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From the Institute of Clinical Chemistry and Laboratory
Medicine,*
the Center of Gynecology and
Obstetrics,
and the Department of
Experimental Tumor Biology,§
University of
Münster, Münster, and the Institute of
Immunology,
University of Witten/Herdecke,
Witten, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3
and 6. In this first study on the behavior of cells and cell
clusters from disaggregated operated cancers in an extravasation
model, we could demonstrate the presence of
c-erbB-2-expressing cell subpopulations within the
individual breast cancers that are presumably of high metastatic
potential.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The c-erbB-2 (HER-2/neu) proto-oncogene encodes a
receptor tyrosine kinase, p185c-erbB-2 or
p185neu, that shares extensive sequence homology with
epidermal growth factor receptor (EGFR).4
Like EGFR,
c-erbB-2 is expressed in various fetal and adult epithelia
and is believed to play an important role in growth and
development.5,6
In cancer, oncogenic amplification and/or
overexpression of c-erbB-2 is found in many different human
primary tumors.7
In human breast cancer, numerous studies
have reported c-erbB-2 amplification and
overexpression,8
and some of them found a positive
correlation with earlier relapse and poorer overall survival of the
patient.9-11
In addition to the reported clinical
correlations, experimental approaches using animal and in
vitro models have provided evidence that the c-erbB-2
oncogene plays an important role in cancer metastasis.8
Yu
and co-workers found that expression of c-erbB-2 promotes
the invasion steps in the metastatic cascade in human lung and breast
cancer cells, such as increased motility, migration through the
extracellular matrix, and secretion of enzymes degrading the basement
membrane.12,13
In a recently published study by Verbeek et
al, overexpression of c-erbB-2 was related to random cell
migration.14
It was also shown that
p185c-erbB-2 interacts with members of the laminin receptor
family (6ß4 and 6ß1 integrins) and that this interaction
might also contribute to generate a locomotive phenotype of carcinoma
cells.15
In a previous study in our laboratory we observed that a high-risk
group of breast cancer patients expressed p185c-erbB-2
within the primary tumor and on blood-borne epithelium-derived
cells.16
The onset of progressive disease was related to
the occurrence of blood-borne epithelium-derived cells positively
stained for c-erbB-2 receptor protein. Here we report on the
evaluation of the biological features of
p185c-erbB-2-positive cells and cell clusters from fresh
breast cancer tissue by in vitro extra-vasation
experiments. For our study, we designed an in vitro model
for the venular wall that consists of an endothelial monolayer of human
umbilical vein endothelial cells (HUVECs) growing on porous membranes
covered with extracellular matrix basement membrane. Thus, the
transendothelial penetration by breast cancer cells followed by
invasion of the underlying basement membrane can be examined. We
evaluated our in vitro model using four breast cancer cell
lines expressing different levels of p185c-erbB-2. One of
them was transfected with the human c-erbB-2 cDNA. We then
tested cells from disaggregated surgical breast tissue in our model to
explore the invasion capacity of cells from benign and malignant breast
tissues and the role of c-erbB-2 for their invasive
potential. For the breast cancer cell lines we found that the level of
c-erbB-2 expression correlated positively with the cells'
extravasation potential. Our results for fresh breast cancer tissues
provide evidence that c-erbB-2 expression is characteristic
of cell populations with high locomotive capability that pre-exist
within the primary tissues. Further immunocytochemical analysis of the
invasive cell populations revealed the expression of proteins that are
likely to be involved in the metastatic invasion process (matrix
metalloproteinase MMP-2, CD44, and integrins vß3 and 6). We
conclude that we can select cell subpopulations in breast cancer
tissues that are presumably of high metastatic potential.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUVECs were isolated from human umbilical cord veins as described by Gimbrone et al17 with modifications according to Friedl et al.18 The cells were grown on gelatin-coated flasks and passaged four to six times in a medium containing equal volumes of Iscove's modified Dulbecco's medium (IMDM) and Ham's F12 nutrient mixture (Life Technologies, Eggenstein, Germany) supplemented with 10 µg/ml sodium heparin (Boehringer Ingelheim, Heidelberg, Germany), 5 µg/ml transferrin, 2.5 ng/ml basic fibroblast growth factor (bFGF; Sigma, Deisenhofen, Germany), 5 µmol/L ß-mercaptoethanol, 2 mmol/L L-glutamine (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B (Sigma), and 15% fetal calf serum (FCS; PAA Laboratories, Linz, Austria).
Breast cancer cell lines MCF-7, SK-BR-3, and MDA-MB-468 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in Dulbecco's modified Eagle's medium (DMEM; ICN, Eschwege, Germany) supplemented with 2 mmo/L L-glutamine, antibiotic drugs as above, and 10% FCS. For culture of transfectants, cells were grown under the same conditions except for addition of 400 µg/ml Geneticin (G418) (Sigma) to the culture medium.
Cell Transfection and Selection
MDA-MB-468 cells were stably transfected with the plasmid vector pCVN/HER-2 (generously provided by A. Ullrich, Martinsried, Germany) using the LIPOFECTIN-Reagent (Life Technologies) as described in the technical protocol. For control experiments, cells were transfected with plasmid vector pCVN lacking the c-erbB-2 cDNA insert. Two days after transfection, cells were harvested and selected with 400 µg/ml G418. For a second selection step, G418-resistant cells were positively sorted using immunomagnetic beads (Dynabeads, Dynal, Hamburg, Germany) coated with p185c-erbB-2-specific antibody c-neu (Ab-5, Oncogene Research Products, Cambridge, MA). Sorted MDA-MB-468/HER-2 cells were >99% p185c-erbB-2-positive as detected by FACS analysis (FACScalibur, Becton-Dickinson, Heidelberg, Germany).
Patients, Tissue Collection, and Disaggregation
Human mammary tissue specimens were received from the operating room in sterile tubes containing DMEM with 10% FCS and antibiotic drugs and were kept on ice until disaggregation. We examined 20 primary breast carcinomas (5 stage I and 15 stage IIA/IIIB) and 3 lymph node metastases (2 stage IIIB and 1 stage IV). Tumor histology was classified according to conventional criteria, and all identifiable lymph nodes were histologically examined. Mammary tissues from five patients with benign breast diseases (four with cystic mastopathia and one with fibroadenoma) were chosen as negative controls. Breast tissue was mechanically disaggregated by means of automated tissue disaggregator Medimachine (DAKO, Hamburg, Germany). For this purpose, tumor tissue was cut into small pieces and placed into a disposable disaggregation chamber (Medicon; DAKO) together with 1.5 ml of serum-free invasion medium (DMEM with 2 mmol/L L-glutamine, antibiotic drugs, and 0.1% bovine serum albumin (BSA; Sigma)). The Medicon was inserted into the motor unit of the machine and run for 2 minutes at a pre-fixed rotation speed of approximately 80 rpm. An aliquot of the cell suspension containing single cells and cell clusters up to 30 cells was examined microscopically for cell viability by Trypan blue exclusion. Cells were counted in a Neubauer hematocytometer.
Invasion Assays
Originally, our in vitro invasion assay was based on the procedure of Albini et al.19 Cell culture inserts with 8-µm porous polyethylene terephthalate (PET) membranes were placed in 12-well plastic tissue culture plates (Becton Dickinson Labware, Franklin Lakes, NY). The membranes were coated with basement membrane extracellular matrix (ECM; Harbor Bio-Products, Norwood, MA) at a concentration of 125 µg/cm2 by drying an appropriate ECM dilution overnight under a laminar flow hood. Dried ECM was rehydrated with 500 µl of HUVEC culture medium (see above) for 1 hour. HUVECs were seeded onto the coated membranes in a concentration of 2 x 105 cells/well. After culturing for 2 days at 37°C in a humidified atmosphere of 5% CO2, HUVECs formed confluent monolayers, which was verified by panoptic staining (Diff-Quik; Baxter Health Care Co., Miami, FL). Cell culture inserts were used for up to 3 days after endothelial cells reached confluence.
For the invasion assays of breast cancer cell lines, cells were harvested with 0.25% trypsin/2 mmol/L EDTA (Life Technologies) and adjusted to a density of 2 x 105 cells/ml with serum-free invasion medium (see above), and 2 x 105 cells were placed onto the HUVEC monolayer on the ECM-coated membrane. The invasion assays of primary breast cancer cells were performed applying approximately 106 disaggregated cells to the membrane. The invasion medium was placed into the wells under the bottom sides of the membranes as well. Invasion assays were incubated for 48 hours at 37°C in 5% CO2.
At the end of the invasion assay, the HUVEC monolayer and noninvading cells on the upper surface of the membrane were removed by cotton swabs and thorough rinsing with phosphate-buffered saline (PBS; pH 7.4). Invading cells on the bottom side of the membrane were fixed in 4% paraformaldehyde and characterized using double immunocytochemistry (see below).
Immunocytochemistry
Membranes with invasive cells were double stained by applying a combined immunogold-enzymatic technique according to Riesenberg et al20 with slight modifications. All antibodies were diluted in PBS containing 10% AB serum (Biotest, Dreieich, Germany) and 0.1% acetylated BSA (BSA-C; Aurion, Wageningen, The Netherlands), and each incubation step was followed by three 3-minute washes with PBS.
After fixation (see above), cells were permeabilized by incubation in 0.1% Triton X-100 in PBS for 10 minutes. Cells were blocked for 20 minutes in 10% AB serum with 0.1% BSA-C. Cells were incubated with rabbit polyclonal antibody c-erbB-2 oncoprotein (DAKO), followed by incubation with 5-nm-gold-conjugated goat anti-rabbit antibody (Paesel & Lorei, Hanau, Germany). After a second fixation step with 2% glutaraldehyde in PBS and a second blocking step, mouse monoclonal biotinylated antibody to human cytokeratin 8 (Progen, Heidelberg, Germany) was applied. Samples were then incubated with alkaline-phosphatase-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA).
For detection of metastasis-associated proteins, mouse monoclonal
antibodies to matrix metalloproteinase MMP-2 (Oncogene Research
Products), to CD44 (phagocytic glycoprotein-1 or hyaluronic acid
receptor; Chemicon, Temecula), and integrin vß3 (vitronectin
receptor; Chemicon), and to integrin 6 (component of the laminin
receptors; Novocastra Laboratories, New Castle, UK) were applied.
Samples were then incubated with rabbit anti-mouse immunoglobulins
(DAKO) as a link antibody, followed by the next incubation step with a
complex of calf intestinal alkaline phosphatase and mouse monoclonal
anti-alkaline phosphatase (APAAP; DAKO).
The antibody binding to cytokeratin, MMP-2, CD44, and integrins
vß3 and 6, respectively, was visualized immunoenzymatically by
using the DAKO Newfuchsin substrate system (DAKO) according to the
manufacturer's protocol. Silver enhancement of colloidal gold
particles was performed with a silver enhancement kit (IntenSE)
purchased from Amersham (Braunschweig, Germany). After rinsing the
samples with distilled water, freshly prepared silver enhancement
mixture was applied for ~20 minutes while monitoring the reaction
under the microscope. To abrogate the enhancement reaction, membranes
were rinsed with distilled water. Samples were counterstained using
Mayer's hematoxylin (Merck, Darmstadt, Germany). Finally, membranes
were separated from the insert assembly and mounted with Aquamount
improved (BDH Laboratory Supplies, Poole, UK) on microscope glass
slides. To exclude nonspecific staining, unrelated rabbit IgG (Sigma)
and mouse myeloma proteins (MOPC21; Sigma) served as a negative control
for immunocytochemistry. Enzymatic and immunogold staining with silver
enhancement was viewed by light microscopy and epipolarization using a
fluorescent microscope (Laborlux S, Leica, Wetzlar, Germany) with an
IGS filter (Leica).
Transmission Electron Micrography
For transmission electron microscopy (TEM), PET membranes with HUVEC monolayers were washed once with PBS and fixed with 2% glutaraldehyde. The samples were embedded in Epon 812, and ultra-thin sections were mounted on 200-mesh copper grids. The specimens were examined with a Philips EM at 60 kV.
Western Blot Analysis
For preparation of cell lysates, confluent cell monolayers were washed three times with PBS and scraped from 75-cm2 tissue culture flasks in ice-cold lysis buffer containing 150 mmol/L NaCl, 5 mmol/L EDTA, 10 mmol/L Tris/HCl (pH 7.2), 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and protease inhibitors (cocktail tablets Complete; Boehringer, Mannheim, Germany). Breast tissue was homogenized in the same lysis buffer. After 15 minutes on ice, the lysate was centrifuged at 4°C and 12,000 x g for 1 hour. Protein concentration was determined using the BCA protein assay reagent (Pierce, Rockford, IL), and defined amounts of protein were electrophoresed on a 7.5% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred onto a polyvinylidenfluoride membrane (Roth, Karlsruhe, Germany), and nonspecific binding sites were blocked with 10% nonfat dry milk (De-Vau-Ge Gesundkostwerk, Lüneburg, Germany) in PBS with 0.1% Tween 20. Blots were probed with antibody c-neu (Ab-3) for detection of p185neu (Oncogene Research Products), followed by incubation with horseradish-peroxidase-conjugated goat anti-mouse antibody (Amersham). Both antibodies were used at a concentration of 0.1 µg/ml. Bands were visualized with the enhanced chemiluminescence (ECL) system (Amersham) with an exposure time of 3 minutes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a system that mimics the in vivo situation in blood
capillaries, we used an in vitro model consisting of a
porous PET membrane coated with extracellular matrix and a monolayer of
HUVECs (Figure 1, A–C)
. Confluence of
HUVEC monolayers was verified by panoptic staining (Figure 1A)
. The
size proportions of the HUVEC monolayer (1), basement membrane layer
(2), and PET membrane (3) are revealed by TEM (Figure 1C)
.
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,
MDA-MB-468 cells express no detectable levels of
p185c-erbB-2, and only very few cells penetrated the HUVEC
monolayer and the basement membrane after incubation for 48 hours.
SK-BR-3 cells expressing high p185c-erbB-2 levels showed
high transendothelial invasiveness, and MCF-7 cells expressing moderate
amounts of p185c-erbB-2 showed increased invasiveness
compared with MDA-MB-468 cells. To ensure that differences in the
extravasation capacity were due to differences in the
c-erbB-2 expression levels, and not to a different genetic
background, we transfected low-invasive MDA-MB-468 cells with the
plasmid vector pCVN/HER2, containing the full-length wild-type human
c-erbB-2 cDNA,21
to generate the
MDA-MB-468/HER-2 transfectants. As shown in Figure 2B
, their
c-erbB-2 expression level is between the one of MCF-7 and
SK-BR-3. We used the MDA-MB-468/neo cell line, which was
established by transfecting MDA-MB-468 cells with the empty pCVN
vector, as a control. As for parental MDA-MB-468 cells,
p185c-erbB-2 is not detectable in MDA-MB-468/neo
cells. Again, we compared the c-erbB-2 expression level with
the number of cells on the bottom side of the membrane and found
significantly more cells from the MDA-MB-468/HER2 cell line with high
c-erbB-2 expression, than MDA-MB-468/neo cells,
which exhibited the same low extra-vasation capacity as the parental
cells (Figure 2A)
.
|
. In the case of cells from
disaggregated breast cancer tissue (see below), cytokeratin analysis
was useful to characterize invasive cells with regard to their
histological origin. As shown in Figure 3, A and C
, for both
c-erbB-2-overexpressing cell lines (wild-type SK-BR-3 and
MDA-MB-468/HER-2), double immunocytochemistry revealed an accumulation
of p185c-erbB-2 in membrane protrusions (arrows) indicated
by the black silver stain, whereas cytokeratin (red color) was mainly
localized in the core of the cells. In Figure 3, B and D
, the same
cells are viewed by epipolarization, which enhances the sensitivity of
the silver staining and makes the stain shine with a fluorescent-like
bluish glow against a dark background.
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To investigate whether cells from fresh breast cancer tissues are
capable of extravasation in our model and whether c-erbB-2
plays an important role for this phenomenon, we tested 23 mechanically
disaggregated malignant tumor tissues (20 primary breast cancers and
three lymph node metastases; Table 1
).
Using double immunocytochemistry (see above), invasive cells were
characterized with regard to their epithelial origin and
c-erbB-2 expression; 12 of 16 primary breast cancer tissues
contained c-erbB-2-positive, predominantly clustered cells
capable of invasion after 48 hours. There was no tumor that contained
only single invasive cells. We never observed invasive cell clusters
that were positive for p185c-erbB-2 and negative for
cytokeratin. In case a tumor contained c-erbB-2-expressing
invasive cell clusters, all of the cells within the invasive clusters
were positive for p185c-erbB-2.
|
, invasive cells/cell clusters
from surgical breast cancers characterized by double
immunocytochemistry are shown. Figure 4A
illustrates a single cell on
the bottom side of the PET membrane. In Figure 4, C and E
, typical
invasive cell clusters are shown. The black silver stain on the
membrane visualizes the c-erbB-2 expression. In Figure 4, B, D, and F
, the cells are viewed with epipolarization (see above). In
some cases we observed that the invasive cells detached from the bottom
of the membrane, attached to the plastic surface of the culture well,
and proliferated for several days (not shown).
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and Table 1
) contained invasive cell
clusters or cells. Each of the three lymph node metastases contained
invasive cells, and in two cases invasive cells/cell clusters were
positive for p185c-erbB-2, too. Initially, it seems to be
paradoxical that four mammary carcinoma tissues, which were negative
for the c-erbB-2 protein, presented
p185c-erbB-2-positive invasive cells/cell clusters in the
extravasation assay. This phenomenon can be explained by the presence
of only very few c-erbB-2-expressing cell populations within
a heterogeneous tumor, not detectable by Western blotting, that were
capable of transendothelial migration. Interestingly, four breast
carcinomas that did not contain invasive cells/cell clusters were
pathologically staged lymph node negative. Those carcinomas did not
express c-erbB-2. For several reasons, quantification of
invasive cells from fresh breast tissue was substantially more
difficult than for the cell lines (see previous section). First, there
were remarkable differences in cell viability after disaggregation
(between 10% and 60%). Second, tumors consist of varying proportions
of carcinoma cells and surrounding stromal cells, eg, fibroblasts. And
third, we observed the vast majority of the invasive cells to be
organized in dense clusters (see Figure 4, C and E
), which made
determination of cell numbers very difficult. In a semiquantitative
analysis, we classified the tissues we had tested in the three
categories: (+), representing 1 to 10; +, representing 10 to 100; and
++, representing >100 invasive entities, considering a cell
cluster or single cell expressing the epithelial marker cytokeratin as
one entity (see Table 1
). Regarding the correlation between
c-erbB-2 expression and motility, as observed using the cell
lines (Figure 2)
, a similar trend was also evident for the tumor
tissues applied to our extravasation model. There were five tumors with
a high expression of p185c-erbB-2 as determined by Western
blot analysis (breast carcinomas 10, 17, 18, and 20 and lymph node
metastasis 21 in Figure 5
), and four of them contained >100 invasive
entities. The five tumors expressing medium or low levels of the
receptor (breast carcinomas 3, 6, 13, and 14 and lymph node metastasis
23 in Figure 5
) contained 10 to 100 invasive entities, whereas 1 to 10
invasive entities occurred only in tumors without detectable expression
of p185c-erbB-2.
|
. As
mentioned above, in contrast to this benign tissue, there was no
malignant tumor that contained only single invasive cells, but at least
a combination of single and clustered cells or solely clustered cells
that managed to migrate through the model vessel wall.
For further analysis of the invasive c-erbB-2-expressing
entities from fresh breast cancer tissue with regard to their
biological relevance in the metastatic process, we performed
immunocytochemistry with monoclonal antibodies directed against matrix
metalloproteinase MMP-2, CD44 (hyaluronic acid receptor), and integrins
vß3 and 6, which are suggested to be involved in the metastatic
invasion process.2,22-25
In four of four tumors with
p185c-erbB-2-expressing invasive cell clusters, the
invasive entities also expressed CD44; in three of four, they expressed
vß3 integrin; and in two of four, they expressed MMP-2 and 6
integrins.
In Figure 6, A–D
, examples of
transendothelial invasive cell clusters characterized by double
immunocytochemistry to p185c-erbB-2 and MMP-2, CD44, and
vß3 and 6 integrins, respectively, are shown. Again, the
brown/black silver stain visualizes the c-erbB-2 expression,
whereas the red color reveals the expression of the respective
metastasis-associated protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By applying several breast carcinoma cell lines to the model, our extravasation assay was evaluated. We found a strong positive correlation between the level of p185c-erbB-2 expression and the numbers of cells that had penetrated the barrier after 48 hours. It should be noted that all cell lines used for our experiments derive from metastatic breast carcinomas and that increased expression of c-erbB-2 augmented the invasive capacity in our model. This was clearly demonstrated by the c-erbB-2 cDNA transfection of the low-invasive MDA-MB-468 cell line. Simultaneously, we demonstrated that our in vitro model is suitable to identify cancer cells that are likely to succeed extravasation. Furthermore, our results from testing the breast cancer cell lines support the data of Yu et al12 and Tan et al13 who found that the introduction of the human c-erbB-2 gene into lung and breast cancer cells promotes the invasion steps in the metastatic cascade, such as increased motility, migration through extracellular matrix, and secretion of basement-membrane-degrading enzymes. We extended their results in that we demonstrated that the migration through an endothelial monolayer grown on extracellular matrix is also enhanced by the increased expression of c-erbB-2.
Our finding that all tumor tissues that express c-erbB-2
contained invasive entities (Table 1)
and that the invasive cell number
tends to be related to the level of c-erbB-2 expression
underlines the important role of the c-erbB-2 receptor for
the transendothelial invasiveness of the cells and correlates with our
results for the breast carcinoma cell lines. We observed a high ratio
of p185c-erbB-2-positive, highly motile cells and cell
clusters that derived from disaggregated breast cancer tissues. Thus,
we assume an important functional role of the c-erbB-2
receptor in the metastatic steps that require cell migration, eg, the
extravasation process.
Recent studies have shown that overexpression of c-erbB-2 is sufficient to induce cell migration.14 It was reported that a critical level of p185c-erbB-2 seems to be necessary to achieve transformation,27 which can be explained by a model in which there is an equilibrium between monomeric and dimeric forms of c-erbB-2.28 As the quantity of p185c-erbB-2 increases by overexpression, the equilibrium is shifted to the dimeric state resulting in constitutive activation of the tyrosine kinase and inappropriate cellular signaling, subsequently leading to a locomotive phenotype.28 Thus, it was not necessary to add an agonist of the erbB receptors (eg, epidermal growth factor, EGF) to our extravasation assay to stimulate cell motility.
The process of cell migration requires the coordinated activation of both growth factor and adhesion receptor signaling.14 It has been described that signals downstream of c-erbB-2 can modulate integrin-mediated processes.29 The reported interaction of p185c-erbB-2 with members of the integrin family15 may also contribute to generate a more invasive phenotype in carcinoma cells.
This is the first study of the behavior of cells and cell clusters from disaggregated fresh breast cancers in an extravasation model. Our finding of predominantly clustered carcinoma cells that managed to migrate through our model vessel wall is consistent with data from Friedl et al30 that suggests that locomoting highly polarized clustered cells are prime candidates for precursors of metastasis formation. It also backs up our data that mainly cytokeratin/p185c-erbB-2-positive clustered cells are found in the peripheral blood of breast cancer patients, which we assumed to be the metastasis-forming entities.16
Our discovery of c-erbB-2 expression in the majority
(14 of 19, Table 1
) of transendothelial invasive cell clusters and
single cells from surgical breast cancers supports the assumption that
p185c-erbB-2 indicates even small subpopulations with high
invasion potential within the primary tumor. This is substantiated by
our finding that four tumors with no detectable expression of
c-erbB-2 by Western blotting contained cells/cell clusters
with high locomotive potential that expressed p185c-erbB-2.
Moreover, the immunocytochemical detection of several presumably
metastasis-associated proteins (MMP-2, CD44, and integrins vß3 and
6),2,22-25
which are expressed by the invasive
cells/cell clusters selected in our extravasation model, further
supports this view.
As mentioned above, metastasis results from the preferential survival and growth of a few subpopulations of cells that pre-exist within the parent tumor.26 Clinical data support this view.31 Overexpression of c-erbB-2 even on a focal basis in cancer tissues has been taken as indicating patients who are poorly responsive to adjuvant chemotherapy.31 The detection of those entities in a primary tumor that are less sensitive to chemotherapy may be achieved by our assay. In addition, the more detailed characterization of the metastatic subpopulations could be essential for devising new therapeutic approaches.
Our extravasation assay allows an analysis of individual breast tumors in vitro and may help to identify those patients who are prime candidates for more aggressive chemotherapeutic regimens, whereas other patients can be protected from the harm of an aggressive chemotherapy. Thus, by creating this assay we made a contribution to the individualization of the clinical management of breast cancer.
Thus, our findings may close a gap in that we selected the same cells with regard to their extravasation capacity we would have later isolated from the patients' bloodstream. Moreover, our experimental results complement the findings of Pantel et al32 and Niehans et al33 who reported that p185c-erbB-2-positive cells from breast cancer patients lodged in the bone marrow or spread to distant organs. Our view is also supported by the finding that the four breast tumors that did not contain invasive entities in our model were derived from patients without detectable lymph node metastasis. Nevertheless, because of the small sample number in this study and the short follow-up time, we were not able to detect further correlations between the tumor staging and/or prognosis and invasive potential of the cells from the disaggregated tumors.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Deutsche Forschungsgemeinschaft and from FONDS der chemischen Industrie to Dr. A. Barnekow. This article contains part of the Ph.D. thesis of A. Roetger.
Accepted for publication August 31, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6ß4 and 6ß1 integrins associate with ErbB-2 in human carcinoma cell lines. Exp Cell Res 1997, 236:76-85[Medline]
6 and v integrins in metastasis. Curr Top Microbiol Immunol 1996, 213:195-203
vß3. Cell 1996, 85:683-693[Medline]
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