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Tumor Biology |
Departments of Radiation Oncology [I. D., J. Z. S., B. F., W. M. L., P. O.], Pediatrics [P. K.], and Medicine [W. M.], University of Rochester School of Medicine, Rochester, New York 14642
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To be effective, antiangiogenic therapy with endostatin in tumor-bearing mice requires prolonged administration and high doses of recombinant protein (8 , 14) . In addition, production of the functional polypeptide has proven difficult, perhaps because of its physical properties and because of variations in the purification procedures utilized by different laboratories (15 , 21) . However, some preliminary data have shown that local or systemic administration of endostatin is an effective means of application in cancer therapy. A few groups have demonstrated that antiangiogenic gene therapy with viral vectors is a potentially useful approach for inhibiting tumor growth in mouse models (22, 23) . Although viral vectors have high transfection efficiency and are commonly used in experimental systems, safety issues and the toxicity of these viral vectors likely precludes their use as an i.v. agent. In contrast, we and others have shown that systematically delivered endostatin plasmid possesses low toxicity and high effective antitumor action (17, 18, 19) . In the current study, we investigated whether intratumoral injection of endostatin plasmid once a week for 2 weeks inhibited mammary tumor growth. We also explored the underlying physiological and molecular mechanisms of endostatin-mediated antitumoral effects.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and purified by alkaline lysis and chromatographic
methods using Endofree kit (Qiagen, Valencia, CA). Purified
plasmid had the following specifications: <50 Eu/mg
endotoxin; <1% protein: and <5% (wt/wt) chromosomal DNA.
Murine Mammary Tumor Models and Treatment.
Isotransplants of the murine mammary carcinoma MCa-4 were used. Frozen
MCa-4 tumor cells were inoculated i.m. into the hind limbs of
6–8-week-old female BALB/c (nu/nu) mice (National Cancer Institute,
NIH, Frederick, MD). Tumors were selected for endostatin plasmid
treatment when they reached volumes of between 150 and 250
mm3
(as measured by calipers and the formula:
Volume = Diameter3
/6). Endotoxin-free
endostatin plasmid (45 µg) was injected once a week for 2 weeks into
the tumor in the right thigh. All of the tumors (right and left) were
averaged. Equal volumes of saline and vector were injected as controls
in separate mice. Mice were sacrificed 7 days after the second
injection. Tumors were removed, examined, and frozen for later
immunohistochemistry or RNA isolation. Guidelines for the humane
treatment of animals were followed as approved by the University
Committee on Animal Resources.
Measurement of Anatomical (CD31), Perfused (DiOC7),
and Angiogenic (CD105) Blood Vessels.
Immunohistochemistry methods have previously been described in detail
(24)
. To visualize blood vessels open to flow, an i.v.
injected stain, DiOC7 (Molecular Probes, Engene, OR), was
utilized. Injections were administered i.v. at a concentration of 1.0
mg/kg, 1 min prior to freezing. This dose and schedule has been shown
to provide optimal visualization of tumor vasculature by preferentially
staining cells immediately adjacent to blood vessels.
DiOC7-stained vessels emit green fluorescence when excited
by blue light. CD31 antibody staining (PharMingen, San Diego,
CA) was used for visualizing total structural vessels. In
addition, an anti-CD105 antibody (PharMingen) that was recently
reported to specifically identify vascular neogenesis was also
utilized. CD105 is strongly expressed in activated endothelial cells of
various human tumors, including breast cancer, but is either
undetectable or only weakly present in mature blood vessels of normal
tissues (25, 26)
.
EF5/Cy3 Hypoxia Marker.
Localized areas of tumor hypoxia were assessed in frozen tissue
sections by immunohistochemical identification of sites of
2-nitroimidazole metabolism. A pentafluorinated derivative of
etanidazole (EF5) was injected i.v. 1 h before tumor freezing.
Protein conjugates of EF5 have been previously used to immunize mice
from which monoclonal antibodies were developed (27)
.
These antibodies are extremely specific for the EF5 drug adducts that
form when the drug is incorporated by hypoxic cells, and one of these,
ELK3-51, has been well characterized. Regions of high EF5 metabolism in
tumors (hypoxic regions) were visualized and the area of staining
quantified immunochemically using a fluorochrome (Cy3) conjugated to
the ELK3-51 antibody and computerized imaging techniques.
Imaging and Image Analysis.
For each frozen tumor, 4.0-µm sections were cut using a cryostat. The
stained sections were imaged using an epi-fluorescence-equipped
microscope, digitized (3-CCD camera), background-corrected, and
image-analyzed using Image Pro software (Media Cybernetics) and a
450-MHz Pentium computer. Color images from adjacent microscope fields
were automatically acquired and digitally combined to form 4 x 4 montages of the tumor cross-section (using a motorized stage
and controller). For each section, two peripheral and two interior
image montages were obtained. Each section was then scanned under each
of three staining conditions. First, epi-illumination images of the
fluorescent green DiOC7 staining (perfused vessels) were
obtained immediately after the 4.0-µm sections were sliced on the
cryostat. After the immunohistochemical staining procedures were
completed, the same tumor section used for the DiOC7
imaging was returned to the microscope stage and automatically
rescanned using the same coordinates as for the initial 4 x 4 montages. Using transmitted light, matching montages of the
CD31 (anatomical vessels) and CD105 (angiogenic vessels) were obtained.
Briefly, the image montages were processed to enhance the contrast
between background and either CD31 staining or DiOC7
staining. The quantitative vascular information was analyzed using
custom FORTRAN programs to perform a "closest individual" analysis.
Briefly, the distances from computer-superimposed sampling points to
the nearest blood vessel were determined. The cumulative frequency
distribution of these distances provides the probability of
encountering vessels within any specified distance from the tumor
cells. Median distances to the nearest anatomical or perfused blood
vessel were used for statistical comparisons.
RNase Protection Assay.
Tumor tissue RNA from each treatment group (n = 4–6 mice) was isolated by pulverizing the frozen tissue and
dissolving it in TRIzol reagent (MRC, Cincinnati, OH). RNAse protection
was performed using established multiprobe template sets (PharMingen)
as described previously (27)
.The apoptosis set includes: antiapoptotic genes: Bcl-2, bcl-X,
bcl-W, and bfl-1; and apoptotic genes: Bax,
Bak, and Bad. Two internal controls, L32 and
GAPDH,3
were used to monitor RNA loading. The quantitation
of mRNA expression level tested for each sample was measured using a
Cyclone PhosphorImager (HP Company, Meriden, CT). Relative mRNA
expression levels were ratios of targeted gene divided by
L32 or GAPDH genes.
Western Blot Analysis.
Cell lysates were prepared from frozen tumor tissues
(n = 2) and separated by SDS-PAGE on 12%
(w/v) polyacrylamide gels and electrotransferred onto Millipore
polyvinylidene difluoride membranes in 25 mM Tris, 192
mM glycine. Membranes were blocked in Tris-buffered saline
(20 mM Tris-HCl, 137 mM NaCl) containing 0.05%
Tween 20 and 5% nonfat dry milk for 60 min at room temperature. The
blots were then incubated overnight at 4°C in blocking solution with
the 1:1000 goat-antimurine endostatin antibody (R&D Systems,
Minneapolis, MN). Thereafter, membranes were washed in Tris-buffered
saline and incubated with horseradish peroxidase-conjugated antigoat
IgG (1:3000) in blocking solution for 1 h. The blots were
visualized by chemiluminescence using the Pierce ECL system
(Pierce, Rockford, Illinois).
In Situ Hybridization.
For in situ localization of VEGF and it receptor gene
expression in tumor tissue, we followed procedures previously published
from our laboratory (28)
. Briefly, tumor tissue was fixed
in 10% formalin or 2% paraformaldehyde, and cross-sections were then
cut. Sections of tissue were placed on specially prepared slides
(acid-washed and T3-aminopropyl triethoxysilane-coated), then
deparaffinized, rehydrated, proteinase Kdigested, and hybridized
with VEGF and VEGFR riboprobes labeled with [33P]UTP.
After washing, sections were prepared for autoradiography using NBTII
emulsion. After autoradiography and staining, slides were analyzed by
bright- and dark-field microscopy. Backgrounds for these studies were
determined by using the sense stand RNA probe. As a positive control
for hybridization, some sections were hybridized for a constitutively
expressed mRNA, such as GAPDH, analyzed for cell-specific expression of
the molecule of interest. The location of overexpression was identified
histologically.
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RESULTS |
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. There was a substantial tumor growth delay in endostatin-treated tumors
compared with saline- or vector-treated controls (Fig. 1A)
.
Tumor growth inhibition was only slightly greater in the right
(directly treated) compared with the left thigh tumors, and because the
differences did not achieve statistical significance, the data were
combined. This is consistent with our previous observation that
endostatin expressed in local tissues can release into the circulation
and elicit systemic inhibition of tumor growth and metastasis
(18
, 19)
. Inhibition of tumor growth was statistically
significant beginning 7 days after injection of endostatin plasmid
(P < 0.05). All of the mice were killed 14
days after the first endostatin treatment. There was a significant
reduction of tumor weight in endostatin-treated animals compared with
controls (Fig. 1B)
. Repeating the first experiment with the
same number of mice and treatment schedules again demonstrated
significant reduction in MCa-4 tumor growth. As in the original
experiment, the treated tumors (right leg) had slightly greater growth
inhibition than the opposite leg (left) tumors.
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, endostatin-treated tumors had a significant reduction in total
anatomical vessels and angiogenic vessels. Tumor proliferation and
necrosis index were not significantly altered. In contrast, decreased
blood vessel density was associated with a significant increase in
tumor cell apoptosis. The increase in apoptotic index was 1.7-fold
[3.2 ± 0.45 versus 1.9 ± 0.3 for saline (P < 0.05); 2.0 ± 0.3 for vector-treated). An RNase protection assay also showed
that endostatin-treated tumors had a significant (28%) reduction in
bfl-1 mRNA, as well as 21–26% decrease of
bak and bad mRNA gene expression
compared with controls (P < 0.05) as shown
in Fig. 2
. No other differential mRNA expression was observed in the other
apoptotic gene (bax), or antiapoptotic genes
(bcl-2, bcl-2, and bcl-X) between
endostatin-treated and control animals.
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, endostatin-treated tumors had a 13% increase in median distance to the
nearest perfused vessel compared with saline controls
(P = 0.053). In the same experiment, we also
observed that endostatin-treated tumors showed a significant reduction
in CD31- and CD105-stained vessels (P < 0.01). Vector-treated tumors also had decreased numbers of perfused
vessels compared with saline controls, most likely because of the
larger sizes of the tumors (3.4 ± 0.8 g for vector
versus 2.5 ± 0.5 g for saline) in this
treatment group (Fig. 1B)
. Unpublished studies have shown an
inverse relationship between perfusion and tumor volumes in numerous
tumor
models.4
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The reduction in number of perfused vessels and the increase in tumor
hypoxia were associated with an elevation of local tumor VEGF and VEGFR
mRNA expression, as detected by in situ hybridization (Fig. 4)
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. Both thrombospondin-1 (Fig. 5B)
and endostatin protein (Fig. 5A)
were detected in cellular (a–c) or stromal
(a'–c') compartment.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Application of antiangiogenic growth factors for gene therapy has been recently used in several tumor models (17 , 18) . We initially reported the systemic inhibition of tumor growth and metastasis by i.m. administration of the endostatin gene formulated with synthetic polymer in murine Renca and Lewis lung carcinomas, and tumor volume was 40% of control at 13-day posttreatment (18) . More recently, we reported that i.v. injection of a mixture of liposome with endostatin plasmid inhibited tumor growth and metastases (19) . A similar study has been reported by Chen et al. in human mammary MDA-MB435 xenograft (17) . Preclinical gene therapy models, therefore, suggest that this approach to antiangiogenic growth factor therapy will be efficacious. In contrast, achieving tumor regression with recombinant protein has been difficult and requires frequent high-dose injections in animal tumors. Our results and those of others have demonstrated that local or systemic administration of nonviral endostatin plasmid significantly inhibits the growth of several tumor types, including lung, breast, kidney, and sarcoma tumors (17, 18, 19) . Response, however, is limited to the slowing of tumor growth without complete regression for established tumors. The efficacy may be dependent on tumor type, vector type, and plasmid administration route and dose, as well as the formulation of agents.
Regression of transplanted tumors is a more difficult undertaking than preventing tumor formation when using any cytotoxic or antiangiogenic therapy. Using a transgenic mouse model of a spontaneous pancreatic ß-islet cell tumor, Bergers et al. (29) recently reported that recombinant endostatin effectively prevented the promotion from hyperplastic lesion to small tumor formation but poorly inhibited established tumor growth (progression stage). In our transplantable MCa-4 tumor and others, endostatin succeeded in slowing tumor growth in established tumors as described previously (17, 18, 19, 20) . The antitumoral action of endostatin should, therefore, be further investigated at different stages of carcinogenesis. Ultimately, we may find that the greatest utility for endostatin is in preventing metastases rather than in inhibiting established tumors (18, 19) .
Endostatin is believed to specifically inhibit endothelial cell proliferation rather than tumor cell growth (12, 13, 14, 15) . The underlying molecular mechanisms of antiangiogenesis are presumably related to an increase in endothelial cell apoptosis (13) , or an alteration of cell cycle (21) . In our animal model, the effects of endostatin-mediated antiangiogenesis are consistent with previous studies. In addition, we quantitatively demonstrated that endostatin-treated tumors showed a clear decrease in perfused vessel density and an increase in tumor cell hypoxia, and that the effects were long lasting, with physiological effects still manifesting 14 days after initial treatment. Direct inhibition of tumor structural vessels, angiogenic vessels, and perfused vessels can explain the observed endostatin-mediated antitumoral effects. Although we believe that the tumor cell apoptosis was secondary to ischemia, our observation leaves open the possibility that endostatin has direct apoptotic effects on tumor tissue. Likewise, tumor necrosis and apoptosis may have been attributable to ischemia, reperfusion injury, or some as-yet-unexplained indirect endostatin-induced cytotoxicity.
We favor a direct effect of endostatin on tumor vascularity, resulting
indirectly in tumor growth reduction. Consistent with this hypothesis,
endostatin-treated MCa-4 tumors had a similar proliferative rate to
that of controls as measured by mitotic figures (Table 1)
, which
suggests that the tumor cell cycle was not altered by endostatin.
Reduced vascularity, however, can deprive tumors of their nutrient
supply, and, thus, proliferation can result in environmentally deprived
progeny, leading to apoptosis or necrosis after division. Kirsch
et al. (30)
showed that recombinant
angiostatin-treated gliomas caused both neoplastic and endothelial cell
apoptosis. They concluded that endothelial cell apoptosis results from
vessel thrombosis or regression of pre-existing vessels. In our study,
we counted only the tumor cell apoptotic numbers, because very few
endothelial cells displayed an apoptotic appearance on the basis of
morphology. Thus, we cannot confirm (or refute) the observation of
Kirsch et al. (30)
. Regarding molecules
important in promoting or inhibiting apoptosis, there were mixed
responses. The antiapoptotic gene bfl-1 decreased, but so
did the apoptotic genes bad and bak.
Ischemia and reperfusion injury, therefore, appear more important in
generating the necrosis and apoptosis than any hypothetical direct
endostatin-mediated apoptosis. It is possible, however, that
endothelial cells from different tumor types may respond differently to
endostatin (20)
.
Endostatin may exert biological effects directly or indirectly by altering expression of other growth-related molecules. One possible mechanism is a down-regulation of angiogenic molecules by endostatin. Because multiple cell types, such as tumor cells, endothelial cells, activated macrophages, and tumor fibroblasts, in tumors produce angiogenic and antiangiogenic growth factors, and because many angiogenic growth factors can also be mobilized from the extracellular matrix, endostatin-mediated regulation of angiogenic growth factor gene expression is extremely complex and difficult to assess. Kirsch et al. (30) treated three types of malignant gliomas and found a significant reduction of VEGF mRNA by Northern analysis, but an elevation of FGF2 3 weeks after angiostatin treatment. They hypothesized that angiostatin-mediated antiangiogenesis in gliomas may be secondary to down-regulation of certain angiogenic growth factors or to up-regulation of antiangiogenic factors. We found an up-regulation of VEGF and VEGFR mRNA as well as up-regulation of thrombospondin-1 protein expression in endostatin-treated MCa-4 breast tumors. In contrast to Kirsch et al. (30) , our results demonstrated that VEGF and VEGFR were up-regulated rather than down-regulated. These differences could suggest that: (a) the balance of angiogenic and antiangiogenic growth factors as well as their gene regulation may be time-dependent during endostatin treatment. Sampling at different times after endostatin treatment may result in differential gene expression; (b) expression of angiogenic or antiangiogenic growth factors may be tumor size- or tumor histologic type-dependent; (c) endostatin may exert its effects through different mechanisms in different tumors, animal strains, or species; and (d) expression of angiogenic/antiangiogenic factors may be responding to independent stimuli. We believe that tumor hypoxia induced the VEGF and VEGFR mRNA expression, and the up-regulation of thrombospondin-1 may be triggered by endostatin through specific signaling pathways.
In summary, endostatin inhibits tumor growth by reducing structural, angiogenic, and perfused tumor vessels. A lack of adequate blood supply leads to tumor hypoxia and probably accounts for tumor cell apoptosis and the up-regulation of VEGF and VEGFR. Thrombospondin-1 also increased, which suggests that endostatin may regulate this antiangiogenic peptide. However, the molecular mechanisms for endostatin-mediated antiangiogenesis are still unknown at present. Different mechanisms may exist for in vitro and in vivo models. In vitro, several issues need to be addressed: (a) is there a receptor or endostatin-related cell surface molecule on endothelial cells? and (b) if so, what are the signal transduction pathway and target genes? If not, how does it inhibit angiogenesis and is it via stromal effects? In animal models, we also need to consider: (a) does endostatin act on tumor cells through direct or indirect pathways? (b) if by indirect pathways, is it by regulation of other angiogenic or antiangiogenic factors in endothelial, inflammatory, or tumor cells? and (c) can endostatin induce tumor regression rather than just slow growth, and under what circumstances? These mechanistic questions are now under investigation in our laboratory.
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FOOTNOTES |
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1 Supported by NIH Grants CA11051-25A2 (to P. O.,
J. Z. S., W. M. L., and I. D.), CA52586 (to B. F.), and
University of Rochester starting fund (to W. M.). 
2 To whom requests for reprints should be
addressed, at Center for Cardiovascular Research, Department of
Medicine, Box 679, University of Rochester, Rochester, NY 14642.
Phone: (716) 273-1499; Fax: (716) 275-9895; E-mail: Wang_min{at}urmc.rochester.edu
3 The abbreviations used are: VEGF,
vascular endothelial growth factor; VEGFR, VEGF receptor; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
Received 4/ 3/00. Accepted 11/ 8/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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