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Animal Model |
From the Lombardi Cancer Center,*
Department of
Pharmacology,
Department of
Pathology,§
and School of
Nursing,
Georgetown University Medical
Center, Washington, DC; the Laboratory of
Pathology,¶
National Cancer Institute,
Bethesda, Maryland; and Southern Research Institute,||
Birmingham, Alabama
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have developed a model tumor system in which stable transfection of a particular angiogenic growth factor, fibroblast growth factor (FGF)-4, (also called kFGF/hst-1), FGF-1 (acidic FGF), or vascular endothelial cell growth factor (VEGF/VPF), into MCF-7 breast carcinoma cells has resulted in the acquisition of a more tumorigenic and/or more metastatic phenotype. Whereas parental MCF-7 cells require estrogen supplementation for tumorigenicity in ovariectomized nude mice, the FGF transfectants are capable of estrogen-independent growth. Additionally, in contrast to parental MCF-7 cells, the FGF transfectants are capable of growth in antiestrogen-treated mice and are reliably metastatic to lymph nodes and lungs of tumor-bearing mice.10-12 The VEGF/VPF-transfected MCF-7 cells do not form tumors in ovariectomized mice, but when injected with matrix material, they form large, well vascularized, metastatic tumors in both estrogen- and tamoxifen-treated mice.13 As all of the transfectants are derived from common parents, genetic variation other than expression of the transfected angiogenic factor cDNA is minimized. Thus, this model tumor system may be useful to study the process of tumor-induced angiogenesis in situ. Elucidation of steps in the process of tumor-induced angiogenesis using this model might reveal differences between tumor-induced angiogenesis and other types of angiogenesis that might be used to target therapy specifically to tumor blood vessels.
Studies that link maximal microvessel density to prognosis in breast cancer have typically quantitated microvessel density in hot spots of tumor vasculature, most often in the stroma surrounding foci of tumor cells.14 The correlation of density of these particular vessels with prognosis implies functional significance of these vessels in metastasis. Therefore, vessels that comprise hot spots might be the particular vessels in which to study molecular mechanistic features of angiogenesis in a human breast cancer specimen. Because xenografts in the mouse mammary fat pad do not have the same morphology as human breast tumors, there is no location of tumor-associated vessels corresponding to hot spots. Identification of particular vessels for study of gene expression in a xenograft model may therefore not be straightforward. Therefore, although our long-term goal in these studies is to uncover molecular mechanistic features of tumor-induced angiogenesis, we felt it was important to first precisely identify blood vessels of interest and develop ways to study tumors during their initial growth when differences in tumor size are not evident but when neoangiogenesis would be most active. Surprisingly, we found that regardless of the identity of the transfected angiogenic factor, edge-associated and intratumor microvessel density was increased under growth-promoting conditions when compared with growth-inhibiting conditions. Thus, we believe we have identified particular vessel types in the xenograft that are functionally important to the growth of these tumors and that might correspond to vessels in hot spots of human tumors. In addition, we show that the bromodeoxyuridine (BrdU) labeling index can identify particular tumors that are growing rapidly at early time points before differences in tumor size become apparent. Therefore, study of gene expression in edge-associated and intratumor microvessels, in particular, tumors with high BrdU labeling index using this xenograft model system, may identify genes of which expression is important in the functionality of hot spot blood vessels in affecting prognosis in human cancer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ML-20 cells, which have been previously
described,10,11
are a clonal line of wild-type MCF-7 cells
transfected with an expression vector containing the cDNA for bacterial
lacZ. We find their in vivo behavior to be
indistinguishable from wild-type MCF-7 cells. MKL-F cells are ML-20
cells transfected with an expression vector encoding FGF-4. This clonal
cell line forms large tumors in both ovariectomized and
tamoxifen-treated mice, but larger tumors arise in estrogen-treated
mice15,16
(Table 1)
. MKL-4
cells are a clonal line of wild-type MCF-7 cells first transfected with
an expression vector encoding FGF-4 and subsequently transfected with
an expression vector encoding bacterial lacZ. This clonal
cell line also forms tumors in ovariectomized mice under all hormonal
conditions (no treatment, estrogen, or tamoxifen) but the largest
tumors arise in tamoxifen-treated mice10,11
(Table 1)
.
Hence, the MKL-4 cells and MKL-F cells are both clonal cell lines
derived from wild-type MCF-7 cells that contain the same two expression
vectors but that are oppositely affected in vivo by estrogen
or tamoxifen treatment (Table 1)
. Clone 18 cells are a clonal line of
ML-20 cells transfected with an expression vector encoding FGF-1. Clone
18 cells also form large tumors under all three treatment conditions,
but the largest tumors arise in estrogen-treated mice12
(Table 1)
. MV165-14 cells are ML-20 cells transfected with an
expression vector encoding the 165-kd form of VEGF/VPF. These cells do
not form tumors in ovariectomized animals but are tumorigenic and
metastatic in estrogen-treated animals or in tamoxifen-treated animals
when injected with Matrigel13
(Table 1)
. MPCX cells are
ML-20 cells transfected with the expression vector for VEGF/VPF lacking
the VEGF/VPF sequences. Thus, this cell line is the control
transfectant line for MV165-14.13
In our studies of
microvessel density in tumors and tumor nodules, we found its
behavior to be quite similar to its parent line ML-20, and results for
these two lines are pooled for these studies. The in vivo
growth characteristics of these cell lines are summarized in Table 1
.
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Ten million cells/0.2 ml were injected into the mammary fat pad of ovariectomized nude mice as previously described.10-12 For the VEGF/VPF-transfected MV165-14 cells and their control line MPCX, cells were mixed 1:1 with matrix material (Matrigel, Collaborative Biomedical Products, Bedford, MA) before injection. Some mice were treated with 60-day sustained release pellets containing 17ß-estradiol (0.72 mg) or tamoxifen (5 mg, Innovative Research, Sarasota, FL). Eight hours before sacrifice and tumor harvest, 1 mg of BrdU was injected intraperitoneally. At indicated time points, tumors were harvested with adjacent stroma by cutting through the skin and peritoneum in a circular incision circumscribing the tumor. The tumor was fixed for 48 hours in 10% buffered formalin and subjected to X-gal staining as previously described10-12 to delineate borders of the tumor. Stained tumors were trimmed of extraneous skin and muscle, cut in half to reveal a section from skin to muscle, and fixed in 10% buffered formalin for 48 hours, followed by dehydration, paraffin embedding, and sectioning.
Immunohistochemistry
Immunohistochemistry for murine PECAM-1 was accomplished using the rat monoclonal antibody Mec 13.3,17 which was initially generously provided by Elisabetta Dejana, Rolande Berthier, and Annunciata Vecchi. Subsequently, this antibody became available from Pharmingen (01951D), San Diego, CA. After deparaffination in xylenes and rehydration in graded alcohols, slides were digested in 0.1% trypsin in phosphate-buffered saline (PBS) for 30 minutes at 37° and washed in running distilled water for 10 minutes. Endogenous peroxidases were quenched in 0.3% H2O2 in methanol for 30 minutes. Blocking solution (2% normal rabbit serum, 5% bovine serum albumin (Sigma (St. Louis, MO) A7888) in PBS) was applied for 1 hour. Primary rat anti-murine PECAM-1 antibody was applied for 2 hours at room temperature in blocking solution at a concentration of 5 µg/ml. After PBS washes, secondary rabbit anti-rat antibody coupled to biotin (Vector (Burlingame, CA) BA4001) was applied for 1 hour. Slides were washed three times in PBS, and ABC reagent coupled to peroxidase (Vector Elite kit PK6001) was applied for 1 hour. After three PBS washes, slides were incubated with 0.05% diaminobenzidine and 0.01% H2O2 for 15 minutes. Slides were then briefly counterstained with Harris acidified hematoxylin, washed, dehydrated in graded alcohols, and coverslipped from xylenes. Immunohistochemistry for BrdU incorporation followed the same protocol as for PECAM-1 except that before the trypsin digestion, slides were placed in 2 N HCl for 30 minutes and washed thoroughly in deionized water. The primary antibody was a rat monoclonal anti-BrdU antibody (Accurate Antibodies, Westbury, NY, MAS250b) used at a dilution of 1:32 in blocking solution and incubated for 1 hour at room temperature. After incubation with the same secondary antibody as for PECAM-1, slides were incubated with an ABC reagent coupled to alkaline phosphatase (Vector AK5000). Slides were then incubated with the Vector red alkaline phosphatase substrate (Vector SK-5100) according to the manufacturer's directions. For double PECAM-1 and BrdU immunohistochemistry, PECAM-1 staining with the peroxidase ABC kit and diaminobenzidine was performed first. After the wash step after diaminobenzidine disclosure, slides were left overnight in PBS and begun the next morning at the HCl step of the BrdU protocol.
Cell Death Assays
Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) was performed using the TACS 2 TdT kit (Trevigen, Gaithersburg, MD) according to the manufacturer's instructions.
Analysis of Tumor Sections
Analysis of tumor sections was done in a blinded fashion. The
parameters for inflammation or microvessel density were scored on a
rating system of 0 to 4, with 4 being the highest and 0 denoting
absence of the parameter. Inflammatory infiltrates were evaluated from
hematoxylin and eosin (H&E)-stained sections and scored as acute
(primarily neutrophilic) or chronic (primarily lymphocytic), in
addition to being given a numerical rating. Neovascularization was
assessed using slides stained with immunohistochemistry for PECAM-1,
both singly and in combination with staining for BrdU incorporation.
Microvessels were defined as small vessels without visible smooth
muscle. The densities of four microvessel types were rated
subjectively, using standard slides for each numerical rating and
vessel type. Vessel types rated were 1) tumor edge-associated
microvessels, 2) intratumor microvessels, 3) ectatic (dilated vessels
without associated smooth muscle) stromal microvessels, and 4) normal
stromal microvessels. (Because the density of each vessel type was
rated separately, microvessel densities obtained here cannot be
compared with microvessel counts or Chalkley scores obtained by
others.) For the time courses depicted in Figures 1 and 2
and Table 1
,
a single observer (P. R. Vezza) was used. For correlation of BrdU and
TUNEL labeling indices with edge-associated or intratumor microvessel
density, a second observer's (A. C. Filie) scoring was added for
sections harvested between 10 and 20 days after tumor cell injection.
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RNAse Protection
Parental or transfected cells were grown to 60% confluence in medium containing 5% fetal bovine serum. RNA was harvested using Triazol (Life Technologies, Gaithersburg, MD), and ribosomal bands were determined to be intact by agarose gel electrophoresis followed by ethidium bromide staining. The riboprobe vector for human GAPDH has been previously described.10 The riboprobe for human VEGF/VPF produces a 432-bp probe that contains plasmid sequences and a 389-bp fragment identical to the partial sequence of the mRNA that gives rise to the 165-amino-acid form of VEGF/VPF. When hybridized to VEGF/VPF-165 mRNA, a 389-bp protected fragment is produced. When hybridized to mRNA from VEGF/VPF-189 or -121, which share mRNA sequences with VEGF/VPF-165, a 216-bp fragment is protected. VEGF/VPF-189 transcripts also protect a 173-bp fragment. Thirty micrograms of total RNA was subjected to RNAse protection as previously described10 using 32P-labeled antisense riboprobes for human VEGF/VPF and human GAPDH followed by denaturing polyacrylamide gel electrophoresis and autoradiography. The VEGF-165 isoform and the GAPDH band were quantitated by phosphorimaging, and VEGF-165/GAPDH ratios were calculated and compared.
Statistics
For analysis of data shown in Figures 2 and 3
and Table 2
, data
were for the most part not normally distributed. In those cases, the
Kruskal-Wallis test was used to compare microvessel scores in tumors
produced by transfected cell lines with those produced by
parental/control cell lines. In those data sets that were normally
distributed, a one-way analysis of variance was performed. Scores for
the various vessel types before day 5 were excluded from the analyses
as those scores seemed atypical of the rest of the scores and probably
reflect vessels formed in response to inflammation produced by
injection of tumor cells. A Mann-Whitney rank sum test was used to
compare vessel scores between estrogen- and tamoxifen-treated control
tumors, as these data were also not normally distributed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We began these studies with the hypothesis that the phenotypic
progression observed upon transfection of angiogenic factors into MCF-7
breast carcinoma cells to an estrogen-independent or
tamoxifen-resistant phenotype could be explained, at least in part, by
the angiogenic activity of the transfected factors. To summarize this
phenotypic progression, in vivo growth characteristics of
the cell lines used in this study are given in Table 1
. Because
differences in tumorigenicity between the FGF transfectants and the
parental and VEGF/VPF-transfected cells are most marked under
conditions of estrogen-independent growth, we first examined
characteristics of tumors in a time course from 1 day to 5 to 6 weeks
after tumor cell injection. Under these conditions, the parental or
VEGF/VPF-transfected cells form small nodules (~15 mm3
or
less) that regress over ~2 to 3 weeks, whereas the FGF-transfected
cells form progressively growing tumors. For these initial studies, we
used the parental line ML-20, the FGF-4-transfected cell line MKL-F,
the VEGF/VPF-transfected line MV165-14, and its control line MPCX. We
were able to harvest extremely small tumor nodules produced by the
parental/control cells or VEGF/VPF-transfected cells at late time
points because they express bacterial LacZ, enabling them to
be identified in the mammary fat pad by X-gal staining.
At 1 to 10 days after tumor cell injection, neovessels were present in
stroma surrounding and infiltrating the tumor cells in tumor nodules
produced by all cell lines (examples of day 6 in Figure 1, A and E
). At later time points,
however, in tumors produced by the FGF transfectants, small
microvessels were closely associated with the tumor periphery and
extended into the tumor (examples of day 15 and 35 in Figure 1, F and G
) whereas in tumor nodules produced by parental cell injection, blood
vessels remained at the periphery of the nodule, in some cases
separated from the edge of the nodule by an avascular space (examples
of day 15 and 35 in Figure 1, B and C
). Moreover, some of the vessels
in parental cell nodules gradually assumed a dilated, ectatic
appearance with irregular contours (Figure 1, B and C)
. At very late
time points after tumor cell injection, tumors produced by the FGF
transfectants continued to exhibit intratumor microvessels as well as
large numbers of tumor edge-associated microvessels (Figure 1G)
.
Vessels in parental cell nodules at these time points were almost
exclusively peripheral to the tumor (Figure 1C)
.
Because it seemed that regressing tumor nodules produced by the
parental cells were not lacking in blood vessels, but instead exhibited
blood vessels with different characteristics when compared with those
of the progressively growing tumors produced by the FGF transfectants,
we further characterized these sections with regard to microvessel
topography and morphology in tumor sections produced by the parental
and transfected cells. Sections were rated by a blinded observer for
abundance of four distinct morphologies of microvessels noted in the
tumor sections. These were 1) tumor edge-associated microvessels
(Figure 1, F and G)
, 2) intratumor microvessels (Figure 1, A, E, F, and G)
, 3) ectatic (dilated) stromal microvessels (Figure 1, B and C)
, and
4) normal stromal microvessels (Figure 1, B, C, and G)
.
When tumors or tumor nodules produced in ovariectomized mice by
parental/control or transfected cells were compared over the entire
time course with respect to abundance of these four vessel types, it
was found that edge-associated microvessels were more abundant in the
progressively growing tumors produced by the FGF-transfected cells
(Figure 2B
and not shown) than in tumor
nodules produced by the parental or VEGF/VPF-transfected cells (Figure 2, A and C)
. Statistical analysis of scores for edge-associated
microvessels at time points 5 days or later after tumor cell injection
revealed that scores for tumors produced by the tumorigenic
FGF-4-transfected MKL-F cell line were significantly higher than
those for tumor nodules produced by the ML-20 and MPCX
control lines (P < 0.05), whereas
edge-associated microvessel scores for tumor nodules produced by
the nontumorigenic VEGF/VPF-transfected cell line MV165-14 were not
different from control. Thus, edge-associated microvessels were
significantly more abundant in tumors produced by tumorigenic MKL-F
cells in ovariectomized nude mice than in tumor nodules produced by
cell lines that are nontumorigenic under these conditions.
Intratumor microvessels also appeared more abundant in tumors produced
by the FGF-transfected cells when compared with the parental cells
(Figure 3)
in ovariectomized mice.
Statistical analysis of intratumor vessel scores at time points 5 days
or later after tumor cell injection again revealed that tumors produced
by the FGF-4-transfected cell line MKL-F had significantly higher
intratumor vessel scores than the parental/control lines ML-20 and MPCX
(P < 0.05). The intratumor vessel scores of
tumor nodules produced by the VEGF/VPF-transfected line MV165-14
appeared higher than control, but this apparent difference did not
achieve statistical significance. As the VEGF/VPF transfectants do not
form tumors under these conditions, significant intratumor blood vessel
abundance was correlated with estrogen-independent growth.
No significant differences were observed between tumors produced by FGF- or VEGF/VPF-transfected cells and parental cells in densities of ectatic or normal stromal microvessels (data not shown). Taken together, these data suggest that edge-associated and intratumor microvessels may be important in promoting estrogen-independent tumor growth in ovariectomtized nude mice.
Hormonal Treatments That Affect Tumor Growth also Produce Different Topography and Morphology of Tumor-Induced Blood Vessels
We extended our study to include tumors produced from injection of
FGF- or VEGF/VPF-transfected or parental/control cells into
ovariectomized mice treated with sustained-release pellets of
17ß-estradiol or tamoxifen. To differentiate effects that were
hormonally produced from those produced by tumor growth, we
additionally used a clonal line of FGF-4-transfected MCF-7 cells
(MKL-4), which is growth inhibited by estrogen treatment of
tumor-bearing mice and growth stimulated by tamoxifen treatment of
similar mice10
(Table 1)
.
Findings in this study confirmed a correlation of increased
edge-associated microvessels with hormonal conditions that promote
tumorigenicity or increased tumor size. Tumors produced by
FGF-transfected cells grown under conditions of estrogen treatment
exhibited increased abundance of edge-associated microvessels when
compared with parental cell tumors (Table 2)
. This effect was statistically
significant for two of the three FGF-transfected cell lines
(P < 0.05). These two lines (MKL-F and clone
18) produce larger tumors under conditions of estrogen treatment than
the control lines (Table 1)
. Tumors produced by VEGF/VPF-transfected
cells with estrogen treatment had comparable scores for edge-associated
microvessels compared with the parental cell tumors (Table 2)
, which
also correlates with their comparable eventual tumor size compared with
the control line (Table 1)
. Parental cell tumor nodules produced under
conditions of tamoxifen treatment had a significantly reduced abundance
of edge-associated microvessels when compared with estrogen-treated
parental cell tumors (P = 0.045; Table 2
). This
was not true of tumors produced in tamoxifen-treated animals by the FGF
or VEGF/VPF transfectants, which exhibit tamoxifen-resistant growth
(Table 2)
. Moreover, the edge-associated microvessel abundance in
tumors produced by the same two transfected lines (MKL-F and clone 18)
was significantly greater in tamoxifen-treated animals than that in
tumors produced by control cells (P < 0.05).
These data again imply that edge-associated vessels contribute
positively to tumor growth under conditions of estrogen or tamoxifen
treatment.
Intratumor vessel abundance was not significantly increased in the
tumors produced by the FGF-transfected and VEGF/VPF-transfected cells
when compared with the parental cells with estrogen treatment (Table 2)
, a condition in which all lines are tumorigenic. Although there was
a slightly decreased abundance of intratumor vessels in
tamoxifen-treated parental cell tumor nodules, this effect did not
reach statistical significance. However, intratumor microvessel
abundance in tumors produced by all of the transfected cell lines was
significantly increased when compared with control/parental cell tumor
nodules under conditions of tamoxifen treatment
(P < 0.05; Table 2
). This was particularly
evident in the VEGF/VPF-transfected cells (Figure 1, D and H)
. Thus,
these data and data on intratumor vessels under estrogen-free
conditions suggest that intratumor vessels may have an important role
in promoting growth under conditions where the parental cells are not
tumorigenic, especially in the case of tamoxifen-resistant growth.
There were no differences in abundance of ectatic stromal or normal stromal vessels between different cell lines or treatment conditions. These data combined with the data from tumors produced in ovariectomized animals imply that these vessel types do not influence tumor growth.
Sustained Angigogenesis Produced by Tumor Cell Injection Is Not Due to a Prolonged Inflammatory Response
Although breast tumor xenografts in nude mice are a commonly used
tumor model, injection of tumor cells into the mammary fat pad does not
mimic early stages of growth of a spontaneously arising breast tumor.
We were therefore concerned that the early angiogenic response might be
due to inflammation as a result of the tumor cell injection and
therefore would not be representative of spontaneous tumor
angiogenesis. We examined inflammatory infiltrates over the entire time
course, above, and found that, although in a few tumors high levels of
acute, primarily neutrophilic, inflammation were initially present,
after fewer than 10 days, this rapidly declined to baseline levels
(Figure 4)
. Chronic, primarily
lymphocytic, inflammatory infiltrates remained constant throughout the
time course (not shown). There was no difference in the degree of acute
or chronic inflammatory reaction produced by different cell lines
(Figure 4
and not shown). Moreover, injection of the medium used as a
vehicle for the tumor cell injections did not produce
neovascularization or inflammation when the injection sites were
harvested 10 days later (data not shown). In addition,
immunohistochemistry of tumor sections showed negligible expression of
the inflammatory adhesion molecule ICAM-1 in tumor-associated blood
vessels under all treatment conditions despite robust expression in
several adjacent lymph nodes harvested coincidentally with the tumor
(data not shown). In some tumors produced by all transfected cells, but
more frequently in ones produced by VEGF/VPF-transfected cells, there
was sporadic expression of ICAM-1 in blood vessels in adjacent tissues
not intimately associated with the tumor, such as overlying skin (data
not shown). As VEGF/VPF has been shown to increase ICAM-1
expression,21
and FGF-2 (basic FGF) can either up-regulate
or down-regulate ICAM-1 in cultured endothelial cells,21,22
this finding may be due to an effect of diffusion of the transfected
FGF or VEGF/VPF away from the tumor into surrounding tissues and
actually argues for an ICAM-1-suppressing mechanism within the tumor.
In any case, we conclude that sustained tumor-associated angiogenesis
occurring after the initial period in xenografts produced by parental
or transfected MCF-7 cells is not due to inflammation.
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Recently it has been reported that transfection of mammary
epithelial cells or MCF-7 cells with FGF-4 induces expression of
VEGF/VPF.23,24
In addition, FGF-2 induces VEGF expression
in cultured endothelial cells.25
As the validity of our
model depends on our being able to ascribe angiogenic effects of the
transfections to the transfected factors, we examined RNA from the
parental and FGF-transfected cells for mRNA encoding VEGF/VPF. RNAse
protection assays revealed that neither transfection with FGF-4 nor
FGF-1 induced expression of any of the detected isoforms of VEGF/VPF
(Figure 5)
. We also examined RNA
extracted from tumors produced by the same cell lines harvested 15 days
after tumor cell injection. At this point, the tumors were actively
growing but still small enough so there was no detectable necrosis. As
with cells grown in tissue culture, there was no increase in VEGF mRNA
expression in the tumors produced by the FGF-transfected cells when
compared with a tumor produced by the control ML-20 cells (not shown).
These data, in addition to the different in vivo phenotypes
of the FGF-transfected and VEGF/VPF-transfected cell lines (Table 1)
,
argue that the angiogenic effects of FGF transfection in these cell
lines are not due to increased expression of VEGF/VPF.
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Our data, above, validate the importance of particular
microvessels in producing active tumor growth. As mentioned, our goal
is to study gene expression in these microvessels at early time points
in tumor growth when we believe angiogenesis would be most active. From
the time course experiments depicted in Figures 2 and 4
and from our
previous work,10,12,13,16,26
this would seem to be in the
time range of 10 to 20 days after tumor cell injection, after the acute
inflammatory response has subsided, but just before or concurrent with
a substantial increase in tumor growth rate. Thus, we know which
vessels to study and when to study them. However, tumor cell injection
of any cell line under any conditions produces a range of tumor sizes
or no tumor at all. Therefore, to correlate endothelial expression of
particular genes with active tumor growth, it is important to know
whether an individual early tumor is actively growing before
differences in tumor size become apparent. In other words, we want to
know which tumors to study. As tumor growth is thought to be due to the
algebraic sum of cell proliferation and cell death, we asked whether
measurements that estimate either of these parameters could identify
actively growing tumors versus static or regressing ones. As
the individual cell lines have different responses to hormonal
treatment in vivo (Table 1)
, we were able to use conditions
of estrogen or tamoxifen treatment of tumor-bearing animals to answer
this question.
For these studies, we used the FGF-4-transfected cell line MKL-4, which
produces tumors that are stimulated by tamoxifen treatment of
tumor-bearing mice, the FGF-1 transfected cell line clone 18, and the
VEGF/VPF-transfected cell line MV165-14 as well as the ML-20/MPCX
parental/control lines (Table 1)
. Our hypothesis was that if
proliferation, as measured by BrdU incorporation,27
were a
good in situ predictor for future tumor growth, the pattern
of the mean BrdU labeling indices for tumors produced by a given cell
line under conditions of estrogen or tamoxifen treatment would have the
same relationship as the known growth pattern (measured at much later
time points) for that cell line as influenced by estrogen or tamoxifen
(Table 1)
. Conversely, as TUNEL labeling is one measurement of cell
death, if it were a good in situ predictor of future tumor
growth, the mean TUNEL labeling index for a given cell line under
conditions of estrogen or tamoxifen treatment would have the opposite
pattern to the growth pattern produced by estrogen or tamoxifen
treatment for that cell line. We were not interested in explaining
growth characteristics of tumors produced by the different cell lines
as the algebraic sums of proliferation and cell death. This would be a
very difficult task because of the substantial differences in time
sensitivity of the two assays, the probability that TUNEL, which is
only one of several in situ measurements of apoptosis,
identifies only a subset of apoptotic cells and may also stain necrotic
cells,28-30
and the possibility of entry of apoptotic
cells into S phase,31,32
which might produce positive
staining for both BrdU incorporation and TUNEL in some cells.
Representative tumor sections from tumors produced by FGF- or
VEGF/VPF-transfected cells or parental cells in estrogen- or
tamoxifen-treated mice harvested between 10 and 20 days after tumor
cell injection were stained for BrdU incorporation or TUNEL labeling.
Labeling indices for each section were determined by image analysis.
For tumors produced by the ML-20/MPCX parental or control cell lines,
the BrdU labeling index pattern showed the same trends as the tumor
growth pattern, and the TUNEL labeling index pattern was weakly inverse
to the growth pattern (Figure 6)
. Thus,
for these cell lines, BrdU labeling index is the best in
situ predictor of future tumor growth. For the FGF-transfected
MKL-4 cell line, the BrdU labeling index pattern again matched the
tumor growth pattern and the TUNEL labeling index was opposite to the
growth pattern, so that either a high BrdU labeling index or low TUNEL
labeling index could serve as an in situ predictor of future
tumor growth for this cell line (Figure 6)
. For the clone 18 and
MV165-14 cell lines, BrdU is a good predictor, but TUNEL is not, as the
BrdU patterns correlate with the growth patterns, but the TUNEL
patterns are not inverse. Thus, for the parental/control and all the
transfected cells, BrdU incorporation is the best in situ
predictor of tumor growth as it most consistently correlated with
growth patterns of the transfected cell lines under conditions of
estrogen and tamoxifen treatment. Moreover, rapidly growing tumors
produced by individual cell lines can be identified as those for which
BrdU labeling indices exceed means of the growth-stimulated tumors in
these experiments, and slowly growing tumors can be identified as those
for which BrdU labeling indices are lower than means of the
growth-inhibited tumors in these experiments. These data will therefore
assist us in assessing the effects of expression of particular genes on
individual tumors as our studies of gene expression progress.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or TGF-ß, is regulated
in MCF-7 cells by estrogen and tamoxifen.33
Therefore,
effects of hormonal treatment on microvessels may be due to factors
elaborated by the tumor cells in response to the hormonal treatment. On
the other hand, many endothelial cell types have been shown to have
estrogen receptors34-36
and to respond to estrogen by
altering their gene expression,36,37
so a direct effect of
estrogen or tamoxifen on the endothelial cells comprising these
microvessels cannot be ruled out. The FGF-4-transfected MKL-4 cell line, the tumors of which are growth inhibited by estrogen treatment and growth stimulated by tamoxifen treatment, enables us to differentiate effects of hormonal treatment on blood vessels from those associated with growth. The correlation of BrdU labeling index and edge-associated vessel score for this cell line also underscores this point, as sections produced in untreated, estrogen-treated, and tamoxifen-treated animals were included in this analysis. We have also reported a second FGF-4-transfected MCF-7 cell line, MKS-13, which produces tamoxifen-resistant tumors and which is growth inhibited by estrogen treatment of tumor-bearing mice10 (not shown). As these lines produce approximately equivalent or smaller amounts of mRNA for FGF-4 than the MKL-F cell line the tumors of which are not stimulated by tamoxifen16 (not shown), the opposite responses to estrogen and tamoxifen in these clonal cell lines are probably not due to differences in FGF-4 production.
As mentioned, we wish to study molecular mechanisms of tumor-induced
angiogenesis produced by the different transfected angiogenic factors.
Therefore, the data of others showing that VEGF/VPF expression has been
increased by virtue of FGF-4 transfection23,24
or that
FGF-2 can up-regulate VEGF/VPF expression in endothelial
cells25
were disturbing because they implied that the
phenotypic changes of FGF transfections were due to induced VEGF/VPF
expression. However, we do not find VEGF/VPF expression increased in
any of our FGF transfectants (Figure 5)
or in tumors produced by them
(not shown). Because we observe necrosis in very few of our tumors
after the first few days after tumor cell injection, there is probably
not significant hypoxia that might induce VEGF/VPF expression. In
addition, the growth patterns and patterns of neovascularization of the
FGF- and VEGF/VPF-transfected cells are different (Tables 1 and 2
;
Figures 2 and 3
), implying that the blood vessels are responding to
different factors. Collectively, these data argue against substantial
differences in VEGF/VPF expression in tumors produced by the
FGF-transfected lines when compared with the parental cell tumors as
being responsible for the estrogen-independent, tamoxifen-resistant
phenotype of the FGF transfectants.
As others have reported anti-angiogenic activity of tamoxifen,38-41 our findings in tamoxifen-treated animals are of interest. The patterns of increased edge-associated and intratumor vessels observed in the tumors produced by the transfected cell lines when compared with the parental cell tumors could be explained by hypothesizing that in this tumor system, there is no direct negative effect of tamoxifen on endothelial cells or that the transfected angiogenic factors can overcome that negative effect. In any case, we find abundant vascularization of tumors produced by transfected cells under conditions of tamoxifen treatment. Additionally, our collective data10,12,13,16 (including the present study) imply that tamoxifen resistance in human breast cancer may come about because of increased angiogenic ability of the tumor.
The purpose of our measurements of BrdU and TUNEL labeling indices as
in situ predictors of future growth was only to enable us to
identify actively growing tumors for future study. However, it is of
interest that others have also found increased TUNEL staining under
angiogenic conditions,42
as we showed with the clone 18 and
MV165-14 cells (Figure 6, G and H)
. Thus, the failure of TUNEL staining
to qualify as a good predictor of future growth is probably due to the
multifactorial nature of both growth and apoptosis, as well as the
complexity of the apoptotic process and the inability of one test to
identify all apoptotic cells.
The function of tumor-associated blood vessels is presumably to provide oxygen and nutrients to the tumor cells to support their metabolism and proliferation. It could be hypothesized that those tumors cells near blood vessels would be the ones proliferating most rapidly or that tumors with the highest microvessel density would have the highest rate of growth. However, correlation of microvessel density with tumor cell proliferation has been difficult to achieve.43,44 Some studies have shown a correlation between tumor cell proliferation and position at the edge of a tumor or near a blood vessel,45-48 although others have not.44,49 We do observe areas of high and low labeling in our BrdU-labeled tumors, but highly labeled areas of tumor cells are not reliably associated with the tumor's edge or with tumor cords surrounding intratumor blood vessels (not shown). It may be that intratumor vessels represent previously edge-associated vessels that have become surrounded by tumor and that their abundance is sufficient to support tumor cell proliferation in the tumor's center. This would imply that intratumor vessels represent stable vessels. Indeed, BrdU labeling in endothelial cells has been shown to be more frequent at edges of tumors than at their centers,44,46 supporting the idea that neoangiogenesis occurs first at the edge of the tumor. Although we have performed double-label immunohistochemistry for BrdU incorporation and PECAM-1 in our tumor sections, we observe a very low incidence of BrdU-positive endothelial cells (not shown), making such analyses in our tumors technically difficult. Thus, the processes that result in a high abundance of edge-associated or intratumor vessels in our model and their purpose in promoting the tumor's growth are not clear. These questions will form the basis for further study.
The dilated, irregularly shaped, ectatic vessels we observed in all tumors have a morphology similar to that of regressing vessels associated with healing wounds. Thus, it is possible that the initial neovascularization observed in all our tumors and subsequent appearance of these morphologically distinct vessels are at least partially due to the initial acute inflammatory reaction and represent an attempt by the host to heal the wound of injection. A role for the immune system in the process of tumor regression is not ruled out, either by our inability to find a difference between inflammatory infiltrates in growth-stimulated versus growth-inhibited tumors or by the absence of ICAM-1 expression in tumor-associated vessels. Studies that examined natural killer cell activity in lymphocytes isolated from the tumor or assessed the production of various inflammatory cytokines within the tumor might be more enlightening. However, the contribution of the immune system to tumor regression in immunodeficient mice may be quite different from that in immunocompetent humans, making such studies of doubtful relevance to human breast cancer.
In conclusion, this study using a tumor system of FGF- or VEGF/VPF-transfected MCF-7 cells in ovariectomized or hormonally treated nude mice has provided morphological and topographic evidence for effective patterns of neovascularization that occur in actively growing tumors, regardless of the hormonal treatment or angiogenic growth factor. This evidence provides support for the dogma that the ability to stimulate a functionally effective network of neovessels is an extremely important attribute of a tumor and validates this xenograft model as a vehicle for studies of tumor-induced angiogenesis in breast cancer. Our identification of edge-associated and intratumor vessels as being associated with actively growing tumors paves the way for future studies that address gene expression in the endothelial cells that compose these particular neovessels. These future studies may in turn shed light on the mechanisms by which effective tumor neovascularization occurs in the presence of various forms of hormonal therapy in human breast cancer.
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Acknowledgements |
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Footnotes |
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Supported by American Cancer Society starter grant IRG-93 (S. W. McLeskey), NIH grant R29 CA66154 (S. W. McLeskey), a Career Development Award DAMD17-94-4173 from the U.S. Army Breast Cancer Program (S. W. McLeskey), and NIH grant R01 CA50376 (F. G. Kern). Animal work and image analysis were done in the Lombardi Center Animal Research Resource and the Microscopy and Imaging Shared Resource, respectively, supported by NCI Center grant P30 CA51008.
Accepted for publication August 22, 1998.
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