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Department of Pathology and Molecular Medicine, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Angiostatin has been shown to be a potent inhibitor of endothelial cell proliferation and can inhibit primary and metastatic tumor growth (2, 4, 5). Angiostatin inhibits both endothelial cell proliferation and migration through cytotoxic effects that result in cell apoptosis.
IL-12 is a heterodimeric Th1 cytokine that promotes the proliferation
of T cells, NK cells, and tumor-infiltrating lymphocytes
(6). Numerous studies have demonstrated IL-12’s ability
to facilitate tumor regression (7, 8, 9, 10). In addition to
developing strong antitumor CTL responses, IL-12 can induce a cascade
of other cytokines including IFN-
and the chemokine IFN-
inducible protein 10 (IP-10),3
which possesses significant antiangiogenic properties (6, 11).
In this study, we have used a human type 5 adenovirus (Ad) expressing the cDNA for murine angiostatin. The biological activity of this vector had been characterized using the artificial extracellular matrix Matrigel, examining changes in endothelial cell infiltration and cell morphology. Here we demonstrate the local overexpression of angiostatin and murine IL-12 in a mouse mammary carcinoma model (7, 9, 12). Our results indicate that 5- to 10-day expression by intratumoral injection of the vector expressing angiostatin by itself can delay, but cannot eradicate, tumor growth. The direct intratumor administration of IL-12 can delay tumor growth and induce regression in 13% of treated animals. Notably, when used in combination with the angiostatin vector, total regression is seen in 54% of the animals, with cured mice developing a strong CTL protection against tumor rechallenge.
These results are the first to demonstrate the usefulness of the combination of angiostatin and immunotherapy by a gene-therapeutic approach with intratumor administration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old female FVB/n mice were purchased from Charles River Breeding Laboratories (Troy, NY) and housed under pathogen-free conditions at the McMaster Central Animal Facilities. All in vivo procedures were approved by the McMaster University animal ethics research board in accordance with the Canadian Council of Animal Care.
Cell lines used include the following: 293, adenoviral E1-transformed human embryonic kidney cells maintained in F-11 medium supplemented with 10% FBS (complete F-11) (13); 293N3S, 293 suspension cells; and primary polyomavirus middle T (PyMidT) Ag-transformed murine mammary epithelial cells maintained in complete F-11 (14, 15). Splenocyte culture was conducted in RPMI 1640 supplemented with 10% FBS, 20 mM HEPES, and 50 µM 2-ME. All cell culture media contained 100 µg/ml penicillin and 100 U/ml streptomycin. Cells were cultured at 37°C with 5% CO2.
All cell culture media and reagents including 
MEM, MEM F-11, RPMI
1640, FBS, and penicillin/streptomycin were products of Life
Technologies (Burlington, Ontario, Canada).
Statistical significance of differences was tested using Student’s t test.
Adenoviral vectors
Ads were grown on 293 cells and purified by cesium chloride centrifugation (16). The construction and characterization of the Ad vector expressing murine angiostatin has been described previously.4 Briefly, the cDNA for murine angiostatin was constructed by ligating the endogenous signal sequence and the sequence encoding the first 32 amino acids of murine plasminogen to amino acids 98–458, encompassing the four kringle regions of plasminogen (17). This cDNA was cloned into the shuttle plasmid pACCMV and cotransfected with the rescue plasmid pJM-17 in 293 cells (16). Expression of the angiostatin in the viral vector (Ad-angiostatin) is driven by the human CMV immediate early promotor with a terminal SV40 polyadenylation sequence. Construction and characterization of the Ad vector expressing murine IL-12 (Ad-IL-12) has been described earlier (9). The control vector Ad-dl70, without an expressed gene product, was previously described (11).
PyMidT tumor studies
Transgenic mice expressing the PyMidT Ag under the transcriptional control of the mammary tumor virus long terminal repeat spontaneously develop adenocarcinomas of the mammary epithelium in 8–10 wk (12). Tumors were removed and processed to a single-cell suspension with mechanical disruption conducted in 100 ml PBS in the presence of 25 mg collagenase (Life Technologies). The cells were then placed in complete F-11 medium and cultured for 48 h.
The PyMidT tumor cells were harvested, and 5 x
105 cells in 200 µl PBS were injected s.c. into
the right flank of a syngeneic female FVB/n host. Approximately 18–21
days later, a palpable tumor (
150 mm3) had
developed in all mice that were injected. The tumors were injected with
control virus Ad-dl70 (1 x 109 PFU),
Ad-angiostatin (5 x 108 PFU), Ad-IL-12
(5 x 108 PFU), and a combination of
Ad-angiostatin plus Ad-IL-12 (5 x 108 PFU
of each vector). Total viral load was made up to 1 x
109 PFU with the addition Ad-dl70 in a final
volume of 50 µl to compensate for the effect of Ad vectors and
particles.
Tumors were measured (in millimeters) using Vernier calipers at the time of virus injection and at weekly intervals. Tumor volumes (millimeters3) were calculated from the longest diameter and average width, assuming a prolate spheroid (18). Those mice that underwent a total tumor regression on the right flank were rechallenged with a similar tumor dose in the left flank 8 wk later. Failure of the second tumor challenge to grow was deemed to be a total regression. Mice were sacrificed when any single or combined tumor linear measurement exceeded 20 mm.
CTL assay
A single-cell suspension was made from the spleens of mice that had undergone a total tumor regression and were resistant to rechallenge following the coinjection of Ad-angiostatin and Ad-IL-12. Splenocytes were separated from RBC using a Ficoll gradient (Amersham Pharmacia Biotech, Uppsala, Sweden) and cocultured with irradiated (5000 rad) 516 MT3 cells expressing the PyMidT Ag at an E:T ratio of 50:1. Five days later, the activated CTLs were harvested and tested for activity against 51Cr-labeled (500 µCi) 516 MT3 or control PTO516 cells. The assay was conducted in a volume of 50 µl using a 250-µl V-bottom 96-well plate at E:T ratios of 90:1, 30:1, and 10:1 for 3 h. Maximum release and spontaneous release was determined by adding 1M HCl or medium alone to the target cells. The percentage of specific lysis was calculated as follows: 100 x (experimental cpm - spontaneous release cpm)/(maximal cpm - spontaneous release cpm).
Anti-CD31 immunostaining of tumor vasculature
Palpable PyMidT tumors in the right flanks of FVB/n mice were treated with Ad vectors in a manner identical with that described above. One week following vector administration, the tumors were removed, embedded in OCT compound, and frozen rapidly in isopentane, which was precooled in liquid nitrogen to -180°C and stored at -70°C.
The frozen tumors were cryosectioned to 5 µm at -20°C and were allowed to air-dry overnight. The slides were fixed in cold acetone for 10 min, air-dried for 30 min, and were treated with 1% H2O2 for 10 min at room temperature to remove endogenous peroxidases. Slides were rinsed three times for 5 min with PBS, and nonspecific binding was blocked using Powerblock (Biogenex Laboratories, San Ramon, CA) for 6 min, followed by another PBS wash cycle. The sections were then incubated with anti-CD31 Ab (BD PharMingen, San Diego, CA) at a 1:800 dilution in Ab-diluting fluid (Dako, Carpinteria, CA) for 1 h. Following three washes in PBS, the secondary biotinylated anti-rat IgG (Vector Laboratories, Burlingame, CA) was added at a 1:400 dilution for 1 h. The slides were washed in PBS and incubated in a 1:60 dilution of avidin-biotinylated enzyme complex (ABC; Vector Laboratories) for 1 h followed again by three washes with PBS. The substrate chromogen 3-amino-9-ethylcarbazole (Vector Laboratories) was added to the sections for 30 min. The sections were counterstained in 50% Mayer’s hematoxylin and coverslipped in glycerin gelatin. Corresponding frozen tumor sections were also stained with hematoxylin and eosin (H&E) using standard techniques.
Anti-CD31 vessel quantification
Vessel quantification of CD31-stained tumor sections was conducted using a Leica (Deerfield, IL) Laborlux microscope equipped with a Sony (Tokyo, Japan) CCD digital camera. Five medium-power fields (x200) were examined per section in a blinded manner. Vessels were counted using Northern Exposure V2.9 imaging software (Empix Imaging, Mississauga, Ontario, Canada).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our own previous studies have shown that the intratumoral
injection of an Ad expressing murine IL-12 at 5 x
108 PFU can cause total regression of the PyMidT
tumor in 10–30% of tumor-bearing mice, and these "cured" mice
remain tumor free (7). The results in the current study
are consistent with our earlier work, as administration of Ad-IL-12
induced a 13% cure rate in tumor-bearing mice that were subsequently
resistant to rechallenge with PyMidT tumor (Table I
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), and all mice in this group grew
tumors and were finally sacrificed. We established criteria for partial
regression as stasis or decrease in tumor volume 1 wk after the
administration of a vector, as compared with the original tumor volume
before treatment.
Administration of the control virus Ad-dl70 did not delay the growth of
the PyMidT tumor. None of the tumors injected with control virus had
any response and none of the mice were cured, a result that we had
reported previously (7). Tumor volumes for this group
expanded at a near-linear rate as determined by weekly measurements,
and mice in this control group were sacrificed 20 days after injection
due to excessive tumor volumes (Fig. 1A; data from a representative
experiment). In contrast, the kinetics of PyMidT tumor growth in
Ad-IL-12-treated and Ad-angiostatin-treated animals was delayed as
compared with the control-treated animals. This delay extended the
survival of the mice in the treatment groups by 14 days on average
(Fig. 1B). However, none of the Ad-angiostatin-treated
animals were protected, with all animals succumbing to tumor
proliferation. The mechanism for this growth delay is likely a
reflection of angiostatin’s inhibitory effects on proliferation of
tumor vasculature via apoptosis of endothelial cells. Moreover, the
inhibition of tumor growth over this period may be directly related to
the transient expression of angiostatin from the Ad vector. Once the
virus is cleared from the animal, there is no longer transgene
expression and the tumors resume normal and rapid growth.
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Coinjection of Ad-angiostatin with Ad-IL-12 increased the number of
mice that showed an initial/partial tumor regression to 96%, and 54%
showed total regression and were protected against challenge with
further PyMidT tumor cells. In those mice that underwent total tumor
regression, the tumors were completely absent 3 wk after the injection
of the virus combination. Mice that had a relapse of tumor growth after
initial regression had a 2- to 3-wk increase in survival time
comparable to the kinetics seen with angiostatin alone (Fig. 1B). Our results indicate that injection of the vector
expressing angiostatin disrupts the tumor endothelium and, in
combination with the immunostimulatory potential of IL-12, produces a
synergistic effect that dramatically reduces tumor size and leads to
increased total regression.
Anti-tumor immunity in mice injected with a combination of Ad-angiostatin plus Ad-IL-12
Two mice from the cured combination Ad-angiostatin and Ad-IL-12
group were sacrificed and their spleens removed 8 wk after rejecting a
second challenge of 5 x 105 PyMidT cells in
the contralateral flank. Splenocytes were cocultured with irradiated
516 MT3 cells, which express the PyMidT Ag (15), for 5
days to activate tumor-specific lymphocytes. These activated cells
demonstrated a high degree of specific killing against
51Cr-labeled 516 MT3 cells, but not the
PyMidT-negative PT516 control cell line (Fig. 2). CTL killing was 32% at an E:T ratio
of 10:1 and 43% at 30:1, with no measurable background.
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The effects of angiostatin, IL-12, and the coinjection of the two Ad vectors on the tumor vasculature was determined by immunohistochemical staining. The vessels within the tumor were stained with an Ab against CD31, a mouse endothelial cell marker that is involved in endothelial cell-to-cell adhesion and leukocyte transmigration (19, 20).
The photomicrograph of the PyMid T tumor section injected with control
Ad-dl70 shows very extensive CD31 staining of vessels (Fig. 3, A and C).
Examination of the tumor tissue shows robust tumor growth with no
indication of necrotic or apoptotic regions in either the CD31- or
corresponding H&E-stained section (Fig. 3, B and
D).
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, E–G). Disruption of the tumor as a result of probable tumor
necrosis and/or apoptosis are markedly evident in the tumors treated
with angiostatin and IL-12 alone or in combination in both the CD31-
and H&E-stained section (Fig. 3, E–H).
Vessel quantification of the CD31-stained sections is shown in Fig. 4. In the tumors injected with the
control virus, 1.8% (±0.65) of the total surface area within the
randomly selected fields stained positive for endothelial cells. In
contrast, the Ad-angiostatin and Ad-IL-12 alone or in combination
significantly (p > 0.005) reduced the tumor
vasculature compared with the control, as measured by CD31 staining
(Fig. 4). Within the treatment groups that can affect angiogenesis, no
significant difference was found between the number of CD31-stained
regions by treatment with angiostatin or IL-12 alone or in
combination.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies have used Ad expressing IL-12 alone (9) or in combination with other immune-stimulating molecules such as IL-2 (15) and/or B7-1 (21) to obtain regression in the transgenic breast cancer model. Viral targeting of human and murine angiostatin has been shown to be effective in delaying the growth of glioblastoma and human breast cancer cell lines in athymic mice (22, 23) for the duration of viral expression.
Although many cytokines, including IL-12, IP-10, monokine induced by
IFN- (MIG), and TNF-, have strong antiangiogenic properties that
can inhibit tumor growth, the mechanisms by which endothelial cell
inhibition occurs is not completely known and is complex (24, 25). Recent evidence has shown that IP-10 and MIG exert
angiostatic activity through binding to the CXC chemokine receptor 3 on
endothelial cells (26). TNF- has been demonstrated to
inhibit endothelial cell growth in vitro (27) and can
promote intravascular thrombosis within tumors (28).
However, the immune-activating component of TNF- results in
macrophage- or leukocyte-induced angiogensis (27, 29).
Moreover, we have previously reported that the toxicity associated with
TNF- (28) may also be a limiting factor in using this
cytokine at high enough doses to achieve a successful attack on tumor
vasculature.
The use of the angiogenesis-specific inhibitor angiostatin has the advantage over a cytokine-based approach in that no toxicity has been associated with administration of this protein (2, 4). Angiostatin, an internal fragment of the plasma protein plasminogen, does not appear to possess any immunological function or enzymatic activity. The results in our study agree with previous findings. No mice receiving Ad-angiostatin had any noticeable gross side effects, and there was no mortality associated with the administration of this vector at concentrations as high as 1 x 109 PFU (data not shown).
The use of Ad-angiostatin in the transgenic PyMidT breast cancer model
demonstrates the ability of this vector to limit tumor growth by
attacking tumor vasculature. Tumor growth was substantially reduced in
mice treated with angiostatin as compared with the control-treated
group. The kinetics of tumor growth over the first 3 wk in the
Ad-angiostatin-treated mice was similar to that of the Ad-IL-12 group
(Fig. 1A). This reduction in the rate at which tumors
expanded increased the survival of the angiostatin-treated mice by 14
days in most cases, as compared with control-treated animals.
These data illustrate the limitations of first-generation adenoviral vectors and the transient nature of protein expression. The reduction in tumor growth appears to coincide with the kinetics of clearance of the Ad from the animal. Once the vector is gone, transgene expression of angiostatin is also lost and the tumor again progresses at a high growth rate. Similar findings were report by Griscelli (22) and Tanaka (23) using Ad expressing human and murine angiostatin, respectively, in glioma and breast cancer models. A major advantage to using adenoviral-delivered angiostatin over recombinant material produced from Pichia pastoris or Escherichia coli has recently been reported (30). Angiostatin from the later sources may be sensitive to physical manipulation, leading to a rapid loss of activity. In addition, recombinant protein may not be correctly glycosylated, resulting in a shorter circulating half-life (2). In contrast, angiostatin derived from the Ad is produced directly in the infected cell, is properly glycosylated, and is not subject to any physical treatment or purification procedures.
Intratumoral injection of Ad-IL-12 into the PyMidT breast cancer
model has been previously described by us and others (7, 31, 32). These reports concentrated on the immunological properties
of IL-12 to activate IFN-, CD8+ T cells, and
NK cells for the rejection of the tumor. Data from this current study
indicate that in the first 3 wk after vector administration, vascular
disruption within the tumor by IL-12 may be as significant in delaying
initial tumor growth as the development of a T cell-mediated antitumor
immune response. However, this does not diminish the role of the immune
system in the delay in growth. On the contrary, if the mechanism of
IL-12-induced inhibition of angiogenesis is taken into account, then NK
cells, IFN-, and IP-10 may account for this phenomenon because they
attack the endothelial cells within the tumor (11).
Indeed, in a recent study using a combination of IL-12 with the IP-10
and MIG vectors, we see synergistic activity on tumor regression
(33). Therefore, the initial response of the tumor to the
administration of Ad-IL-12 may include inhibition of vascular growth
followed by a cell (NK or CD4/CD8)-mediated response to
tumor-associated Ags (TAA). Conceivably, the necrosis and or apoptosis
seen in the CD31-stained tumor sections may enhance the availability of
TAA and make it more accessible to the surveillance of the immune
system. The cell-mediated cytotoxic response is necessary for inducing
long-term protection and is responsible for the 13% total cures seen
with the IL-12 treatment alone (7).
The direct injection of a combination of angiostatin and IL-12 gene therapy vectors appears to have synergistic benefits in this tumor model with an intial/partial tumor regression, with 96 and 54% of the mice showing total regression. These mice developed a strong CTL response and were resistant to rechallenge with the tumor.
The combination therapy was well tolerated by all of the mice injected. The relatively safe nature of Ad-angiostatin allows for the addition of Ad-IL-12 at its optimal efficacious and toxicity-limiting dose of 5 x 108 PFU (7), such that the total virus injected did not exceed 1 x 109 PFU.
The proposed mechanism of the combination therapy may be a reduction of
tumor vasculature, leading to tumor necrosis and apoptosis and followed
by an immune response to released TAA. The process may be initiated
following administration of Ad-angiostatin, leading to a decrease in
tumor vasculature and resulting in disruption of the tumor environment
to a necrotic or apoptotic state. This action may not only serve to
reduce the number of viable tumor cells, but at the same time may
enhance the availability of TAA for presentation by scavenging APCs.
The inclusion of the Ad-IL-12 activates immune effector cells,
including dendritic, NK, and T cells, and also initiates an
IFN--induced cytokine cascade, resulting in antiangiogenic and pro-T
cell chemotactic chemokines being generated. Moreover, the
amount of remaining viable tumor may be reduced sufficiently by the
initial vascular disruption such that the immune system can mount a
response to a much smaller viable tumor burden.
In conclusion, our study has demonstrated that a short-term course of local antiangiogenic therapy combined with immunotherapy can effectively shrink a solid tumor and vaccinate the animal against rechallenge. The approach is novel in that it uses the intratumoral delivery of the genes encoding both the angiogenesis inhibitor angiostatin and the immune stimulatory cytokine IL-12 via adenoviral vectors. The combination Ad-angiostatin with cytokine-expressing vectors is attractive because of the lack of toxicity associated with angiostatin. This approach may be useful in the treatment of many other solid tumors.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jack Gauldie, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
3 Abbreviations used in this paper: IP-10, IFN- inducible protein 10; Ad, adenovirus; PyMidT, primary polyomavirus middle T; H&E, hematoxylin and eosin; MIG, monokine induced by IFN-; TAA, tumor-associated Ags.
4 S. Gyorffy, K. Palmer, and J. Gauldie. 2001. Adenoviral vector expressing murine angiostatin inhibits a model of breast cancer metastatic growth in the lungs of mice. Submitted for publication.
Received for publication October 18, 2000. Accepted for publication March 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity 9:34.
, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc. Natl. Acad. Sci. USA 84:5277.. Gene Ther. 4:1181.[Medline]
. Nature 329:630.[Medline]
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; n = 5),
Ad-IL-12 (
; n = 5), or a combination of
Ad-angiostatin plus Ad-IL-12 (
; n = 5). Final
viral load was adjusted to 1 x 109 PFU with control
virus Ad-dl70 (•; n = 5). Tumors were measured at
the time of injection and then weekly using Vernier calipers. *,
Regression obtained with a combination of Ad-angiostatin plus Ad-IL-12
was significant compared with Ad-dl70 (day 21, p <
0.005). B, Long-term survival of FVB/n mice bearing
PyMidT tumors following treatment with control virus Ad-dl70 (•;
n = 5), Ad-angiostatin (
