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[Cancer Research 61, 5231-5241, July 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

An Intact Overexpressed E-cadherin/{alpha},ß-Catenin Axis Characterizes the Lymphovascular Emboli of Inflammatory Breast Carcinoma1

James S. Tomlinson, Mary L. Alpaugh and Sanford H. Barsky2

Department of Pathology, University of California Los Angeles School of Medicine, Los Angeles, California 90024


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The step of intravasation (lymphovascular invasion), a rate-limiting step in metastasis, is greatly exaggerated in inflammatory breast carcinoma (IBC). Because nearly all human breast carcinoma cell lines grow as solitary nodules in nude/severe combined immunodeficient mice without manifesting lymphovascular invasion, this step has been difficult to study. We captured the essence of the IBC phenotype by establishing a unique human transplantable IBC xenograft, MARY-X, which manifests florid lymphovascular emboli in severe combined immunodeficient/nude mice. Comparing MARY-X with common non-IBC cell lines/xenografts, we discovered an overexpressed and overfunctioning E-cadherin/{alpha},ß-catenin axis. In MARY-X, the E-cadherin and catenins were part of a structurally and functionally intact adhesion axis involving the actin cytoskeleton. In vitro, MARY-X grew as round compact spheroids with a cell density 5–10-fold higher than that of other lines. The spheroids of MARY-X completely disadhered when placed in media containing absent Ca2+ or anti-E-cadherin antibodies or when retrovirally transfected with a dominant-negative E-cadherin mutant (H-2Kd-E-cad). Anti-E-cadherin antibodies injected i.v. immunolocalized to the pulmonary lymphovascular emboli of MARY-X and caused their dissolution. H-2Kd-E-cad-transfected MARY-X spheroids were only weakly tumorigenic and did not form lymphovascular emboli. A total of 90% of human IBCs showed increased membrane E-cadherin/{alpha},ß-catenin immunoreactivity. These findings indicate that it is the gain and not the loss of the E-cadherin axis that contributes to the IBC phenotype.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The step of intravasation (lymphovascular invasion) is characterized, in part, by the formation of lymphovascular emboli. This step is thought to be a rate-limiting step in metastasis (1 , 2) and a step distinct from angiogenesis. IBC3 is a poorly understood disease that is one of the most lethal forms of breast cancer and a disease that manifests an exaggerated degree of lymphovascular invasion (3) . It should be realized, however, that lymphovascular invasion to a less exaggerated degree is a property exhibited by many metastasizing carcinomas. Furthermore, early micrometastases from many carcinomas can be thought of as a manifestation of lymphovascular invasion because early micrometastases consist of tumor emboli located exclusively within lymph node subcapsular sinuses or visceral organ capillary beds (4) . A knowledge of the molecular mechanisms of lymphovascular invasion and lymphovascular emboli formation might lead to better therapies aimed at detection and treatment of micrometastases in general and IBC in particular.

Because the vast majority of human carcinoma cell lines grow as solitary nodules in nude/SCID mice without manifesting overt lymphovascular invasion, this step has been very difficult to study experimentally. Taking advantage of the fact that IBC is a disease that manifests an exaggerated degree of lymphovascular invasion and lymphovascular emboli formation, we, in a previous study (5) , established the first human inflammatory carcinoma xenograft, MARY-X, which recapitulated this phenotype. Because the inflammatory carcinoma phenotype displayed by MARY-X was characterized by homophilic tumor emboli present within lymphovascular spaces, we reasoned that the mechanism likely involved adhesion molecules on tumor cells and/or angiogenic factors and/or proteolytic enzymes released by tumor cells, all of which might facilitate intravasation. Our initial study (5) compared MARY-X with aggressive non-IBC xenografts MDA-MB-231-X and MDA-MB-468-X and human myoepithelial xenografts HMS-X and HMS-3X, the latter of which have been established by our laboratory (6) . Our studies revealed that two of the major differences were both in the adhesion class of molecules, namely, marked increased expression of MUC-1 and E-cadherin in MARY-X. The observation of increased E-cadherin, in particular, was surprising because numerous previous studies had observed that the expression of E-cadherin and related adhesion molecules such as H-cadherin and neural cell adhesion molecule (7 , 8) was lost in malignant progression. Because the E-cadherin axis might be disrupted at some other point, such as in its {alpha},ß-catenin plaque complex, during malignant progression, we felt it important to investigate the entire E-cadherin axis in MARY-X in the present study to see whether it was indeed contributing to the IBC phenotype of lymphovascular invasion (lymphovascular emboli).


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Establishment of MARY-X.
MARY-X was established from a patient with IBC and exhibited the phenotype of florid lymphovascular invasion and florid lymphovascular emboli formation in nude/SCID mice (5) . In MARY-X, the murine component was considerable (~30%). This murine component could essentially be eliminated by producing a MARY-X "shake" according to previously described methods (5) . The shake consisted of spheroids that grew in suspension culture. These spheroids were maintained by suspending the MARY-X shake in KSFM with supplements (Life Technologies, Inc., Gaithersburg, MD) or in MEM with 10% FCS (Life Technologies, Inc.). A murine-specific COT-1 probe verified that the MARY-X shake and the MARY-X spheroids were 99% human (5) .

Other Cell Lines and Xenografts.
Our rationale for choosing the following cell lines and xenografts as comparisons (controls) was as follows. Because we were investigating the importance of the E-cadherin axis in IBC and in MARY-X, we chose common E-cadherin-positive (MCF-7, MCF-10, and MDA-MB-361) and -negative (MDA-MB-231 and MDA-MB-468) breast carcinoma cell lines for comparison studies with MARY-X and the MARY-X spheroids. We also used the malignant COLO-205 cell line as a control because it overexpressed MUC-1 but did not manifest spontaneous metastasis. We also wanted to compare MARY-X with normal cells found in the breast: epithelial and myoepithelial cells. We used HMECs (Clonetics, San Diego, CA) as our epithelial control and our recently established myoepithelial cell lines and xenografts (HMS-1-3 and HMS-X-3X; Ref. 6 ) as our normal myoepithelial surrogates. We also selected the prostatic PC-3 cell line (9) because it was known to express an abnormal E-cadherin/ß-catenin axis, in which {alpha}-catenin was absent, and E-cadherin was overexpressed but not functional in adhesion. We thought it to be a good comparison with MARY-X spheroids for that reason. The MCF-7, MCF-10, MDA-MB-361, MDA-MB-231, MDA-MB-468, COLO-205, and PC-3 cell lines were obtained from the American Type Culture Collection, Manassas, VA). Our myoepithelial cell lines (HMS-1-3) and HMECs were grown in KSFM with supplements (Life Technologies, Inc.); all other cell lines were grown in MEM containing 10% FCS and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) at 37°C in a 95% air:5% CO2 atmosphere at constant humidity.

Antibodies.
The antibodies used included monoclonal antibodies to {alpha}-catenin and ß-catenin and an antiphosphotyrosine monoclonal antibody (PY-20 and mouse IgG2b; Transduction Laboratories, Lexington, KY), each at a concentration of 1–2 µg/ml; E-cadherin (IgG1; clone HECD-1) at a concentration of 1–10 µg/ml (Zymed Laboratories, San Francisco, CA); actin (Sigma Chemical Co., St. Louis, MO; IgG2a; clone AC-40) at a 1:500 dilution; control mouse IgG1 (Dako, Glostrup, Denmark) at a concentration of 50 µg/ml; MUC-1 (clone HMPV, mouse IgG1; PharMingen, San Diego, CA) at a concentration of 1–100 µg/ml; CD44s (IgG1-clone DF1485; Zymed Laboratories) and ß-actin (a gift of Dr. Judy Berliner, UCLA, Los Angeles, CA).

Western, Northern, and Southern Analysis.
The cell lines were harvested and then frozen immediately. The xenografts were excised, frozen, and pulverized with mortar and pestle to a fine powder. Both were then extracted with buffer (1% Triton X-100, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 100 mM NaF, 2 mM sodium orthovanadate, 150 mM NaCl, 10 mM sodium phosphate, and 10 mM EDTA) for 4 h at 4°C with gentle agitation. The samples were then centrifuged at 13,000 x g at 4°C for 15 min. Protein concentrations were determined using the Bio-Rad reagent (Bio-Rad Laboratories, Hercules, CA.). Samples containing equal protein were boiled in 1x Laemmli buffer under reducing conditions, run on a 7.5% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane that was incubated with the primary and secondary antibodies, and signal was detected with the enhanced chemiluminescence detection system (Amersham Life Sciences, Arlington Heights, IL). A monoclonal antibody to ß-actin was used to normalize for protein loading. Genomic DNA was extracted using standard methods; digested overnight with BamHI, HindIII, or PstI (Life Technologies, Inc.); and run on a 1.0% agarose gel. Total RNA was extracted using Trizol reagent (Life Technologies, Inc.), 20 µg of total RNA were loaded onto a denaturing formaldehyde/1.2% agarose gel, and Northern blot analysis was performed. The E-cadherin cDNA, a 2.7 kb of the 3' region, was excised from plasmid PERF-2 (pCMV-NeoPoly2; a gift from Dr. David Rimm, Yale University, New Haven, CT), and the insert was cut from the plasmid using a combination of restriction enzymes EcoRV and XhoI (Stratagene, La Jolla, CA) and labeled by random priming (Megaprime DNA labeling system; Amersham Life Sciences). The housekeeping probe 36B4 (a gift of Dr. Judy Berliner) was used to normalize for RNA loading. All of the above-mentioned methods were also performed on the MARY-X shake, the enriched population of MARY-X spheroids.

Triton X-100 Solubility Assay.
The MARY-X shake was obtained as described previously (5) , except that a sterile screen filter was used to select for the smallest tumor cell aggregates (spheroids). In the assay to investigate the effect of Ca2+ on the partitioning of E-cadherin, the shake was incubated in either 1x PBS containing 1 mM CaCl2 or 1x PBS without calcium for 1 h at room temperature. The shake was then subjected to either a short (10-min) or a long (30-min) extraction. The shake was suspended in extraction buffer [0.5% Triton X-100, 300 mM sucrose, 10 mM PIPES (pH 6.8), 50 mM NaCl, and 3 mM MgCl2] and gently agitated at 4°C for either 10 or 30 min. The samples were then centrifuged at 10,000 x g for 15 min at 4°C. The supernatant was removed as the soluble fraction, and the particulate fraction was resuspended in 2x Laemmli buffer under reducing conditions (volume equal to supernatant). Equal volumes of the soluble and particulate fractions were then loaded onto 7.5% SDS-PAGE. PC-3 cells were grown to confluence on 100-mm plates, and the cells were washed with 1x PBS and 500 µl of extraction buffer and treated similarly.

Immunoprecipitation and Phosphotyrosine Studies.
Because phosphorylation of the E-cadherin adhesion complex, especially ß-catenin, might influence the adhesion state of the complex, we measured the degree of phosphorylation by immunoprecipitation and Western blot studies. Coimmunoprecipitation of ß-catenin in tumoral and cellular extracts (MARY-X spheroids, HMS-3X cells, and HMECs) was achieved by incubation with an anti-E-cadherin antibody followed by precipitation with a rabbit antimouse antibody conjugated to Sepharose A (Transduction Laboratories). The precipitate was resuspended in Laemmli buffer and run on a 7.5% SDS-PAGE followed by Western blot transfer. Antiphosphotyrosine antibodies were used to probe the blot, followed by a horseradish peroxidase-conjugated rat antimouse IgG2b antibody (Zymed Laboratories). The phosphotyrosine band intensity of the 92-kDa band thought to be ß-catenin was measured and normalized against ß-catenin protein levels. The degree of phosphorylation of ß-catenin in the MARY-X spheroids was compared with the levels observed in HMS-3X cells and HMECs. A second direct immunoprecipitation strategy using anti-ß-catenin instead of anti-E-cadherin was used to compare results.

Retroviral Transfection Studies.
Retroviral plasmids (2.0 pmol) containing either the GFP reporter gene (pMSCV-GFP; Clontech, Palo Alto, CA), a dominant-negative E-cadherin mutant (pBabe-H-2Kd-E-cad), or its control (pBabe-H-2Kd-E-cad{Delta}C25; gifts of Dr. Fiona Watt, Imperial Cancer Research Fund, London, United Kingdom; Ref. 10 ) were used in conjunction with a packaging plasmid (1.2 pmol; pCL-Ampho; Imgenex, San Diego, CA; Ref. 11 ) to transiently transfect 293T cells (106 cells) via a calcium phosphate transfection method (10 , 11) . The 293T cells were a derivative of the adenovirus-transformed E1A-expressing human embryonic kidney cell line that had previously been transfected with a SV40 large T-antigen to amplify the transfected pCL-DNA. Harvesting of the viral supernatants in conditioned media (Iscove’s modified Dulbecco’s medium with 10% FCS) was begun at 36 h posttransfection, and supernatants were collected every 4–6 h for up to 72 h. Viral supernatant (25 ml) was filter-sterilized through a 0.45-µm-pore size filter and used in subsequent experiments. This approach produced high titers (106–107 colony-forming unit/ml) of helper-free retrovirus containing the desired constructs. The dominant-negative E-cadherin mutant (H-2Kd-E-cad) encoded a 66-kDa chimeric protein consisting of the extracellular domain of H-2Kd (297 amino acids) linked to the COOH-terminal 191 amino acids of mouse E-cadherin, which comprised 16 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains containing the catenin binding site. As a control, a construct (H-2Kd-E-cad{Delta}C25) was derived from this dominant-negative mutant in which the catenin binding site had been destroyed by a 25-amino acid deletion in the cytoplasmic domain (10) . The filtered undiluted retroviral supernatants were used immediately in some experiments or aliquoted and stored at -70°C for later use. Spheroids (103–104) of MARY-X (average size, 100–200 µm in diameter) were plated onto 60-mm dishes in 2 ml of viral supernatant in the presence of Polybrene (8 µg/ml) according to the parameters defined previously for the retroviral infection of spheroidal aggregates in suspension (12) . Infection was carried out in a humidified incubator at 37°C in a 95% air:5% CO2 atmosphere at constant humidity over 3 h. After this the viral supernatant was removed, the spheroids were placed in DMEM with 10% FCS. After 48 h and over the next 72 h, the spheroids were observed for gene expression and/or phenotypic changes. GFP expression was determined with an inverted Nikon fluorescence microscope. The presence of H-2Kd (13) was determined by incubating the spheroids with a FITC-conjugated rat antimouse H-2Kd monoclonal antibody (Seikagaku Co., Tokyo, Japan) at 1:10 to 1:100 dilutions at room temperature for 1–2 h followed by thorough washings. In some experiments, the spheroids were permeabilized with absolute methanol for 20 min at -20°C before incubation with antibody. H-2Kd expression was determined with an inverted Nikon fluorescence microscope as described above.

In Vitro Spheroid Disadherence Assays.
MARY-X spheroids were placed individually into wells of a 96-well plate containing 50 µl of media (KSFM + 1 mM CaCl2, calcium-free KSFM, or 50–100 µg/ml E-cadherin antibody in KSFM + CaCl2). Anti-MUC-1 antibody, anti-CD44 antibody, and mouse IgG1 were used separately as control antibodies in the same concentrations. The spheroids were monitored for disadherence by visualization under a phase-contrast microscope at successive time points over a 2–144-h period. The cell density of the spheroids was determined by counting the total number of cells liberated. The retrovirally transduced spheroids that were transfected with the GFP reporter construct, the dominant-negative E-cadherin mutant (H-2Kd-E-cad), or its control (H-2Kd-E-cad{Delta}C25) were similarly monitored for disadherence. When indicated, repeat retroviral transfections were carried out. Control xenograft and cell line aggregates derived from E-cadherin-negative (MDA-MB-231 and MDA-MB-468) and E-cadherin-positive (MCF-7, MCF-10, MDA-MB-361, HMS-1, HMS-X, HMS-3, and HMS-3X) sources were used in comparative studies.

Murine Tumorigenicity, Histopathology, Immunolocalization, and Embolic Dissolution Studies.
We had determined the numbers and sizes of pulmonary lymphovascular emboli that MARY-X spontaneously gives rise to in a previous study (5) . In the present study, we investigated whether anti-E-cadherin antibodies can immunolocalize to the pulmonary lymphovascular emboli of MARY-X. In the present study, we also investigated the dissolution effects of tail vein-injected murine monoclonal antibody to E-cadherin, and we used murine IgG1 monoclonal antibody as our control. In the first set of experiments, mice with known pulmonary lymphovascular emboli of MARY-X received a single i.v. tail vein injection of anti-E-cadherin (20 µg/100 µl) or control (murine IgG1) for pulmonary embolic immunolocalization studies. Ten mice in each group received injections. In the second set of experiments, mice with known pulmonary lymphovascular emboli of MARY-X received daily i.v. tail vein injections of anti-E-cadherin (100 µg/100 µl) or control (murine IgG1) for 5 successive days for pulmonary embolic dissolution studies. Ten mice in each group received injections. In the immunolocalization studies, the mice were sacrificed 90 min after receiving the antibodies; in the embolic dissolution studies, the mice were sacrificed 24 h after the last injections. Removed lungs were inflated, embedded in OCT, sectioned, and subjected to standard immunocytochemical protocols. The number and size of pulmonary lymphovascular emboli/unit area of the lung were tabulated with the assistance of digital image analysis, and the presence of E-cadherin and anti-E-cadherin immunoreactivity was determined. The MARY-X spheroids that had been transfected ex vivo with the GFP reporter, the dominant-negative E-cadherin mutant, or control were reinjected into mice, and tumorigenicity, histopathology, and the presence of primary and pulmonary lymphovascular emboli formation were recorded. In all of the mice that were sacrificed, heart ventricular punctures were performed immediately to retrieve venous blood. We obtained 10–100 µl of blood per mouse. This blood was collected and spun down, and serum was obtained. When appropriate, unconcentrated serum (10 µl) was analyzed by Western blot.

Studies of Human IBC Cases.
Twenty-five cases of IBC were retrieved from archival pathological material and studied immunocytochemically with standard protocols. Non-IBC and normal breast tissues were used as controls.

Statistical Analysis.
Experiments were performed with groups of 10 mice, and results were analyzed with standard tests of significance, including the two-tailed Student’s t test and a one-way ANOVA.

Institutional Certifications.
Informed patient consent and approvals from the UCLA Human Subject Protection Committee, the Chancellor’s Animal Research Committee (Animal Research Certification 95-127-11), and the UCLA Institutional Biosafety Committee were obtained before all studies.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The MARY-X Phenotype.
The MARY-X phenotype recapitulated the phenotype of primary IBC by exhibiting florid lymphovascular invasion (local lymphovascular emboli formation; Fig. 1, a–dCitation ) with occasional pulmonary emboli. In MARY-X, the murine component was considerable (~30%). However, we could effectively separate the human carcinoma component from the murine stromal and vascular component in vitro. We called this separation the MARY-X shake or MARY-X spheroids. When reinjected into nude/SCID mice, MARY-X spheroids recapitulated the complete MARY-X phenotype of florid lymphovascular invasion.



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  		Fig. 1. a, lymphovascular invasion in primary IBC is characterized by numerous tumor emboli in lymphovascular spaces (arrows). b, MARY-X exhibits similar lymphovascular invasion (arrows). c, tumor cell-tumor cell adhesion (double arrows) and tumor cell-endothelial cell adhesion (arrow) within a lymphovascular embolus of MARY-X are suggested by this epon-embedded section. d, tumor cell-tumor cell interface of MARY-X illustrates prominent adherens junctions (arrows), presumed sites of E-cadherin and the plaque proteins, {alpha}-catenin and ß-catenin.

 
Comparisons of MARY-X with Noninflammatory Xenografts.
The overexpression of the two adhesion molecules, MUC-1 and E-cadherin, was even more marked in the MARY-X shake and the MARY-X spheroids than in MARY-X, indicating that the source of this overexpression was the human breast carcinoma cells themselves and not the murine stromal or vascular component. Overexpression of MUC-1 had been observed in many different non-IBCs (14) , but overexpression of E-cadherin was a new finding. Although loss of E-cadherin was the rule in some human breast carcinomas and cell lines, other breast carcinomas and cell lines retain strong E-cadherin expression (15, 16, 17, 18, 19) . For this reason, we compared the levels of E-cadherin expression in the MARY-X spheroids with those in E-cadherin-positive and -negative cell lines/xenografts (Fig. 2)Citation . The MARY-X spheroids showed 3–5-fold greater E-cadherin overexpression than other E-cadherin-expressing breast carcinoma cell lines, normal mammary epithelial cells, and myoepithelial cells (Fig. 2a)Citation . {alpha}- and ß-Catenins were similarly overexpressed (Fig. 3a)Citation . We decided to investigate the significance of these observations further.



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  		Fig. 2. a, Western blot depicting overexpression of E-cadherin in MARY-X spheroids even when compared with E-cadherin-expressing normal mammary epithelial cells (HMEC) and breast carcinoma cell lines with strong E-cadherin expression (MCF-7, MCF-10, and MDA-MB-361) and the E-cadherin-negative cell line MDA-MB-231. Normalization of protein loading was done with ß-actin. b, Northern blot depicting increased steady-state E-cadherin mRNA in MARY-X spheroids compared with E-cadherin-expressing normal mammary epithelial cells (HMEC) and myoepithelial cells (HMS-1 and HMS-3) and E-cadherin-expressing breast carcinoma cell lines (MCF-7 and MDA-MB-361). Normalization of RNA loading was done with the 36B4 housekeeping probe. c, Southern blot of MARY-X spheroids reveals no evidence of E-cadherin gene amplification or rearrangement as the basis for the E-cadherin overexpression. Digestion of high molecular weight genomic DNA of MARY-X spheroids, HMECs, MCF-7, MCF-10, and MDA-MB-231 with PstI and probing with E-cadherin cDNA reveal two bands of identical size and comparable intensity in all of the lanes. Digestion with other six cutters yielded similar results.

 


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  		Fig. 3. Intactness and overexpression of the E-cadherin axis, including the catenin plaque proteins, in MARY-X compared with noninflammatory xenografts MDA-MB-231-X and MDA-MB-468-X. a, Western blot of {alpha}-catenin and ß-catenin; b, Western blot of the Triton X-100 solubility assay performed in the presence of Ca2+ in MARY-X compared with PC-3. In MARY-X with a short (10-min) Triton X-100 extraction, the entire E-cadherin axis, including the plaque proteins {alpha}-catenin and ß-catenin, appears in the particulate (P) fraction along with cytoskeletal actin, suggesting that the E-cadherin axis is intact and functionally adhesive in MARY-X. In contrast, in PC-3, which lacks {alpha}-catenin, the E-cadherin is exclusively in the soluble (S) fraction, its axis is not intact, and it is nonadhesive. c, Western blot of the Triton X-100 solubility assay performed in the presence and absence of Ca2+. In MARY-X with a long (30-min) Triton X-100 extraction in the presence of Ca2+, most of the E-cadherin is still in the particulate (P) fraction; in the absence of Ca2+, E-cadherin is predominantly in the soluble (S) fraction, indicative of its dissociation from the cytoskeleton. In PC-3, which lacks {alpha}-catenin, E-cadherin is exclusively in the soluble (S) fraction even in the presence of Ca2+ because it is unbound to the cytoskeleton. d, coimmunoprecipitation of ß-catenin with anti-E-cadherin followed by antiphosphotyrosine Western blot analysis (top left blot). The band at 92 kDa is ß-catenin and is only weakly phosphorylated. Other bands represent other phosphorylated proteins of the E-cadherin complex. Immunoprecipitation with anti-ß-catenin followed by antiphosphotyrosine Western blot analysis (top right blot) gave similar results. Western blot analysis for ß-catenin on the respective anti-E-cadherin (bottom left blot) and anti-ß-catenin (bottom right blot) precipitates confirmed its identity. These Western blots of ß-catenin were used to normalize and compare the degree of tyrosine phosphorylation of ß-catenin in MARY-X with HMS-3X and HMEC. e, not only was the internal E-cadherin/{alpha},ß-catenin axis intact, but external E-cadherin exhibited no evidence of cleavage or shedding from the cell surface because no circulating E-cadherin could be detected in serum in mice harboring MARY-X. In contrast, MUC-1, which was also overexpressed in MARY-X, could be detected in murine serum. Control xenografts expressing E-cadherin (HMS-X) and MUC-1 (COLO-205) are also depicted. f, prominent E-cadherin membrane immunoreactivity is present in MARY-X. This photomicrograph depicts a high-power (x400) magnification of a 2-cm MARY-X xenograft sectioned and incubated with an anti-E-cadherin monoclonal antibody.

 
In MARY-X, E-cadherin Overexpression Is due to Increased Steady-state mRNA.
Whereas increased MUC-1 expression in breast carcinoma including IBC is due to gene amplification (14) , increased E-cadherin expression in MARY-X was due to increased steady-state mRNA levels that also exceeded the levels of other E-cadherin-positive breast carcinoma lines, normal mammary epithelial cells, and myoepithelial cells (Fig. 2b)Citation . This increase in E-cadherin expression was not due to gene amplification or rearrangement (Fig. 2c)Citation . The increase in steady-state mRNA levels was due to increased transcription as determined by a nuclear run-on assay and not to increased mRNA stability (data not shown). This suggested that undefined, upstream genetic abnormalities related to the IBC phenotype were causing the increase in E-cadherin expression.

In MARY-X, the E-cadherin Axis Is Structurally Intact.
The observation that expression of E-cadherin was 3–5-fold greater in MARY-X compared with strong E-cadherin-positive non-IBC cell lines/xenografts and myoepithelial lines/xenografts merited close scrutiny with regard to its significance. E-cadherin expression has generally been thought to be either lost in malignant progression or rendered nonfunctional through structural changes in the E-cadherin molecule (or associated catenin axis) or cleavage by extracellular proteases (15, 16, 17, 18, 19) . As an adhesion molecule, E-cadherin is found in normal cells as part of a membrane complex with {alpha}-catenin and ß-catenin, which form the adherens plaque. The modular architecture of the E-cadherin molecule consists of five repeats in the extracellular domain, a single transmembrane region, and a single intracellular domain linked to the actin filaments of the cytoskeleton via {alpha}- and ß-catenins (20) . The specific homophilic binding capacity of the extracellular domain of E-cadherin (EC1) translates into stable cell adhesion via antiparallel dimers (17) . Disruption of any part of this axis causes nonadhesion rather than adhesion. E-cadherin that has lost either extracellular domains or cytoplasmic domains, the absence of {alpha}-catenin, or abnormally phosphorylated ß-catenin and the absence of this complex’s binding to the actin cytoskeleton all lead to the nonadhesive state. The E-cadherin adhesive state is also Ca2+ dependent; the absence of Ca2+ disrupts the homodimeric interactions between repeat motifs within the EC1 domains of the E-cadherin molecule (15 , 21 , 22) and, in addition, is probably chaotropic for intracellular interactions with the catenins and the actin cytoskeleton. Some cancers may overexpress a structurally defective E-cadherin molecule or exhibit structural disruption of the E-cadherin axis. For example, in the PC-3 prostatic carcinoma cell line, there is expression of E-cadherin but a complete absence of {alpha}-catenin due to homozygous deletions of the {alpha}-catenin gene (23) . This cell line, as a result, has absent E-cadherin axis function despite the presence of E-cadherin. Therefore, the demonstration that E-cadherin overexpression in MARY-X was actually contributing to increased adhesion and the IBC phenotype of lymphovascular emboli formation would require a number of studies demonstrating that the complete E-cadherin axis was structurally intact as well as overexpressed and overfunctioning. The E-cadherin protein, in fact, was fully intact in MARY-X (full 120-kDa size; Fig. 2aCitation ) and therefore had not lost either the extracellular or cytoplasmic domain. Not only were both {alpha}-catenin and ß-catenin present in MARY-X, but they were also overexpressed (Fig. 3a)Citation . Short Triton X-100 solubility extraction in the presence of Ca2+ resulted in coprecipitation of E-cadherin, {alpha}-catenin, ß-catenin, and cytoskeletal actin in the particulate (membrane) fraction in MARY-X, in contrast to the PC-3 cells, where E-cadherin fractionated in the soluble fraction unbound to cytoskeletal actin (Fig. 3b)Citation . Long Triton X-100 solubility extraction of MARY-X or MARY-X spheroids in the absence of Ca2+ resulted in partitioning of the E-cadherin in the soluble fraction, whereas Triton X-100 extraction in the presence of Ca2+ demonstrated that the E-cadherin was bound in the particulate (membrane) fraction (Fig. 3c)Citation . Coimmunoprecipitation studies with anti-E-cadherin antibodies followed by a Western blot phosphotyrosine analysis of ß-catenin revealed that ß-catenin, when normalized for protein, was phosphorylated to the same degree in MARY-X as it was in HMECs and HMS-3X cells and was certainly not overphosphorylated (Fig. 3d)Citation . In fact, a direct immunoprecipitation strategy using anti-ß-catenin instead of anti-E-cadherin yielded similar results in terms of the degree of ß-catenin phosphorylation (Fig. 3d)Citation . Although this direct immunoprecipitation precipitated more ß-catenin than the E-cadherin coimmunoprecipitation, the ratio (ß-catenin precipitated by the ß-catenin method:the ß-catenin precipitated by the E-cadherin method) was no different in MARY-X, HMS-3X, or HMEC (Fig. 3d)Citation . Because the last two of these represent normal myoepithelial and epithelial cells, respectively, cells in which ß-catenin is thought to function only as a plaque protein and not as a transcription factor, we concluded from this data that in MARY-X, ß-catenin functions only as a plaque protein as well. As has been established, free and abnormally phosphorylated catenins can play a central role in signal transduction and the regulation of gene expression; however, in MARY-X we found no evidence of free cytoplasmic or nuclear ß-catenin and no evidence of abnormal phosphorylation. Furthermore, there was no evidence in MARY-X that the E-cadherin molecule was cleaved or shed from the cell surface because no circulating E-cadherin could be detected in murine serum, even when MARY-X tumors were allowed to grow to a large size (2 cm; Fig. 3eCitation ). The overexpression of E-cadherin in MARY-X was also manifested as prominent membrane immunoreactivity (Fig. 3f)Citation . {alpha}- and ß-Catenin showed a similar pattern of membrane immunoreactivity. ß-Catenin did not show nuclear immunolocalization. The increased membrane but not cytoplasmic localization of E-cadherin/{alpha},ß-catenins supported our notion that this axis was structurally intact in MARY-X. These collective results all suggested that the entire E-cadherin axis in MARY-X was intact. However, the demonstration of structural integrity of the E-cadherin axis in MARY-X did not necessarily demonstrate functional integrity, so studies had to be conducted to address this issue.

In MARY-X, the E-cadherin Axis Is Functionally Intact, Overfunctional, and Uniquely Susceptible to Perturbation.
The MARY-X shake produced the appearance of very tight spheroids that grew in suspension culture (Figs. 4Citation and 5Citation ). The MARY-X spheroids (Fig. 5, a and c)Citation disadhered when placed in media lacking Ca2+ or containing anti-E-cadherin antibodies (Fig. 5, b and d)Citation . The disadherence effect of lack of Ca2+ was in evidence after 2 h and was complete after 6 h; the disadherence effect of anti-E-cadherin antibodies was in evidence after 6 h and was complete after 12–24 h, depending on the size of the spheroid (Fig. 5, e and f)Citation . MARY-X spheroids, in contrast to other noninflammatory carcinoma aggregates, formed round, very compact structures (Fig. 4a)Citation with a cell density 5–10-fold greater (103 cells in a spheroid of 120 µm in diameter) than that of other E-cadherin-expressing breast carcinoma cell lines (data not shown). The MARY-X spheroids remained permanently in suspension and did not form monolayers. All other cellular aggregates (both E-cadherin-positive and –negative aggregates) were less round, less compact, and more loosely associated (Fig. 4, b and c)Citation and did not remain permanently in suspension but formed monolayers. The effects of antidisadherence strategies (absent Ca2+, anti-E-cadherin) were more spectacular with the spheroids of MARY-X, causing a dramatic and eventual total release of individual cells in large numbers (Fig. 5, b and d–f)Citation . These antidisadherence maneuvers had no effect with the E-cadherin-negative cell lines; these maneuvers had some antidisadherence effects with the E-cadherin-positive cell lines, but the effects were less spectacular, resulting in a loosening rather than a total disadherence. Furthermore, with the MARY-X spheroids, anti-MUC-1 antibody, anti-CD44 antibody, and control murine IgG1 did not produce disadherence. These findings indicate that specific and increased homophilic E-cadherin interactions cause the compact spheroid formation observed in the MARY-X spheroids in vitro and suggest that these same E-cadherin homophilic interactions mediate the formation of the tumor embolus within lymphovascular spaces in vivo. This unique functional dependency of MARY-X on the overexpression of its E-cadherin/{alpha},ß-catenin axis establishes a special role for the E-cadherin-catenin axis in MARY-X and the inflammatory carcinoma phenotype. Lymphovascular invasion and the inflammatory phenotype are then governed by the gain rather than the loss of E-cadherin function. The pulmonary lymphovascular emboli in MARY-X retained strong expression of E-cadherin, negating the hypothesis that it is the subsequent loss of E-cadherin that contributes to metastasis (Fig. 6a)Citation . Mice with pulmonary lymphovascular emboli of MARY-X, receiving a single i.v. tail vein injection of anti-E-cadherin (20 µg/100 µl) or control (murine IgG1) demonstrated binding of anti-E-cadherin but not the control antibody to the cell surfaces of pulmonary vascular emboli of MARY-X, presumably to sites of membrane E-cadherin (Fig. 6, b and c)Citation . Mice receiving five successive daily injections of anti-E-cadherin (100 µg/100 µl) but not control showed significant dissolution of their pulmonary vascular emboli (P < 0.01; Fig. 6dCitation ).



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  		Fig. 4. a, MARY-X grows as almost perfectly round, compact spheroids of very high density that remain in suspension. In contrast, non-E-cadherin-expressing breast carcinoma cell lines such as MDA-MB-231 (b) and E-cadherin-expressing cell lines/xenografts such as HMS-3X (c) grow as loose aggregates in suspension culture on their way to forming monolayers.

 


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  		Fig. 5. The MARY-X shake is comprised of tight spheroids of tumor cells that correspond in vivo to the tumor emboli within lymphovascular channels. a and c, in vitro these spheroids will grow and enlarge in suspension; b, in the absence of Ca2+, these spheroids undergo disadherence between 2 and 6 h; d, in the presence of anti-E-cadherin antibodies, these spheroids undergo disadherence between 6 and 12 h. Partial (e) and eventual (f) complete disadherence of the spheroids is observed with either manipulation.

 


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  		Fig. 6. MARY-X produces pulmonary lymphovascular emboli spontaneously and sporadically from the primary s.c. xenograft. a, sectioning the lungs and using a primary anti-E-cadherin antibody on the tissue sections demonstrates intense E-cadherin overexpression in these pulmonary emboli (arrows), excluding the possibility that they represent subclones that have lost E-cadherin expression. b, injecting murine IgG1 antibodies into the tail veins of mice with lymphovascular emboli of MARY-X, sectioning the lungs 90 min later, and omitting to add the primary E-cadherin antibody to the tissue sections but adding the chromogenic antibodies reveals no nonspecific staining (arrows). c, injecting anti-E-cadherin antibodies into the tail veins of mice with lymphovascular emboli of MARY-X and repeating all of the steps enumerated in b demonstrates dramatic peripheral circumferential E-cadherin immunoreactivity (arrows) in the lymphovascular emboli at the sites where anti-E-cadherin antibodies would be expected to immunolocalize. d, daily tail vein injections of anti-E-cadherin for 5 days result in a decrease in both the size and number of pulmonary lymphovascular emboli of MARY-X. The number and size distribution of emboli in a representative midlongitudinal cross-section of lung in E-cadherin-treated animals versus the control group are depicted. Ten sections of each lung were counted, and 20 lungs from 10 mice comprised each group. Results are expressed as the mean ± SE.

 
Dominant-negative E-cadherin Transfection Abolishes Spheroid Formation in Vitro and Lymphovascular Emboli Formation in Vivo.
Retroviral transfection with MSCV-GFP successfully delivered this reporter gene to the outer layer of cells within the MARY-X spheroid after 48 h (Fig. 7, a and b)Citation but caused no disadherence. Retroviral transfection with both Babe-H-2Kd-E-cad (Fig. 7c)Citation and Babe-H-2Kd-E-cad{Delta}C25 produced evidence of chimeric gene expression at 72 h, but only Babe-H-2Kd-E-cad produced disadherence (Fig. 7d)Citation beginning at 96 h. Repeated transfections with Babe-H-2Kd-E-cad resulted in complete and total disadherence of the MARY-X spheroids (Fig. 7e)Citation . With all three in vitro approaches (anti-E-cadherin, no Ca2+, and retroviral dominant-negative E-cadherin transfection), disadherence was uniformly observed in virtually every spheroid examined under a phase-contrast microscope. The effect was dramatic and highly reproducible from experiment to experiment. Smaller spheroids (<50 µm) disadhered completely within 12 h, whereas larger spheroids (>100 µm) disadhered completely after 24 h of anti-E-cadherin treatment. The disadherence effect of lack of Ca2+ occurred at an earlier time point, but the same pattern of smaller spheroid disadherence before larger spheroid disadherence was observed. With respect to the retroviral E-cadherin dominant-negative experiments, total disadherence of the smaller spheroids could be achieved with one or two rounds of transfection, whereas with the larger spheroids, four to five rounds of transfection had to be performed. This was because with the larger spheroids, complete retroviral penetration and transfection of all of the cells within the spheroid could not be achieved on a single or even a second attempt. We and others have observed that replication-defective retroviruses will penetrate only a few of the outer layers of cells in a spheroid conformation such as that exhibited by MARY-X spheroids. With only one or two rounds of transfection, the cells on the inside of the larger spheroid do not receive the dominant-negative mutant and therefore do not undergo disadherence. Repeated transfections gradually penetrate the inner layers of the larger spheroid as the outer layers undergo disadherence, and all cells within the larger spheroid eventually receive the dominant-negative mutant and undergo disadherence. With an ex vivo approach, GFP- and H-2Kd-E-cad{Delta}C25-transfected spheroids (nondisadhered) were 100% tumorigenic in nude/SCID mice and maintained the characteristic MARY-X phenotype of florid local lymphovascular emboli and occasional pulmonary lymphovascular emboli, whereas H-2Kd-E-cad-transfected spheroids (disadhered) were only 10% tumorigenic and produced tumors lacking any local or pulmonary lymphovascular emboli.



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  		Fig. 7. a and b, retroviral transfection of the MARY-X spheroid with MSCV-GFP results in successful delivery and expression of this reporter gene at 48 h after transfection, as evident in the outer layer of cells of the spheroid. Successful retroviral delivery and expression of H-2Kd-E-cad results in (c) cytoplasmic and membrane expression of H-2Kd at 72 h and (d) early disruption (membrane blebbing) of the spheroid at 96 h. e, repeated retroviral H-2Kd-E-cad transfection results in disadherence of the spheroid. Retroviral transfection of H-2Kd-E-cad{Delta}C25 produces expression of H-2Kd but no disadherence.

 
Observations with MARY-X Are Applicable to the Inflammatory Phenotype of Human IBC.
In 25 cases of IBC (Fig. 8a)Citation , E-cadherin membrane immunoreactivity was present in all cases and increased in intensity in 90% of these cases within the lymphovascular emboli compared with adjacent normal ducts and acini (Fig. 8, b and c)Citation and with most non-IBCs. In non-IBCs, E-cadherin immunoreactivity was either lost or decreased in some of the cancers, especially in the infiltrating lobular carcinomas, or retained at relatively normal levels, especially in the well-differentiated and moderately well-differentiated ductal carcinomas, but it was not increased as it was in 90% of the inflammatory carcinomas. Not only was the overall immunoreactivity increased in IBC, but the distribution of immunoreactivity was altered. In the normal ducts and acini, the E-cadherin immunoreactivity was confined to the lateral adherens junctions, whereas in the lymphovascular emboli of IBC, it was distributed circumferentially (Fig. 8d)Citation . {alpha}-Catenin and ß-catenin membrane immunoreactivity was also increased in the vast majority of these IBC cases; nuclear ß-catenin immunoreactivity was not observed. Because MARY-X was established from a human IBC and exhibited the phenotype of florid lymphovascular invasion and emboli formation, and because both MARY-X and actual cases of human IBC showed strong E-cadherin and catenin immunoreactivity, we feel that the experimental observations made with MARY-X are applicable to the majority of IBCs arising in humans.



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  		Fig. 8. Two representative cases of human IBC are depicted. a, lymphovascular emboli of the first case (thick arrow) lie adjacent to a normal duct (thin arrow; H&E-stained sample). b, these emboli (thick arrow) show increased E-cadherin membrane immunoreactivity compared with the normal ductal epithelium (thin arrow). c, lymphovascular emboli of the second case (thick arrow) again show increased E-cadherin membrane immunoreactivity compared with the normal epithelium in a focus of adenosis (thin arrow). d, high-power magnification of this immunoreactivity also shows that within the normal epithelium, the E-cadherin immunoreactivity is confined to the lateral borders between cells (the adherens junctions; thin arrow), whereas in the lymphovascular emboli (thick arrow), the E-cadherin membrane immunoreactivity is distributed circumferentially about the cell (thick arrow).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The discovery of the overexpression of E-cadherin in inflammatory carcinoma is exciting because it views the E-cadherin molecule from a different perspective. Loss of epithelial differentiation in carcinomas is thought to be accompanied by higher mobility and invasiveness, which, in turn, are often a consequence of reduced intercellular adhesion (15) . Although this phenomenon may account for certain aspects of the invasion and metastasis process, the phenomenon has really not been observed or examined in the specific step of intravasation or the formation of tumor emboli within lymphovascular spaces. Furthermore, the loss of E-cadherin that has been observed is usually epigenetic rather than genetic. Although loss of heterozygosity and mutation have occasionally been observed, the preponderance of studies indicate reduced gene expression due to decreased transcription (15 , 19) . These studies have all been performed in cell lines (19) and human cancers in situ, but not in foci of lymphovascular invasion per se and not in IBC. It seems reasonable to reconcile our results with those of others on the basis of the fact that E-cadherin expression is reversible and can be either up- or down-regulated, depending on the stage of tumor progression. For example, in a study of cell lines derived from disseminated cancer cells in the bone marrows of patients with solid epithelial tumors, every cell line derived exhibited strong E-cadherin expression (24) . Our studies have shown that E-cadherin is not only overexpressed but is part of an intact and amplified axis of adhesion where {alpha}-catenin and ß-catenin are present and also overexpressed and bound to the actin cytoskeleton. Through homophilic and homodimeric interactions of E-cadherin, this adhesive network mediates the tumor cell-tumor cell aggregates so characteristic of the lymphovascular emboli observed in lymphovascular invasion. Both lack of Ca2+ and anti-E-cadherin antibodies disrupt in vitro the tumor spheroids derived from the MARY-X shake. In a related recent study (25) , the formation of E-cadherin-mediated multicellular aggregates of HSC-3 (human squamous cell carcinoma) survived, proliferated, and exhibited anchorage independence and resistance to apoptosis. These multicellular aggregates required high levels of extracellular Ca2+ and were inhibited with function-perturbing anti-E-cadherin antibody. In that study, cadherin-mediated intercellular adhesions generated a compensatory mechanism that promoted anchorage-independent growth and suppressed apoptosis. The multicellular aggregates in that study in essence had gained a survival benefit for the tumor from the gain rather than loss of E-cadherin function. We feel that the E-cadherin-mediated spheroid formation of MARY-X and its in vivo manifestation of lymphovascular tumor emboli are of similar survival benefit to this cancer. Tumor cell emboli are more efficient at forming metastases than single cells (26) ; tumor cell emboli are hypoxic in their centers and are therefore more resistant to chemotherapy and radiation therapy than single cells (27) ; tumor cell emboli probably exhibit their surface determinants in a different conformational state than those present on single cells that would escape recognition and destruction by adoptive immunotherapy strategies based on immunizations to single cells. For these reasons, we think it advantageous to disrupt these tumor cell emboli.

One cannot help but draw analogies between the MARY-X spheroid (lymphovascular embolus) and the 8-16 cell embryonic blastocyst. It is interesting that increased E-cadherin expression mediates the formation and compaction of the embryonic blastocyst as well (16 , 17) . The blastocyst is destined for implantation in the uterus, just as the lymphovascular embolus is destined for implantation at its metastatic site.

Our anti-E-cadherin strategy was successful both in vitro and in our xenograft model. Using an intravascular approach, we can successfully immunolocalize the pulmonary vascular emboli (micrometastases) with our anti-E-cadherin antibodies and hence potentially increase the sensitivity of detection of micrometastases; furthermore, we can reduce the vascular embolic burden by causing dissolution of established micrometastases, reducing their number as well as their size. Retroviral transfection of a dominant-negative E-cadherin mutant in the spheroids of MARY-X similarly resulted in the disadherence of the spheroids in vitro, a pronounced decrease in tumorigenicity, and an abolishment of both primary and pulmonary lymphovascular emboli of MARY-X. Dominant-negative cadherin mutants provide a powerful approach to the study of E-cadherin and catenin function. Two types of constructs have been used historically, one in which the cadherin cytoplasmic domain is deleted (28) , and one in which the extracellular domain is deleted (10) . The extracellular domain deletion mutants appear to have a more powerful dominant-negative effect than the cytoplasmic domain deletion mutants (10) , and for this reason, we used them in our study.

The finding of the apparent dissolution of pulmonary lymphovascular emboli in mice with our two different approaches (anti-E-cadherin antibodies and retroviral E-cadherin dominant-negative transfection) is an important surrogate end point. Animal survival could not be used as an appropriate end point because the tumor embolic burden in the lungs of untreated animals with MARY-X does not shorten the life span of the animals.

One might ask where cells go after dissolution of the vascular emboli with the in vivo anti-E-cadherin approach, and how the disadherence of the spheroids ex vivo with the dominant-negative E-cadherin approach results in decreased tumorigenicity and absent lymphovascular emboli formation in vivo. The disruption of the overfunctional E-cadherin/{alpha},ß-catenin axis in the MARY-X spheroids achieved by both approaches and resulting in disadherence could also result in direct alterations of the cell cycle, decreases in the availability of autocrine/paracrine growth factors from neighboring cells, and/or induction of apoptotic pathways, all of which could explain the observed phenotypic alterations. Future studies are planned to investigate which of these mechanisms are operating in vivo.

In the present study, we did not perform a mutational analysis of E-cadherin/{alpha},ß-catenin; therefore, we could not exclude the possibility of point mutations of these genes in MARY-X and therefore in IBC. However, based on the numerous structural and functional studies that we did perform in MARY-X that showed an overexpressed E-cadherin/{alpha},ß-catenin axis that is both structurally and functionally intact, we feel that we can conclude that either there are no mutations present in the catenins and E-cadherin or, if there are any, they are not of structural or functional significance.

Our observations concerning the role of an overexpressed E-cadherin-catenin axis in lymphovascular emboli formation certainly do not explain all aspects of the phenotype of lymphovascular invasion. We certainly have not explained how the cells of MARY-X get inside the vascular space to form the lymphovascular embolus in the first place. Questions such as whether the cells of MARY-X have an increased propensity to actively invade pre-existing lymphovascular channels or stimulate the formation of vascular channels around them via mechanisms of angiogenesis or vasculogenesis remain unanswered, and we hope that the MARY-X model will ultimately answer them. With respect to the unique aspects of E-cadherin function in IBC, we have not shown that an overexpressed E-cadherin axis causes the inflammatory phenotype, only that it is required for it. Because previous studies have shown that tumor emboli are more efficient at forming metastases than single cells (26) and that hematogenous metastases are more likely to originate from the proliferation of attached intravascular tumor cells than extravasated ones (29) , targeting the step of intravasation or the formation of lymphovascular emboli would make early metastatic colonies vulnerable. Because significant numbers of cancer patients are thought to have micrometastases or lymphovascular tumor emboli at the time of initial disease presentation, targeting these emboli should improve micrometastasis detection and treatment.


   ACKNOWLEDGMENTS
 
We thank Siavash Kurdistani and David Dawson for critical review of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant CA BCRP 5JB-0104 from the California Breast Cancer Research Program and Grant 99-003173 from the Susan G. Komen Breast Cancer Foundation. Back

2 To whom requests for reprints should be addressed, at Department of Pathology, UCLA School of Medicine, Los Angeles, CA 90024. Phone: (310) 475-0378; Fax: (310) 441-1248; E-mail: sbarsky{at}ucla.edu Back

3 The abbreviations used are: IBC, inflammatory breast carcinoma; SCID, severe combined immunodeficient; HMEC, human mammary epithelial cell; MUC-1, mucin-1; GFP, green fluorescent protein; KSFM, keratinocyte serum-free medium; UCLA, University of California Los Angeles. Back

Received 1/ 3/01. Accepted 4/30/01.


   REFERENCES
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 RESULTS
 DISCUSSION
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