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From Dipartimento di Fisiopatologia Clinica,*
Dipartimento di Scienze Biomediche ed
Oncologia,
and Dipartimento di Discipline
Medico-Chirurgiche,
Università di
Torino, Italy; 3S-Pharmacological Consultation and Research
GmbH,§
Harpstedt, Germany; and Cattedra di
Nefrologia,¶
Dipartimento di Scienze Cliniche e
Biologiche, II Facoltà di Medicina e Chirurgia, Università
di Pavia, Varese, Italy
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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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and interleukin
(IL)-1.46
Preliminary studies indicate an involvement of
PAF in inflammatory neoangiogenesis occurring in humans.47
In contrast, the potential role of this mediator in tumor angiogenesis
has not yet been explored. The aim of the present study was to evaluate
whether the production of PAF within human breast cancer correlates
with the extent of neovascularization of the tumor and whether
tumor-extracted PAF induces neoangiogenesis in an in vivo
murine model. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specimens from 18 patients with primary invasive breast carcinoma
(11 ductal, 5 lobular, and 2 mixed ductal/lobular carcinomas; age
range, 45 to 85 years; Table 1
) and from
20 controls (7 normal breast tissues and 13 benign breast lesions such
as fibroadenomas, fibrocystic changes, and sclerosing adenosis; age
range, 42 to 75 years) were obtained after surgery. Adjacent cross
sections of specimens were processed for PAF extraction and
histological analysis.
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Samples were fixed in buffered formalin and paraffin embedded. Sections were stained with hematoxylin and eosin (H&E) for histological diagnosis. Representative sections of the tumors including the infiltrating edges were selected and processed for immunohistochemistry. For the purpose of heat-induced antigen retrieval, sections were pretreated in a pressure cooker in a 10 nmol/L citrate buffer (pH 6.0) solution. A standard avidin-biotin-peroxidase complex procedure was applied. The assessment of vascularization was performed using anti-CD31 monoclonal antibody (MAb; diluted 1:30; Dako, Glostrup, Denmark) and anti-CD34 MAb (diluted 1:50; Novocastra Laboratories, New Castle upon Tyne, UK). After staining, sections were scanned at low power for hot spots of angiogenesis. Microvessels were evaluated on three x200 fields. Any endothelial cell or cluster of endothelial cells positive for CD31 or CD34 was counted. Cathepsin D expression was evaluated using MAb M1G8 (diluted 1:1; CIS BIO International, Gif Sur Yvette, France). The percentage of the overall staining contributed solely by carcinoma cells and the percentage contributed only by stromal staining were scored. Hormonal receptor expression was evaluated using estrogen receptor MAb ER-1D5 (diluted 1:50; Dako) and progesterone receptor MAb PGR (diluted 1:50; Abbott Diagnostic, Wiesbaden-Delkenheim, Germany). p53 protein expression was studied using the MAb DO-7 (diluted 1:100; Biogenex, San Ramon, CA), and c-erbB-2 oncoprotein was demonstrated with a MAb HER-2/neu (diluted 1:100; Pabish, Pero, Italy). Only cell membrane immunoreactivity was regarded as specific for c-erbB-2 overexpression. Proliferation rate was assessed using a Ki67-related MAb (MIB1; diluted 1:10; Immunotech, Marseille, France).
Breast Cell Lines
The T-47D and MCF7 breast adenocarcinoma cell lines were obtained from American Type Culture Collection (Manassas, VA) and nontumor mammary gland MCF10A cell line was obtained from the Michigan Cancer Foundation (Detroit, MI). Cells were cultured in RPMI containing 10% fetal calf serum (Sigma Chemical Co, St. Louis, MO). Cells grown at confluence in 35-mm Petri dishes were washed twice and cultured overnight in RPMI containing 0.25% bovine serum albumin (BSA fraction V; Sigma). PAF was extracted from the cell supernatant as previously described.48
Extraction and Quantitation of PAF
Lipids were extracted from breast specimen homogenates or from
culture medium by chloroform/methanol/water acidified to pH 3.0 to 3.5
with formic acid as previously described.47
PAF was
quantified by bioassay on washed rabbit platelets after purification
from the lipid extract by thin layer chromatography (TLC; silica gel
60, F254, Merck; solvent system: chloroform/methanol/water, 65:35:6,
v:v) and high-pressure liquid chromatography (HPLC; µPorasil
Millipore Chromatographic Division, Waters, Bedford, MA; mobile phase:
chloroform/methanol/water, 60:55:5, v:v; flow rate, 1 ml/minute) as
previously described.48,49
The recovery of radioactive
standards (Du Pont-NEN, Brussels, Belgium), submitted to the same
procedures of extraction and TLC and HPLC purification of biological
samples was, respectively, 96 to 98% for
1-O-[3H]-alkyl-PAF C16:O, 96 to 98% for
1-O-[3H]-alkyl-PAF C18:O, and 79 to 82% for
[14C]-acyl-PAF C16:O. The specificity of platelet
aggregation was inferred from the inhibitory effect of 3 µmol/L WEB
2170, a PAF receptor antagonist (Boehringer, Ingelheim,
Germany).50
PAF bioactivity was not inhibited by
phospholipase A1 (Sigma), thus suggesting that it is related to
alkyl-PAF rather than to acyl-PAF, which is known to be more than 1000
times less active than alkyl species of PAF.37
The
bioactive material was further characterized as PAF on the basis of TLC
and HPLC behavior and of physicochemical
characteristics,45,49
such as inactivation by strong bases
and phospholipase A2 (Sigma) but resistance to phospholipase A1 and
acidic treatment. After TLC and HPLC purification, PAF-bioactive
material was also analyzed by a recently developed technique based on
HPLC (normal phase silica column µPorasil, 250 x 4.6 mm
internal diameter (Millipore Waters), eluted under isocratic conditions
at 1.0 ml/minute, with a mobile phase composed of
chloroform/methanol/water, 60:55:5, v:v) tandem mass spectrometry
(HPLC-MS/MS).51
To reduce the mobile phase flow rate to a
level compatible with the MS system, post-column splitting was
performed by connecting a silica capillary of adequate length to the
splitting port of the MS interface. The samples were injected dissolved
in the mobile phase using an injection volume of 250 ml and a 200-ml
sample loop. Mass spectrometric analysis was performed on a
Perkin-Elmer-Sciex (Thornhill, Canada) API III-Plus triple quadrupole
mass spectrometer, equipped with an atmospheric pressure articulated
ion spray source. High-purity nitrogen served both as the nebulizer gas
(operative pressure was 0.5 MPa) and curtain gas (flow rate was 0.8
L/minute). Argon was used as the target gas for the MS/MS experiments,
at a collision gas thickness of 3 x 1015
atoms/cm2. The ion spray needle voltage was set at 5 kV,
the orifice voltage at 50 V, and the MS collision energy at 25 V, which
were previously shown optimal conditions for these
analyses.51
The quantitative analysis was performed in
multiple reaction monitoring (MRM). Parent ion spectra, positive mode,
were obtained from daughter ions with mass-to-charge ratio (m/z) 184
corresponding to phosphocholine fragment; the scanning range was m/z
100 to 600. In the MRM analysis, acquired in positive mode, the study
of different PAF molecular species was done using the following
reactions (parent ions
daughter ions): 552184 (C18-alkylPAF),
524184 (C16-alkylPAF), 524184 (C18-lysophosphatidylcholine
(LPC)), 496184 (C16-LPC, lyso-PAF), 538184 (C16-acylPAF),
566184 (C18-acylPAF), and 594210 (CV3988, used as internal
standard). CV3988 was kindly provided by Takeda (Osaka, Japan). All
other standards were purchased from Sigma.
Murine Angiogenesis Assay
Female C57 mice at 6 to 8 weeks of age were used. Angiogenesis was assayed as growth of blood vessels from subcutaneous tissue into a solid gel of basement membrane (Matrigel, Becton Dickinson Labware, Bedford, MA) containing heparin (64 U/ml; Sigma) and the test sample.52 Matrigel (8 mg/ml), in liquid form at 4°C, was mixed with the experimental substances and injected (0.5 ml) into the abdominal subcutaneous tissue of mice, along the peritoneal midline. The Matrigel used was extracted according to the procedure described by Taub et al,53 which has been previously shown to efficiently deplete Matrigel of basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor I, and platelet-derived growth factor.54,55
The angiogenic effect of 25 µl of TLC-purified lipid extract from breast tumor, control breast, or culture media from breast cell lines was evaluated in the absence or in the presence of WEB 2170. WEB 2170 was included in the Matrigel plug (final concentration, 250 ng/ml) and injected intraperitoneally (i.p.; 10 mg/kg) 30 minutes before the subcutaneous injection and daily for 6 days. In preliminary experiments, these concentrations of WEB 2170 were shown to inhibit angiogenesis induced by 12.5 ng of synthetic PAF.39,47 Moreover, the angiogenic effect of 5 ng of synthetic C16-alkylPAF or C16-acylPAF (Bachem Feinchemikalien, Bubendorf, Switzerland) was assessed. After 6 days, mice were killed and gels were recovered and processed for light or for immunofluorescence microscopy, performed as previously described.39,47 For immunofluorescence studies, rabbit anti-human von Willebrand factor (vWF) antibody (Sigma), anti-mouse T-cell serum MAb, and anti-Mac-1 FITC-conjugated MAb (Cedarlane, Ontario, Canada) were used. Vessel area and the total Matrigel area were planimetrically assessed from stained sections.56 We considered vessels only those structures possessing a patent lumen and containing red blood cells. Results are expressed as percentage ± SE of the vessel area to the total Matrigel area.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The lipid extracts of breast carcinomas and of controls were studied by
HPLC-MS/MS. Figure 2A
shows the
representative HPLC-MS/MS chromatograms of a mixture of phospholipid
standards containing C18-alkylPAF, C16-alkylPAF, C18-LPC, C16-LPC,
lyso-PAF, C16-acylPAF, C18-acylPAF, and CV3988, obtained by MRM
detection at m/z (parentdaughter) 552184, 524184, 524184,
496184, 482184, 538184, 566184, and 594210,
respectively. Figure 2B
shows the representative HPLC-MS/MS
chromatograms of an individual sample of lipid extract from a breast
carcinoma positive for PAF bioassay. Five samples were studied with
similar results. The HPLC-MS/MS chromatographic analysis obtained by
MRM of lipid extracts from breast carcinomas positive for PAF bioassay
showed chromatographic peaks with MS/MS fragmentation and retention
time corresponding to the standards C16-alkylPAF, C18-LPC, C16-LPC,
lyso-PAF, and C16-acylPAF. CV3988 was used as internal standard (Figure 2B)
. In breast carcinomas containing low levels of PAF bioactivity
(<1.5 pg of PAF/mg of dry tissue; five individual samples) and in
normal breast tissues (<0.25 pg of PAF/mg of dry tissue; five
individual samples), the chromatographic peaks corresponding to
C16-alkylPAF and C16-acylPAF were absent (Figure 2C)
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shows the vascularization of breast cancer tissues as
detected by CD31- and CD34-positive cells. The amount of PAF extracted
from all breast specimens (tumors and controls) significantly
correlated with the number of CD34-positive (r =
0.584; P = 0.003) and CD31-positive
(r = 0.661; P = 0.001) cells.
Moreover, Figure 1, C and D
, shows the linear regression analysis
performed between CD34- or CD31-positive cells and concentrations of
PAF extracted from the specimens of breast carcinomas only. In
contrast, no significant correlation was observed versus
tumor markers such as Ki67 (r = -0.053;
P = 0.834), p53 (r = -0.220;
P = 0.380), c-erbB-2 (r =
-0.306; P = 0.216), estrogen receptor
(r = 0.344; P = 0.162),
progesterone receptor (r = 0.288;
P = 0.247), neoplastic cathepsin D
(r = -0.034; P = 0.894), and
stromal cathepsin D (r = -0.246;
P = 0.310); nor was a correlation observed
versus tumor grading. The histotypes of breast carcinomas
containing high levels of PAF bioactivity were, in 6 of 11 patients,
ductal carcinomas, in 2 of 2 patients, mixed ductal/lobular carcinomas,
and, in none of 5 patients, lobular carcinomas (Table 1)
.
As PAF has been shown to induce angiogenesis in vivo, we
evaluated whether the individual TLC-purified lipid extracts of breast
specimens could elicit neoangiogenesis in a murine model of
subcutaneous Matrigel injection. The lipid extracts from specimens of
breast carcinoma containing high levels of PAF bioactivity (37.88
± 9.4 pg of PAF/mg of dry tissue; mean ± SE; n =
6) induced a significant angiogenic response (Figures 3 and 4
, A
and B). In contrast, the lipid extracts from specimens of breast
carcinomas containing low levels of PAF bioactivity (0.57 ± 0.23
pg of PAF/mg of dry tissue; mean ± SE; n = 6,
Figures 3 and 4D
) and from control specimens (0.24 ± 0.07 pg of
PAF/mg of dry tissue; mean ± SE; n = 6, Figures 3 and 4E
) did not induce significant angiogenesis. Matrigel explanted
from mice injected with the PAF extracted from specimens of breast
carcinoma contained several Mac-1, esterase-positive monocytes but only
scattered cells positive for pan-T lymphocyte markers. Infiltration of
polymorphonuclear neutrophils was absent. The presence of endothelial
cells in association with the vessels was confirmed by staining
sections of the gel with anti-vWF antibodies (Figure 4C)
. The treatment
with the PAF receptor antagonist WEB 2170 significantly prevented
neoangiogenesis induced by lipid extracted from specimens of breast
carcinoma (Figures 3 and 4F)
, thus suggesting a role for PAF as an
angiogenic mediator. Control mice (n = 5)
injected with vehicle alone and treated as above with WEB 2170 did not
exhibit any cellular infiltration within Matrigel (data not shown).
Synthetic C16-alkylPAF at concentrations compatible with that observed
in the lipid extracts from specimens of breast carcinomas was able to
induce angiogenesis (Figure 3)
. The angiogenic response elicited by
synthetic C16-alkylPAF was completely prevented by treatment of mice
with WEB 2170. In contrast, synthetic C16-acylPAF induced a slight
infiltration of Matrigel by vWF-positive endothelial cells without
formation of canalized vessels.
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, a significant amount
of PAF was released in basal culture conditions by two breast cancer
cell lines (T47D and MCF7) but not by an immortalized normal breast
cell line (MCF10). When the culture medium of these cell lines was
injected within Matrigel in mice, a significant neoangiogenic response
was elicited by T47D and MCF7 cell lines but not by MCF10 or control
medium. This angiogenic response was partially inhibited by the PAF
receptor antagonist WEB 2170 (Table 2)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor vascularization is crucial for the pathogenesis of solid tumors. In the absence of local neovascular formation, tumors may not grow beyond 2 to 3 mm in diameter.3 Therefore, it has been suggested that tumor neovascularization is a requirement for its growth.3-5 Several studies have suggested that tumor angiogenesis in breast carcinoma is an independent prognostic factor.10-15,17 The intratumor microvessel density has been correlated with tumor invasiveness and formation of metastases. The complex mechanisms involved in the formation of new vessels remain largely unknown. Certain tumors have been shown to produce factors that are directly angiogenic. Other tumors may depend upon vascularization induced by mediators produced from inflammatory cells.21-24 Endothelial cells are the primary target for these mediators and are stimulated to degrade extracellular matrix, to migrate, and to initiate a capillary sprout. The sprout subsequently expands and assumes a tubular structure that, as result of endothelial cell proliferation, progresses in the extracellular matrix with development of loops and then of a functioning circulatory network. It has been shown that an imbalance in the expression of angiogenic and angiostatic factors occurs locally in tumors.5,7 Studies on tumor angiogenesis have primarily focused on the role of angiogenic factors such as vascular endothelial growth factor (VEGF), TNF, IL-8, transforming growth factor-ß, basic fibroblast growth factor, and tissue factor.21-24 Several of these factors have been involved in the growth and in the vascular density of human breast carcinomas.25-36 It has been recently shown that the endothelium in the breast cancers has a mitotic index 50-fold greater than that in nonmalignant tissues and that this proliferation is mainly present at the periphery of the tumor.58 However, it has been suggested that different growth factors regulate the growth of tumor and of endothelium.58
Recent studies indicate that PAF contributes to the angiogenesis
induced by certain polypeptide mediators such as TNF and
HGF.39,40
Recently, we have shown the involvement of PAF in
inflammatory neoangiogenesis in rheumatoid arthritis.47
The
results of the present study indicate that PAF may contribute to
neoangiogenesis occurring in the breast carcinoma. Indeed, we found
that different molecular species of PAF were detectable in the lipid
extracts of breast carcinomas but not in normal breast. These included
the alkyl-PAF C16, which is biologically active on
platelets37
and which is angiogenic in
vivo.41
The presence of PAF in the breast cancer
tissues significantly correlated with the extent of tumor
vascularization detected as number of CD31- and CD34-positive cells.
Although the present study does not provide conclusive information on
the origin of PAF detected in breast carcinomas, one can speculate that
either infiltrating inflammatory cells or endothelial cells contribute
to the synthesis of PAF within the tumor.43,59
Moreover, we
provide evidence that two breast cancer cell lines, but not a nontumor
breast cell line, are able to release in basal condition bioactive PAF.
The supernatants of these two breast cell lines were shown to induce an
angiogenic response in vivo that was, at least in part, due
to the presence of PAF, as inferred by the inhibitory effect of WEB
2170. These results are consistent with the previously reported ability
of breast cancer cells to produce several mediators able to recruit
inflammatory cells and/or to induce angiogenesis.25-36
In
addition, we observed that the lipid extracts of breast carcinomas
containing high levels of PAF bioactivity, but not the lipid extracts
of breast carcinomas containing low levels of PAF bioactivity or the
lipid extract of normal breast, were angiogenic in vivo in
mice. The observation that WEB 2170, a PAF receptor antagonist,
significantly prevented angiogenesis induced by the lipid extracts of
breast carcinomas suggests a key role of PAF in the mechanism of new
vessel formation. The potential role of PAF in the progression of tumor
disease has been recently addressed in an experimental model of
melanoma. In this model, the production of PAF has been correlated with
the formation of pulmonary metastasis.46
In addition, PAF
was shown to mediate the metastasis-promoting activities of TNF- and
IL-1.46
These observations further support the role of
PAF as mediator of several biological activities of certain
cytokines.37,43
Angiogenic factors such as
TNF-,60
IL-8,61
or VEGF (unpublished
observation) are all able to stimulate the synthesis of PAF. In this
context, PAF may act as a mediator of cell-to-cell communication
involved in the amplification of cytokine-triggered signal. PAF may act
either as a chemoattractant for endothelial cells or as a mediator that
amplifies and propagates the reaction by determining the production of
heparin-binding growth factors.41
Indeed, PAF induces
the expression of several angiogenic factors and chemokines including
basic and acidic fibroblast growth factor, placental growth factor,
VEGF and its specific receptor flk-1, HGF, KC, and
macrophage inflammatory protein 2.42,62
In conclusion, the results of the present study suggest that PAF, which is present in the tumors with high microvessel density, may account, at least in part, for the neoangiogenic activity occurring in human breast cancer. The role of polypeptide mediators such as fibroblast growth factor and VEGF in the vascularization of this tumor has been well established,25-29,36 and the contribution of several other cytokines and growth factors has been suggested.30-36 In this context, a lipid mediator such as PAF, which can be produced by either inflammatory or cancer cells, may contribute to amplify the stimuli leading to the formation of new vessels.
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Footnotes |
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Supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and MURST.
Accepted for publication August 5, 1998.
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or interleukin-1. Eur J Immunol 1994, 24:3131-3139[Medline]
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