. Balb/c mice were injected subcutaneously in the abdominal mammary gland with 1 104 viable 4T1 cells.
Mean tumor diameters were determined as described under Materials and Methods. Palpable tumors were injected with 20 l containing either 5.4 105 pfu
of HSV-1 1716 or DMEM (mock-infected) on (A) days 9 and 13 (double therapy; mock, n 24; 1716, n 23) or (B) days 9, 13, and 16 (triple therapy; mock,
n 22; 1716, n 19). Arrows indicate the days when therapy was administered. *Significant differences between mock- and 1716-treated tumors. Error bars
represent standard error.
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FIG. 3. HSV-1 1716 replication in 4T1 tumors. Balb/c mice with established
4T1 tumors were given triple therapy as described for Fig. 2. Two tumors per
time point were harvested on the days indicated post-injection of virus and
prepared as described under Materials and Methods. Tumor lysates were
assayed for HSV-1 1716 levels by plaque assay on Vero cells in triplicate. The
mean pfu at each time point is presented in log scale standard error. Arrows
indicate the days when therapy was administered.
FIG. 4. Survival of mice with HSV-1 1716-treated 4T1 mammary tumors. On day 27 post-injection of tumor cells, tumors were surgically removed from mice
described in Fig. 2 as described under Materials and Methods and the mice were monitored for survival. KaplanMeier survival curves are presented. (A) Double
therapy: mock, n 12; 1716, n 12; P 0.014. (B) Triple therapy: mock, n 12; 1716, n 10; P 0.005.
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TABLE 1: Quantitation of clonogenic lung metastases from mice with HSV-1 1716-treated 4T1 mammary tumors
No. of mice with clonogenic lung metastasesa
b
Therapy
No. of injections
01000
100120,000
20,00150,000
50,000
Mean (ln)e
Mock
11
HSV-1 1716
12
Mock
HSV-1 1716
p-valuef
0.004
0.033
a
Mice described in Figure 2 were sacrificed on day 27 post-injection of 4T1 cells and the lungs were removed, processed, and cultured as described in Materials and Methods. The number
of 6-thioguanine-resistant 4T1 colonies stained with methelene blue were counted for each mouse. The number of mice with lung 4T1 colony numbers within each range is presented.
b
Mock-treated mice received culture media and HSV-1 1716-treated mice received 5.4 105 pfu per injection.
c
Established 4T1 tumors received injections of either HSV-1 1716 or culture media (mock) either two (days 9 and 13 post-injection of 4T1 cells) or three times (days 9, 13, and 16
post-injection of 4T1 cells) as described in Figure 2.
d
Total number of mice per group.
e
Mean number of 6-thioguanine-resistant 4T1 colonies standard deviation. (ln) is the natural log of the mean number of metastases.
f
The natural log (ln) of the number of metastases in the lungs of each mouse was used to determine the p-value for either two or three injections as described in Materials and Methods.
in 29]. To examine further a role for an anti-tumor immune response in HSV-1 1716 therapy of 4T1 tumors, we
performed immunohistochemical analyses on sections of
HSV-1 1716- and mock-treated tumors. Mice with established 4T1 tumors received triple HSV-1 1716 and mock
therapy as described above and we harvested and froze
tumors for sectioning during and after the course of therapy. Tumor sections were then analyzed for histology and
for the presence of CD4 and CD8 T cells. As early as day
12 post-injection of tumor cells, inflammatory cells, such
as neutrophils, could be detected throughout the mass of
HSV-1 1716-treated tumors (Fig. 6). CD4 and CD8 T
FIG. 5. Growth of a second challenge of 4T1 tumor cells after HSV-1 1716
therapy of primary 4T1 tumors. 4T1 tumors were established, received HSV-1
1716 or mock therapy on days 9, 13, and 16, and were measured periodically
as described for Fig. 2. On day 18 post-injection of 4T1 cells, the primary
tumors were surgically removed, and another 1 104 4T1 cells were injected
into the contralateral side. Seven days following the second injection, growth
of tumors was again measured periodically until the endpoint of 38 days. n
10 each group until days 32, 35, and 38 when two mock-treated mice each
day and day 38 when one HSV-1 1716-treated mouse was euthanized because
of illness. Arrows indicate the days when therapy was administered. *Significant differences between mock- and 1716-treated tumors. Error bars represent
standard error.
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cells could also be detected throughout the HSV-1 1716treated tumors. Peak staining for CD4 and CD8 T cells
appeared between day 17 (shown in Fig. 6) and day 20
post-injection of tumor cells and began to subside by day
23 (data not shown). Very few inflammatory cells could
be detected in mock-treated tumors (data not shown).
HSV-1 1716 Therapy Has No Effect on 4T1 Tumor
Growth and Metastases in SCID Mice
We wanted to further confirm the importance of the
immune system in the reduction of metastases by examining HSV-1 therapy of primary 4T1 tumors in immuneincompetent SCID mice, which are deficient in both T
cells and B cells. In addition, we wanted to address the
possibility that, without an adaptive immune response,
HSV-1 1716 would grow uninhibited throughout the tumor and, therefore, more efficiently destroy the tumor. As
above, we injected SCID mice with 1 104 viable 4T1
cells subcutaneously in the abdominal mammary gland.
Mice received intratumoral injections of either HSV-1
1716 or mock therapy on days 9, 13, and 16 post-injection
of cells, and we monitored the mean tumor diameter over
time. Interestingly, there was no difference in the growth
between HSV-1 1716-treated and mock-treated primary
4T1 tumors (Fig. 7). Consequently, there was also no
significant difference in the number of metastatic cells in
the lung (data not shown). These results indicate that an
intact immune response is needed for HSV-1 therapy of
the primary tumor, as well as for the reduction of metastases.
DISCUSSION
The use of replication-restricted HSV-1 vectors has gained
acceptance as plausible for cancer therapy. This acceptance is due to its efficacy in numerous rodent models of
cancer and its lack of toxicity in early clinical trials for
glioma and melanoma [1113]. Here we wanted to determine whether oncolytic HSV-1 therapy of an aggressive,
spontaneously metastasizing mammary carcinoma model
would be effective. It was found that HSV-1 therapy had
only a moderate effect on growth of the primary tumor,
yet primary tumor therapy led to an increase in survival
times and a reduction of metastatic cells in the lungs.
Tumor therapy using 34.5-mutant viruses in only
some models leads to complete regression of the tumor,
demonstrating that the efficacy of HSV-1 therapy will
likely be tumor-type dependent. In the 4T1 mouse mammary carcinoma model, HSV-1 1716 only temporarily
FIG. 6. Presence of immune infiltrates in HSV-1 1716-treated 4T1 tumors. 4T1
tumors were established and received HSV-1 1716 or mock therapy on days 9,
13, and 16 as described for Fig. 2. During and after the course of therapy, mice
were sacrificed and tumors harvested, frozen, and sectioned as described
under Materials and Methods. Sections of HSV-1 1716-treated 4T1 tumors
were either stained with hematoxylin and eosin (H&E) or reacted with antiCD4 or anti-CD8 antibodies and counterstained with methyl green to visualize
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FIG. 7. HSV-1 1716 therapy of 4T1 mammary tumors in SCID mice. 4T1
mammary tumors were established in SCID mice and received either HSV-1
1716 or mock therapy on days 9, 13, and 16 post-injection of tumor cells as
described for Fig. 2. n 10 each. Arrows indicate the days when therapy was
administered. No significant difference was found at any time point. Error bars
represent standard error.
reduced the growth of primary tumors. This lack of efficacy is likely due to the reduced replication kinetics of
HSV-1 1716 in these tumor cells and the poor replication
in 4T1 tumors as determined by plaque assay. Interestingly, wild-type 17 was able to replicate efficiently in
4T1 cells, indicating that 4T1 cells are not able to complement the replication defect of HSV-1 1716.
In human breast cancer models, inefficient HSV-1 replication in vitro also correlated with the poor oncolysis of
tumors in vivo. In these studies, Toda et al. [30] found that
the MDA-MB-435 and MCF-7 breast cancer cell lines are
more susceptible to HSV-1 mutant G207 infection at a
m.o.i. of 0.1 than T47D cells and that MDA-MB-231 cells
were not very susceptible to G207 infection at the same
m.o.i. This differing susceptibility correlated with the
ability of G207 to reduce the growth of MDA-MB-435
tumors but not of MDA-MB-231 tumors. Also similar to
4T1 cells, mouse CT-26 colorectal tumor cells have a reduced ability to support G207 replication at a low m.o.i.
(0.1), but can be efficiently killed at a higher m.o.i. (1.0)
[14,31]. Why HSV-1 has a reduced ability to replicate in
these different cell types has not been determined, yet the
block is not at the point of viral entry, like in B16 melanoma cells [8], since 4T1 and CT-26 cells are capable of
being infected and lysed by HSV-1. Studies by Farassati et
al. [32] have shown that efficient replication of 34.5-
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MATERIALS
AND
METHODS
Cell culture, viruses, and virus titrations. The 4T1 tumor cells were a kind
gift from Dr. Fred Miller, Karmanos Cancer Center (Detroit, MI). 4T1 cells
and Vero cells were cultured in Dulbeccos modified Eagle medium
(DMEM) (GIBCO) supplemented with 5% fetal calf serum and were maintained in 0.05% penicillin/streptomycin, at 37C, in a humidified chamber, and in an atmosphere of 5% CO2. For the studies described here, we
used the HSV-1 1716 virus, which contains a 759-bp deletion within both
copies of ICP34.5, located in the terminal 1 kb of the long repeat region of
the wild-type HSV-1 strain 17 [2]. Wild-type HSV-1 17 and the HSV-1
1716 mutant were propagated in Vero cells, purified, concentrated by
high-speed centrifugation, and titered by black plaque assay as described
elsewhere using standard methods [46].
Replication of HSV-1 in cells. The efficiency of replication of HSV-1 17
and HSV-1 1716 in cells was as previously described [9]. Briefly, both 4T1
and Vero cells at 80 85% confluency were infected at a m.o.i. of 0.1 with
HSV-1 17 and HSV-1 1716. Cells were infected for 1 h at 37C. The
inoculum was removed and the cell monolayers were washed twice with
DMEM and overlaid with 2 ml of culture medium. The zero time point
was taken after washing of the cell monolayers immediately following
infection. At the appropriate time points, the cells were scraped into the
medium and frozen at 70C. Samples were thawed and refrozen twice
prior to titering on Vero cells as described above.
Establishment and therapy of 4T1 mouse mammary tumors. Female
Balb/c mice (Taconic, Germantown, NY) or SCID mice (Wistar Institute
Animal Facility, Philadelphia, PA) at 6 10 weeks of age were injected
subcutaneously in the abdominal mammary gland with 1 104 viable 4T1
cells. Tumors were palpable by day 7 8 post-injection of 4T1 cells and had
a mean tumor diameter of 3 to 5 mm. The mean tumor diameter was
calculated as the square root of the product of two perpendicular diameters
[20]. On days 9 and 13 (double therapy) or days 9, 13, and 16 (triple
therapy), each tumor was injected using a 27.5-gauge needle in two or
three places with a total volume of 20 l containing either 5.4 105 pfu
of HSV-1 1716 or DMEM (mock-infected). The mean tumor diameter was
measured every 2 4 days using a caliper. Tumor diameters of each mouse
were normalized by subtracting the mean tumor diameter measured on
day 8 post-injection of tumor cells from the mean tumor diameter measured on each subsequent day. On day 27 post-injection of 4T1 cells, mice
were divided into two groups. The first group was assayed for survival and
the second group assayed for lung metastases as described below. SCID
mice were sacrificed and assayed for lung metastases as described below on
day 26 post-injection of 4T1 cells.
For the second challenge of tumor cells, 4T1 tumors were established
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ACKNOWLEDGMENTS
We thank Kevin OBrien at East Carolina University (Greenville, NC) for guidance with statistical analyses, Srikanth Yellayi for help with histological analyses, Daniel Ruge for technical assistance, and the members of the Fraser
laboratory for helpful discussions. This work was supported by a grant from the
National Institutes of Health (NS37516) to N.W.F. D.L.T. was supported by a
NRSA (CA93034) from the National Cancer Institute.
23.
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