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Advances in Brief |
Pharmacology Division [K. S., K. K., R. A., M. I., H. W.], Pathology Division [H. T.], and Genetics Division [H. S., M. T.], National Cancer Center Research Institute, Tokyo 104-0045; Department of Clinical Oncology and Clinical Research, National Shikoku Cancer Center, Ehime, 790-0007 [Y. H.]; Department of Surgery, Japanese Foundation for Cancer Research, Tokyo 170-8455 [F. Ka., M. Y.]; Department of Veterinary Anatomy, Hokkaido University, Sapporo 060-8638 [T. I.]; and Department of Surgery, Omiya Medical Center, Jichi Medical School, Omiya 330-0834 [K. S., F. Ko.], Japan
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ABSTRACT |
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vß3,
flt-1, tie-2, vascular epidermal
growth factor, and CD31) were overexpressed in
exposure to tumor cells. The molecular basis and these unique
histological features may be associated with aggressive IBC on
angiogenic and nonangiogenic pathways. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. Immunohistochemically, ER and PgR were absent.
|
Comparative Studies.
Studies of tumorigenicity, growth rate, patterns of metastasis, and
histopathology were conducted on WIBC-9. The results were compared with
those of the established non-IBC xenografts including MC-2, MC-5,
MC-18, and cancer cell line SK-BR3 (American Type Culture Collection,
Manassas, VA; Ref. 11
).
Morphological and Chromosomal Analysis.
H&E, PAS, Giemsa staining of paraffin-embedded specimens and
electron microscopic examinations were performed following a
conventional method. For karyotype studies of the xenograft, the Giemsa
G banding method was performed after 6 and 12 passages. ER and PgR were
assayed by ELISA with ER-EIA and PgR-EIA kits (Dinabot,
Tokyo, Japan).
In Vitro Cell Growth.
The resected xenografts were passed through 200-µm gauge stainless
steel mesh three times after being minced. The cells were resuspended
in a medium containing 20–60% Percoll (Amersham Pharmacia Biotech,
Uppsala, Sweden) and centrifuged at 1500 rpm for 20 min at room
temperature. The cell pellet was collected, and erythrocytes were
removed by treatment with 0.83% ammonium chloride in 10 mM
Tris-HCl (pH. 7.5). The remaining 1 x 106 cells were cultured on a type 1
collagen-coated dish (Asahi Techno Glass, Tokyo, Japan). A total of
five dishes were used for each xenograft.
Semiquantitative RT-PCR Analysis.
Total RNA of each xenograft and cell line was extracted using the
guanidinium thiocyanate-phenol-chloroform extraction method. The
oligonucleotide primers for RT-PCR were designed to amplify specific
mRNAs using published sequences. After 20, 25, or 30 cycles, the
products were stained with ethidium bromide, and the relative
expression levels were calculated as the density of the products
divided by that of each positive control (-<0.3, 0.3 
+<1.0, ++
1.0). cDNA of activated lymphocytes was
prepared from peripheral blood according to the standard procedure with
Lymphosepal (IBL, Fujioka, Japan), lipopolysaccharide
(PharMingen, San Diego, CA) and phytohemagglutinin (PharMingen).
Flow Cytometric Analysis.
Each sample was stained with the primary Abs, anti-EGFR monoclonal Ab
(Oncogene Science, Uniondale, NY), anti-ErbB-2/3/4 monoclonal Ab
(NeoMarker, Freemont, CA). FITC-antimouse Ab (Becton Dickinson, San
Jose, CA) was then applied as the secondary Ab. The amount of each ErbB
receptor was analyzed using a FACSCalibur with CellQuest software
(Becton Dickinson, Mountain View, CA).
ELISA.
The concentrations of human IL-1ß, human IL-8, human bFGF
(PharMingen), human VEGF (Immuno-Biological Laboratories Co., LTD.,
Fujioka, Japan), and murine VEGF (R&D Systems, Inc., Minneapolis, MN)
were measured with immunoassay kits. A 100-µl aliquot of murine serum
was obtained from each BALB/c nude mouse that had received a xenograft
8 weeks earlier and prepared for ELISA. A 1 x 106 cell/ml sample of each of the tumor cells was
maintained in McCoy’s 5a medium containing 10% fetal bovine serum and
5 mM glutamine. After 7 days, a 100-µl aliquot of each
culture supernatant was used for in vitro ELISA. Each assay
was performed with triplicate wells.
Immunohistochemistry.
Frozen sections of the resected tumor samples embedded in OCT Compound
(Mile’s Sankyo, Tokyo, Japan) were assayed with the immunoperoxidase
procedure. Antimouse CD31, antimouse integrin
vß3, antihuman
integrin vß3,
antimouse flt-1, antimouse flk-1, antimouse tie-2, and antihuman
E-cadherin (PharMingen) were used as the primary Abs. The
reaction was visualized using streptavidin-biotin (PharMingen)
techniques.
Statistical Analysis.
All data are expressed as the mean ± SD. StatView
computer software (ATMS Co., Tokyo, Japan) was used for statistical
analysis of tumor volumes and ELISA results for the two different
groups. Two-sided P < 0.05 was considered
statistically significant.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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were transplanted into BALB/c nude mice and SCID mice.
The tumor from the ninth patient, referred to as WIBC-9, induced
erythema in the overlying skin (Fig. 1B)
, thus showing the
features of IBC. All but this case showed regression of the
transplanted human breast tumors and the establishment of murine
lymphomas, a result that has been reported previously (7)
.
Histologically, WIBC-9 grew locally in an expansive manner, forming a
solid nest structure and exhibiting marked lymphatic permeation. In the
center of the solid nests, the tumor exhibited a lack of endothelial
formation and never exhibited central necrosis (Fig. 1, C–E)
. This closely matched the histological features of the
original tumor. Transmission and phase-contrast electron microscopy
clearly visualized blood pooling without a lining of ECs in the center
of the tumor nests (Fig. 1, F and G)
. There was
no vascular structure between tumor cells and erythrocytes. Neither
necrosis nor fibrosis was observed in the tumor nest. Mice with WIBC-9
(1.5 cm or more in diameter) frequently developed metastatic foci in
their lungs, and tumor cell leakage from preexisting vessels was also
observed (Fig. 1H)
. This phenotype remained stable for over
15 transplant generations. A karyotype analysis revealed chromosomal
abnormalities in terms of structure and number. The median chromosome
number was 75 (range, 72–77), and there was aneuploidy
(n = 20; Fig. 1I
).
In Vivo and in Vitro Growth.
Once established, WIBC-9 manifested 100% tumorigenicity, with a
latency of 2 weeks, and grew rapidly (Y = 46.10 x
4.14e-2X R2 = 0.960; Y,
tumor volume; X; days). There was no significant
difference in growth rate between WIBC-9 and MC-5. On a DNA histogram,
analyzed by flow cytometer, the percentage of the S phase of WIBC-9 was
15% or greater, but this was not significant compared with
those of the three other xenografts or
SK-BR3.4
Semiquantitative RT-PCR.
All ErbB receptors’ mRNAs were detected in WIBC-9 cells.
The expression of ErbB-2 was detected only in WIBC-9 and
SK-BR3, which is consistent with gene
amplification.4
Angiogenic factors human acidic
fibroblast growth factor and human TGF- were detected only in
WIBC-9, and higher levels of expression of human bFGF, human
VEGF, and human ANG-1 were detected in WIBC-9
than in the explored cells. Human VEGF-C and human
VEGF-D were not detected in WIBC-9. The VEGF
family receptor murine flt-1 was expressed at a higher level
in WIBC-9, but murine flk-1 and murine flt-4 were
not detected in WIBC-9. Expression of human Flt-1 and
KDR was detected in all xenografts. The human ANG receptors
Tie-1 and Tie-2 were detected only in WIBC-9, and
a higher level of expression of murine tie-2 was detected in
WIBC-9. The cytokine human IL-1ß was detected only in
WIBC-9, and a higher expression of human IL-8 was detected
in WIBC-9. The adhesion molecules human integrin
ß3, murine integrin
ß3, and murine integrin
v were detected at higher levels in WIBC-9.
The intercellular adhesion molecule human E-cadherin was
detected at the same level in all explored cells, and murine
VE-cadherin was not detected in WIBC-9 (Table 1)
.
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.
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. Protein expression level of human bFGF and human VEGF
was significantly higher in WIBC-9 than in MC-5, in both murine sera
and culture media (Fig. 2, C and D)
. In WIBC-9,
murine VEGF concentration was 3250 ± 240 pg/ml, a
31–39-fold amplification of expression in murine serum in response to
the xenografted cells (Fig. 2E)
.
Immunohistochemistry on the Tumor Margin.
Protein expression of murine flt-1 was detected in the endothelia and
migrated cells of both WIBC-9 and MC-5, whereas murine flk-1 was not
detected in WIBC-9 at the tumor margin (Fig. 3)
. The expression levels of murine tie-2 and murine integrin
vß3 were 5.1–7.3-fold
higher in WIBC-9 than in MC-5, and this result was consistent with the
results of the semiquantitative RT-PCR examination. With regard to
microvascular density, WIBC-9 exhibited significantly more intense
immunoreactivity than MC-5 to murine CD31 in the neovascular epithelia
of the tumor marginal area.
|
. The in vitro features were similar to those
observed in the IBC specimen containing erythrocytes (Fig. 4b)
. Note that these tubules are lined externally with tumor
cells, and no ECs are identified. Transmission electron microscopy
visualized the tube-like structures and loops containing erythrocytes
of WIBC-9 in vivo (Fig. 4, c and d)
.
WIBC-9 exhibited PAS-positive components of loops and blood pooling
(Fig. 4e)
. Weak protein expression of human integrin
vß3 was detected in
the tube-like structures (Fig. 4f)
.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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vß3, and were PAS
positive. These tubules are lined externally with tumor cells,
and no ECs were identified. These results suggest de novo
formation of vascular channels by tumor cells in the central area of
the xenografted tumor in the regimes of hypoxia and up-regulated
angiogenic factors. They also suggest that vessel regression has not
been occurring in these tumors and that the blood from ruptured vessels
has not filled tumor-lined lakes or channels. WIBC-9 exhibited the
absence of fibrosis, central necrosis, or lining of endothelia, whereas
MC-2, MC-5, and MC-18 commonly exhibit fibrosis and central necrosis as
the tumor grows. We believe that these findings may be related to the
expression of certain genes in WIBC-9 (i.e., human Flt-1,
human Tie-2, human Tie-1, and human integrin
vß3). This gene
expression may result in the observed endothelial/vascular phenotype
and the putative de novo formation of the vascular channel
by tumor cells. In the tumor margin, WIBC-9 exhibited hypervascularity
and significantly more intense immunoreactivity of murine CD31 in the
neovascular epithelia than non-IBC xenografts. This may explain
endothelial sprouting of new vessels from preexisting vessels as a
result of overexpression of the angiogenic factors.
Inflammatory cytokines, including IL-1, IL-8, and TNF-, are secreted
from macrophages, neutrophils, lymphocytes, ECs, and tumor cells. They
are involved in migration and inactivation of inflammatory cells. It
has been speculated that inflammatory cytokines may modulate
angiogenesis (12)
. Inflammatory cytokines not only exert
direct action on ECs but also exert indirect action on migrating
macrophages and lymphocytes that secrete other cytokines (13
, 14)
. They directly affect angiogenesis and indirectly modulate
adhesion molecules and matrix metalloproteinase. IL-8, which is a
member of the ELR+ CXC chemokine families, has been reported to be an
EC migration accelerator and a bFGF accelerator (15)
.
ANG-1 is a strong inducer of EC sprouting, which is a first step in both angiogenesis and neovascularization (16) . VEGF and bFGF stimulate EC sprouting, and the stimulated ECs, in turn, induce the production of ETS-1, which up-regulates angiogenesis by promoting the activation of protease and integrin ß3 (17) . In WIBC-9, human angiogenic factors (ANG-1, VEGF, and bFGF) and murine angiogenic factors (flt-1, integrin ß3, VEGF, and CD31), were expressed at higher levels than they were in the three non-IBC xenografts. On the host side, murine VEGF exhibited a 31–39-fold amplification of expression in WIBC-9 in response to xenografted cells. Human VEGF-C and human VEGF-D have been postulated to be molecules characteristic of IBC because Flt-4, which is known to be one of the receptors of VEGF-C and VEGF-D, is present in the epithelium of the lymphatic ducts. However, human VEGF-C, human VEGF-D, and murine flt-4 were not detected in WIBC-9.
Integrin vß3, which is
the most well-documented integrin family, introduces angiogenesis in
the endothelium. Blocking of integrin
vß3 results in the
regression of angiogenesis and induces apoptosis of ECs
(18)
. In WIBC-9, murine integrin
v and murine integrin
ß3 were amplified by RT-PCR, and murine
integrin vß3 was
immunohistochemically determined to be overexpressed. In WIBC-9, the
expression levels of human angiogenic factors (VEGF, ANG-1, bFGF, and
IL-8) on the graft side and murine angiogenic factors (integrin
ß3, lt-1, tie-2, VEGF, and CD31) on the host
side were significantly high. This may explain why angiogenic factors
from the tumor promote the production of host angiogenic factors,
including the elevated integrin
vß3 in the epithelium.
Elevated host integrin
vß3 and other factors
derived from migrated host cells up-regulate murine VEGF and other host
angiogenic factors paracrinely among the cells, thus leading to
the formation of a hypervascular tumor on the tumor margin. The
activated form of Flt-1 kinase-transfected fibroblasts forms a
tube-like structure in the basement membrane matrix
(19)
. Possible similarities between the characteristics of
migrated tumor cells and those of activated ECs and endothelial
precursor cells are now under investigation. VE-cadherin is specific to
the endothelium. When the VE-cadherin gene is knocked out, tube
formation is not observed (20)
. In WIBC-9, this molecule
was not detected. This finding may also be related to the fact that
WIBC-9 lacks endothelial formation in the central tumor.
This molecular basis and these unique histological features may be associated with aggressive IBC on angiogenesis and nonangiogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants-in-Aid for the Second-Term
Comprehensive 10-year Strategy for Cancer Control from the Ministry of
Health and Welfare of Japan, Grants-in-Aid for Cancer Research from the
Ministry of Health and Welfare of Japan, and Grants-in-Aid for Cancer
Research from the Ministry of Education, Science, Sports and Culture of
Japan. 
2 To whom requests for reprints should be
addressed, at Pharmacology Division, National Cancer Center Research
Institute, 5-1-1 Tsukiji, Chuo-Ku, Tokyo 104-0045, Japan. Phone:
81-3-3542-2511, extension 4450; Fax: 81-3-3542-1886; E-mail: hwakasug{at}gan2.ncc.go.jp
3 The abbreviations used are: IBC, inflammatory
breast cancer; SCID, severe combined immunodeficient; EGFR, epidermal
growth factor receptor; ER, estrogen receptor; PgR, progesterone
receptor; IL, interleukin; VEGF, vascular epidermal growth factor;
bFGF, basic fibroblast growth factor; ANG, angiopoietin; RT-PCR,
reverse transcription-PCR; EC, endothelial cell; PAS, periodic
acid-Schiff; Ab, antibody; TGF, transforming growth factor.
Received 3/ 2/00. Accepted 11/15/00.
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REFERENCES |
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/ß+, CD4-/8- phenotype after implantation of human inflammatory breast cancer cells in BALB/c nude mice. Jpn. J. Cancer Res., 86: 1086-1096, 1995.[Medline]
vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell, 79: 1157-1164, 1994.[Medline]
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) and MC-5 (
) with ELISA
(B–E). The levels of human IL-8 (B),
human bFGF (C), human VEGF (D), and
murine VEGF (E) in murine sera and culture media were
significantly higher in WIBC-9 than in MC-5. In particular, the
concentration of murine VEGF was 3250 ± 240 pg/ml, a
31–39-fold amplification of expression in WIBC-9.
n = 5 for each group. Statistical
analysis was performed with Student’s unpaired t test.
*, P < 0.05; N.D., not
detected.
