Title: Selection of Potentially Metastatic Subpopulations Expressing c-erbB-2 from Breast Cancer Tissue by Use of an Extravasation Model
Abstract: Overexpression of the c-erbB-2 gene-encoded p185c–erbB-2 is correlated with early onset of metastasis in breast cancer patients. Furthermore, the detection of blood-borne epithelium-derived clustered cells expressing p185c–erbB-2 was related to advanced stages in breast cancer. To further elucidate the receptor's function in the metastatic process of human breast cancers, we analyzed disaggregated cells and cell clusters from freshly dissected breast cancer tissues. We studied whether their capability of extravasation is correlated with their expression of c-erbB-2. A model for the venular wall was constructed by growing human umbilical vein endothelial cells (HUVECs) on porous membranes coated with basement membrane extracellular matrix. In four control breast cancer cell lines (SK-BR-3, MCF-7, MDA-MB-468, and MDA-MB-468, the latter transfected with a full-length c-erbB-2 cDNA vector) producing different levels of the c-erbB-2 receptor, the expression level correlated positively with the invasiveness of the cells. The invasive, predominantly clustered cells from 14 of 23 tumors were positively stained for p185c–erbB-2 by immunocytochemistry. Furthermore, we show that the invasive cell populations express the metastasis-associated proteins matrix metalloproteinase MMP-2, CD44, and integrins αvβ3 and α6. 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. Overexpression of the c-erbB-2 gene-encoded p185c–erbB-2 is correlated with early onset of metastasis in breast cancer patients. Furthermore, the detection of blood-borne epithelium-derived clustered cells expressing p185c–erbB-2 was related to advanced stages in breast cancer. To further elucidate the receptor's function in the metastatic process of human breast cancers, we analyzed disaggregated cells and cell clusters from freshly dissected breast cancer tissues. We studied whether their capability of extravasation is correlated with their expression of c-erbB-2. A model for the venular wall was constructed by growing human umbilical vein endothelial cells (HUVECs) on porous membranes coated with basement membrane extracellular matrix. In four control breast cancer cell lines (SK-BR-3, MCF-7, MDA-MB-468, and MDA-MB-468, the latter transfected with a full-length c-erbB-2 cDNA vector) producing different levels of the c-erbB-2 receptor, the expression level correlated positively with the invasiveness of the cells. The invasive, predominantly clustered cells from 14 of 23 tumors were positively stained for p185c–erbB-2 by immunocytochemistry. Furthermore, we show that the invasive cell populations express the metastasis-associated proteins matrix metalloproteinase MMP-2, CD44, and integrins αvβ3 and α6. 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. Breast cancer metastasis is the major cause of death for patients with breast carcinomas. Metastasis is viewed as a highly selective competition, favoring the survival of a subpopulation of metastatic tumor cells that pre-exists within the heterogeneous primary tumor.1Fidler IJ Hart IR Biological diversity in metastatic neoplasms: origins and implications.Science. 1982; 217: 998-1003Crossref PubMed Scopus (814) Google Scholar, 2Liotta LA Steeg PS Stetler-Stevenson WG Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation.Cell. 1991; 64: 327-336Abstract Full Text PDF PubMed Scopus (2634) Google Scholar Early micrometastasis in patients with small resectable cancer poses a great problem for the treatment of cancer.3Schlimok G Funke I Holzmann B Göttlinger G Schmidt G Häuser H Swierkot S Warnecke HH Schneider B Koprowski H Riethmüller G Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies.Proc Natl Acad Sci USA. 1987; 84: 8672-8676Crossref PubMed Scopus (282) Google Scholar Hence, it is of major importance to understand the molecular mechanisms of cellular processes essential for cancer metastasis as a basis for new therapeutic approaches.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).4Coussens L Yang-Feng TL Liao Y-C Chen E Gray A McGrath J Seeburg PH Libermann TA Schlessinger J Francke U Levinson A Ullrich A Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with Neu oncogene.Science. 1985; 230: 1132-1139Crossref PubMed Scopus (1557) Google Scholar 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.5Nilsen-Hamilton M Growth Factors and Development. Academic Press, London1990Google Scholar, 6Press MF Cordon-Cardo C Slamon DJ Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues.Oncogene. 1990; 5: 953-962PubMed Google Scholar In cancer, oncogenic amplification and/or overexpression of c-erbB-2 is found in many different human primary tumors.7Brandt BH Vogt U Schlotter CM Jackisch C Werkmeister R Thomas M von Eiff M Bosse U Assmann G Zänker KS Prognostic relevance of aberrations in the erbB oncogenes from breast, ovarian, oral and lung cancers: double-differential polymerase chain reaction (ddPCR) for clinical diagnosis.Gene. 1995; 159: 35-42Crossref PubMed Scopus (44) Google Scholar In human breast cancer, numerous studies have reported c-erbB-2 amplification and overexpression,8Yu D Hung M-C The HER-2/neu gene in human cancers.in: Freireich EJ Stass SA Molecular Basis of Oncology. Blackwell Science, Cambridge1995: 131-162Google Scholar and some of them found a positive correlation with earlier relapse and poorer overall survival of the patient.9Slamon DJ Clark GM Wong SG Levin WJ Ullrich A McGuire WL Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.Science. 1987; 235: 177-182Crossref PubMed Scopus (9811) Google Scholar, 10Thor AD Schwartz LH Koerner FC Edgerton SM Skates SJ Yin S McKenzie SJ Panicali DL Marks PJ Fingert HJ Analysis of c-erbB-2 expression in breast carcinomas with clinical follow-up.Cancer Res. 1989; 49: 7147-7152PubMed Google Scholar, 11Wright C Angus B Nicholson S Sainsbury JR Cairns J Gullick WJ Kelly P Harris AL Horne CH Expression of c-erbB-2 oncoprotein: a prognostic indicator in human breast cancer.Cancer Res. 1989; 49: 2087-2090PubMed Google Scholar 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.8Yu D Hung M-C The HER-2/neu gene in human cancers.in: Freireich EJ Stass SA Molecular Basis of Oncology. Blackwell Science, Cambridge1995: 131-162Google Scholar 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.12Yu D Wang S-S Dulski K Tsai C-M Nicolson GL Hung M-C c-erbB-2/neu overexpression enhances metastatic potential of human lung cancer cells by induction of metastasis-associated properties.Cancer Res. 1994; 54: 3260-3266PubMed Google Scholar, 13Tan M Yao J Yu D Overexpression of the c-erbB-2 gene enhanced intrinsic metastasis potential in human breast cancer cells without increasing their transformation abilities.Cancer Res. 1997; 57: 1199-1205PubMed Google Scholar In a recently published study by Verbeek et al, overexpression of c-erbB-2 was related to random cell migration.14Verbeek BS Adriaansen-Slot SS Vroom TM Beckers T Rijksen G Overexpression of EGFR and c-erbB2 causes enhanced cell migration in human breast cancer cells and NIH3T3 fibroblasts.FEBS Lett. 1998; 425: 145-150Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar It was also shown that p185c–erbB-2 interacts with members of the laminin receptor family (α6β4 and α6β1 integrins) and that this interaction might also contribute to generate a locomotive phenotype of carcinoma cells.15Falcioni R Antonini A Nisticò P Di Stefano S Crescenzi M Natali PG Sacchi A α6β4 and α6β1 integrins associate with ErbB-2 in human carcinoma cell lines.Exp Cell Res. 1997; 236: 76-85Crossref PubMed Scopus (179) Google ScholarIn a previous study in our laboratory we observed that a high-risk group of breast cancer patients expressed p185c–erbB-2within the primary tumor and on blood-borne epithelium-derived cells.16Brandt B Roetger A Heidl S Jackisch C Lellé RJ Assmann G Zänker KS Isolation of blood-borne epithelial derived c-erbB-2 oncoprotein positive clustered cells from the peripheral blood of breast cancer patients.Int J Cancer. 1998; 76: 824-828Crossref PubMed Scopus (78) Google Scholar 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 αvβ3 and α6). We conclude that we can select cell subpopulations in breast cancer tissues that are presumably of high metastatic potential.Materials and MethodsCell Lines and CultureHUVECs were isolated from human umbilical cord veins as described by Gimbrone et al17Gimbrone Jr, MA Cotran RS Folkman J Human vascular endothelial cells in culture: growth and DNA synthesis.J Cell Biol. 1977; 60: 673-684Crossref Scopus (606) Google Scholar with modifications according to Friedl et al.18Friedl P Tatje D Czapla R An optimized culture medium for human endothelial cells from human umbilical veins.Cytotechnology. 1989; 2: 171-179Crossref PubMed Scopus (35) Google Scholar 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 β-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 SelectionMDA-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 DisaggregationHuman 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 AssaysOriginally, our in vitro invasion assay was based on the procedure of Albini et al.19Albini A Iwamoto Y Kleinman HK Martin GR Kozlowski JM McEwan RN A rapid in vitro assay for quantitating the invasive potential of tumor cells.Cancer Res. 1987; 47: 3239-3245PubMed Google Scholar 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 × 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 × 105 cells/ml with serum-free invasion medium (see above), and 2 × 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).ImmunocytochemistryMembranes with invasive cells were double stained by applying a combined immunogold-enzymatic technique according to Riesenberg et al20Riesenberg R Oberneder R Kriegmair M Epp M Bitzer U Hofstetter A Braun S Riethmüller G Pantel K Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumor cells.Histochemistry. 1993; 99: 61-66Crossref PubMed Scopus (80) Google Scholar 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 αvβ3 (vitronectin receptor; Chemicon), and to integrin α6 (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 αvβ3 and α6, 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 MicrographyFor 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 AnalysisFor 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 × 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.ResultsEvaluation of an in Vitro Extravasation Model Using Breast Cancer Cell Lines with Different c-erbB-2 ExpressionAs 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).To evaluate the selection capacity of the model, a panel of breast cancer cell lines (SK-BR-3, MCF-7, and MDA-MB-468) expressing different levels of p185c–erbB-2 was tested for their extravasation potential. As shown in Figure 2, 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,21Hudziak RM Schlessinger J Ullrich A Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH 3T3 cells.Proc Natl Acad Sci USA. 1987; 84: 7159-7163Crossref PubMed Scopus (548) Google Scholar 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/neocells. 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).Figure 2Extravasation potential of breast cancer cell lines and expression of c-erbB-2. A: A total of 2 × 105 tumor cells were placed onto the HUVEC monolayer on the ECM-coated membrane. The columns show numbers of invasive cells of indicated breast cancer cell lines after a 48-hour incubation. Each column represents an average counting from five separate experiments.B: Immunoblot analysis for the c-erbB-2-encoded p185 proteins in the cell lysates of the indicated cell lines. Protein (20 μg) from each sample was electrophoresed on a 7.5% SDS-polyacrylamide gel, transferred to PVDF membrane, and probed with a monoclonal anti-human c-neu antibody (Ab-3). The position of p185c–erbB-2(p185) is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For visualization of c-erbB-2 expression in the highly motile SK-BR-3 and MDA-MB-468/HER-2 cells, we characterized the cells on the bottom side of the porous membrane by double immunocytochemistry for epithelial marker cytokeratin 8 and p185c–erbB-2(Figure 3). 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.Figure 3Transendothelial invasive cells from breast cancer cell lines stained by double immunocytochemistry to cytokeratin and p185c–erbB-2. A: An invasive SK-BR-3 cell was fixed on the bottom side of the membrane. Double immunocytochemistry was performed using a mouse monoclonal biotinylated antibody to human cytokeratin 8 and a rabbit polyclonal antibody to c-erbB-2 oncoprotein. C: An invasive MDA-MB-468/HER-2 cell stained as described above. Membrane protrusions of both cell types contain less cytokeratin (red color from the Newfuchsin dye reaction) but accumulations of the c-erbB-2 receptor visualized by the dark brown staining from the silver enhancement procedure (arrows). B and D:When viewing the same cells by epipolarization, the silver granules appear in a fluorescent-like bluish glow against a dark background. Scale bars, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Extravasation Capacity and c-erbB-2 Expression of Cells from Disaggregated Surgical Breast TissuesTo 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.Table 1Extravasation after 48 Hours and c-erbB-2 Expression of Invasive Cells and Fresh Breast TissuesNumber of samplesTissue samplesInvasive entitiesc-erbB-2 expression of invasive entitiesp185c-erbB-2 immunoblot (tissue samples positive)Primary breast carcinomas (n = 20)164++1289+3(+)Lymph node metastases (n = 3)31++221+1(+)Controls, tissues from benign breast diseases (n = 5)11(+)00Invasive entities = invasive clustered or single cells that stained positive for epithelial marker cytokeratin 8. (+), 1 to 10; +, 10 to 100; ++, >100 invasive entities. Open table in a new tab In Figure 4, 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).Figure 4Transendothelial invasive cells and cell clusters from disaggregated breast cancer tissues stained by double immunocytochemistry to cytokeratin and p185c–erbB-2. A: Typical single double-positive cell on the bottom side of the membrane. Scale bar, 10 μm. C and E: Double-positive cell clusters. Scale bars, 20