Cancer Research Versailles No Abst Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shekhar, M. P. V.
Right arrow Articles by Tait, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shekhar, M. P. V.
Right arrow Articles by Tait, L.
[Cancer Research 60, 439-449, January 15, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Interaction with Endothelial Cells Is a Prerequisite for Branching Ductal-Alveolar Morphogenesis and Hyperplasia of Preneoplastic Human Breast Epithelial Cells: Regulation by Estrogen1

Malathy P. V. Shekhar2, Jill Werdell and Larry Tait

Breast Cancer Program, Karmanos Cancer Institute, [M. P. V. S., J. W., L. T.] and Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201[M. P. V. S., L. T.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although there is experimental evidence supporting the involvement of angiogenesis in the pathogenesis of breast cancer, the exact nature and effects of interaction between human breast epithelial cells (HBECs) and endothelial cells (ECs) have not been described thus far. This approach requires an assay system that permits growth and differentiation of both epithelial and endothelial cells. Here, we report the development of a three-dimensional in vitro culture system that supports growth and functional differentiation of preneoplastic HBECs and ECs and recapitulates estrogen-induced in vivo effects on angiogenesis and the proliferative potential of MCF10AT xenografts. MCF10A and MCF10AT1-EIII8 (referred to as EIII8) cell lines used in this study are normal or produce preneoplastic lesions, respectively. When MCF10A or EIII8 cells are seeded on reconstituted basement membrane (Matrigel), both lines organize into a three-dimensional tubular network of cells; however, tubes produced by EIII8 cells appear multicellular in contrast to unicellular structures formed by MCF10A cells. However, when MCF10A or EIII8 cells are cocultured with human umbilical vein endothelial cells (HUVECs) on Matrigel, rather than interacting with extracellular matrix, the ECs exhibit preferential adherence to epithelial cells. Although both MCF10A and EIII8 cells provide preferential substrate for EC attachment, only EIII8 cells facilitate sustained proliferation of ECs for prolonged periods that are visualized as "endothelial cell enriched spots," which express factor VIII-related antigen. At regions of endothelial-enriched spots, preneoplastic HBECs undergo branching ductal-alveolar morphogenesis that produce mucin, express cytokeratins, and proliferating cell nuclear antigen. The presence of actively proliferating and functional endothelial cells is essential for ductal-alveolar morphogenesis of preneoplastic HBECs because without ECs, the epithelial cells formed only tubular structures. This ability to establish functional ECs and ductal-alveolar morphogenesis is facilitated only by preneoplastic HBECs because normal MCF10A cells fail to elicit similar effects. Thus, a cause-effect relationship that is mutually beneficial exists between EC and preneoplastic HBECs that is critical for generation of functional vascular networks and local proliferative ductal alveolar outgrowths with invasive potential. Both these processes are augmented by estrogen, whereas antiestrogens inhibit these processes. Induction and maintenance of angiogenic phenotype is associated with up-regulation in expression of interleukin 8 and matrix metalloproteinase-2 and estrogen-induced increases in vascular endothelial growth factor and vascular endothelial growth factor receptor 2. This three-dimensional culture model offers a unique opportunity to study endothelial- and epithelial cell-specific factors that are important for ductal-alveolar morphogenesis, angiogenesis, and progression to malignant phenotype.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth and formation of capillary blood vessels or neovascularization is an essential component of solid tumor growth (1 , 2) . Every increase in the tumor cell population must be preceded by an increase in new capillaries that converge upon the tumor, and this angiogenesis has been directly correlated with tumor growth and metastasis (3) . Products derived from both tumor cells and a variety of nonneoplastic mediator systems have been implicated in this vasoproliferative response (2 , 4) . Several growth factors, cytokines, and extracellular matrix molecules have been reported to induce or regulate endothelial cell growth or migration in vitro (5) . These include several well-characterized polypeptide growth factors, proteolytic enzymes, IFN, cyclic nucleotides, prostaglandins, heparin, lowered oxygen tension, histamine and other vasoactive amines, and several low molecular weight endothelial mitogens and chemotactic factors (5) .

VEGF3 (6) or vascular PF (Ref. 7 ; VEGF/PF) is an endothelial-specific mitogen that mediates developmental, physiological, and pathological neovascularization (8) . VEGF has been reported to act as a survival factor, preventing the apoptotic death of microvascular endothelial cells (9 , 10) . The human VEGF gene encodes a dimeric glycoprotein comprising four possible monomers as a result of differential splicing of eight exons that make up the gene product. The four VEGF subtypes are 121, 165, 189, and 206 amino acids in length (11) . The smaller forms are secreted, whereas VEGF189 and VEGF206 are retained close to the membrane of producing cells bound to heparan proteoglycans. Receptors for VEGF, VEGFR-1 (Flt-1), and VEGFR-2 (Flk-1/KDR) bind VEGF, whereas VEGFR-3 (Flt-4) appears to be specific for VEGF-C (11) . Expression of Flk-1/KDR is confined to endothelial cells, accounting for the selective nature of VEGF-induced mitogenesis (11) . VEGF is expressed at high levels in a wide range of tumors and tumor cell lines (12) and is believed to be a key mediator of tumor angiogenesis (13, 14, 15) and the high blood vessel permeability characteristic of tumors (16 , 17) . Expression of VEGF in the uterus has been shown to be rapidly and strongly stimulated by estrogen (18) , suggesting that VEGF mediates the normal, estrogen-induced increase in vascular permeability and blood vessel growth in the uterus. Similarly, expression of VEGF is rapidly induced by E2 in DMBA-induced estrogen-dependent mammary tumors (19) .

Using the MCF10AT1 xenograft model for human proliferative breast disease, we have demonstrated previously that E2 exerts a growth-promoting effect on benign or premalignant ductal epithelium by enhancing the speed of transformation from simple/mild hyperplasia (grades 0/1) to atypical hyperplasia (grade 3) and ductal carcinoma in situ (grade 4; Ref. 20 ). Much of this growth-promoting effect appears to arise from effects of E2 on angiogenesis because lesions from unsupplemented animals are either simple or hyperplastic without atypia and lack angiogenesis (20) . The dramatic increase in growth and advanced histological grades of progression, concomitant with its remarkable effect on angiogenesis, suggests that one of the mechanisms by which E2 acts as a breast cancer promoter could be through its effect on expression of angiogenesis-regulating factors.

The ECM acts locally to modulate the responsiveness of endothelial and mammary epithelial cells to external factors. Besides providing a scaffolding during capillary morphogenesis, the ECM, by virtue of its ability to mediate both biochemical and biomechanical signaling events, has been shown to exert complex local controls on the functions of endothelial cells (21) and growth, differentiation, and apoptosis of normal murine and human breast epithelial cells (22 , 23) . Collagenolytic degradation of endothelial and parenchymal basement membranes is an essential step in the process of tumor invasion and angiogenesis (24) . Proteolysis and interruption of the basement membrane and ECM require the activation of specialized MMPs, the type IV collagenases or gelatinases, which degrade basement membrane collagens types IV and V (25) . Two species of MMPs, the Mr 72,000 species (MMP-2, gelatinase A) and the Mr 92,000 species (MMP-9, gelatinase B), have been cloned and sequenced (25, 26, 27) . Both MMP-2 and MMP-9 are secreted as latent proenzymes and require removal of an 80- and 87-amino acid NH2-terminal domain, respectively, for activation (28 , 29) .

Using a three-dimensional basement membrane assay system, we demonstrate the existence of a direct cause-effect relationship between endothelial and preneoplastic MCF10AT1-EIII8 HBECs that is integral for generation of active angiogenesis and ductal-alveolar morphogenesis, two processes that are regulated by E2 at the molecular and cellular levels. Furthermore, we show that the abilities to undergo ductal-alveolar morphogenesis and establish an active angiogenic process are dependent on the cellular genotype of the breast epithelial cells, because untransformed parental MCF10A breast epithelial cells lack both of these abilities. Finally, our data suggest that active angiogenesis is integral for growth and proliferative potential of "ductal-alveolar structures," which in turn may determine the malignant phenotype.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
The MCF10AT system is a xenograft model of early human breast cancer progression (30) . MCF10AneoT cells are T24 Ha-ras-transformed cells derived from MCF10A human breast epithelial cells (31) . MCF10A cells do not form persistent lesions in immune-deficient mice, whereas MCF10AneoT cells do (30) . MCF10AneoT and lines derived by alternating in vivo transplantation and in vitro culture (MCF10ATn) are collectively known as the MCF10AT system (32) . The lesions formed by lines of the MCF10AT system are composed of a heterogeneous spectrum of ductular tissues with a range of morphology that includes mild to moderate hyperplasia, atypical hyperplasia, carcinoma in situ, moderately differentiated carcinoma, and undifferentiated carcinoma, as well as histologically normal ducts (30) . Thus, the MCF10AT system provides a transplantable, xenograft model of human proliferative breast disease with proven neoplastic potential. These studies used parental MCF10A cells and the following lines of the MCF10AT xenograft model: MCF10AT1 and MCF10AT1-EIII8. MCF10AT1 represents the first transplant generation of the MCF10AT xenograft model (30) . MCF10AT1-EIII8 (referred to as EIII8) cells are breast preneoplastic epithelial cells that were derived from lesions of MCF10AT1 cells arising in E2-supplemented animals (20) and respond to E2 with increased growth in vitro and in vivo. MCF10A and MCF10AT-derived cells were maintained in phenol red-free DMEM-F12 medium supplemented with 0.1 µg/ml cholera toxin, 10 µg/ml insulin, 0.5 µg/ml hydrocortisone, 0.02 µg/ml EGF, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5% horse serum. Charcoal-stripped serum was not used because it reduces the proliferative capacity and/or viability of MCF10A cells, possibly because of removal of essential growth factors. The only sera used routinely were those that were unable to support growth of the estrogen-dependent cell line, MCF-7, indicating absence of biologically significant levels of E2 or other estrogenic compounds.

HUVECs (purchased from American Type Culture Collection) at passage 13 were maintained in Endothelial Serum Free Basal growth Medium (SFM; Life Technologies, Inc., Grand Island, NY) supplemented with 10 ng/ml EGF, 20 ng/ml bFGF, and 10 µg/ml fibronectin. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 in air.

Homotypic and Heterotypic Three-dimensional Basement Membrane Culture of MCF10A, EIII8, and HUVEC Cells.
For homotypic three-dimensional cultures, 10 x 104 cells were seeded as a single-cell suspension in eight-well chamber slides coated with Matrigel (Collaborative Biomedical Products, Bedford, MA) in DMEM-F12-supplemented media for MCF10A cells and its derivatives or in SFM-supplemented media for HUVEC cells. For heterotypic three-dimensional cocultures, 50 x 103 MCF10A, MCF10AT1, or EIII8 cells were mixed with an equal number of HUVEC cells and seeded onto chamber slides coated with Matrigel as described for homotypic cultures. Typically, heterotypic cocultures were performed in SFM supplemented with EGF and bFGF because it allows optimal viability, growth, and three dimensional organization of both HUVEC and MCF10A cells. Heterotypic cocultures and homotypic epithelial cell cultures (MCF10A, MCF10AT1, or EIII8) were routinely maintained up to 3 weeks, and morphological development was analyzed by phase contrast microscopy.

The interaction between EIII8 and HUVEC cells was determined by prelabeling them with the fluorescent cationic membrane tracers, DiI and DiO (Molecular Probes, Inc., Eugene, Oregon), respectively, prior to coculturing. These brightly fluorescent dyes diffuse laterally within the plasma membrane, resulting in uniform staining of the entire cell, and because transfer of these probes between intact membranes is negligible and cytotoxic effects are minimal, they permit long-term cell tracking in the three-dimensional cultures.

Preparation of Conditioned Media.
EIII8 or MCF10A cells (50 x 103) were seeded alone or mixed with an equivalent number of HUVEC cells on Matrigel in SFM media as described above. Cells were incubated for 6 h to attach, and media were replaced with fresh SFM. After appropriate incubation, the culture media from homotypic and heterotypic cocultures were collected, centrifuged to remove debris, and stored at -20°C. After removal of culture media, matrix containing the three-dimensional structures was either solubilized for SDS-PAGE and Western blot analysis or fixed in buffered formalin for evaluation of morphology and distribution of functional markers.

Effect of Conditioned Medium on Proliferation of HUVEC Cells.
HUVEC cells (10 x 103) were plated in SFM supplemented with EGF, fibronectin, and bFGF in 24-well plates. After 8 h of plating, various volumes of unconcentrated conditioned media prepared from homotypic EIII8 or MCF10A cultures, or heterotypic MCF10A-HUVEC or EIII8-HUVEC cocultures, were added. For the inhibition experiments, polyclonal antibodies to human VEGF (recognizes the COOH terminal epitope found in VEGF165, VEGF189, and VEGF206; Oncogene Science, Cambridge, MA), mouse monoclonal antibody to human Flk-1/KDR (epitope not known; Chemicon International, Inc., Tamecula, CA), or polyclonal antibody to human IL-8 (which has <5% cross-reactivity with Gro{alpha}, Groß, and Gro{gamma}; R&D Systems, Minneapolis, Minnesota) was diluted in SFM and added at 10 µg/ml. Appropriate nonimmune IgG was used at 10 µg/ml. Cultures were incubated at 37°C in 5% CO2-95% O2 for 5 days with medium change and supplementation of conditioned media or appropriate antibodies every other day. Cells were released by trypsinization, and viable cells, as demonstrated by trypan blue exclusion, were counted in a hemocytometer. All cell counts were done from triplicate wells, and results were expressed as the mean ± SE from three independent experiments.

Western Blot Analysis.
Analysis of expression of VEGF, IL-8, ER, and Flk-1/KDR proteins was carried out by Western analysis with the specific antibodies. Aliquots of unconcentrated conditioned media or lysates of three-dimensional cultures containing 20 or 40 µg of total protein, respectively, were collected at indicated times of culture, separated by SDS-PAGE on 12.5% (VEGF), 17% (IL-8), or 7% (ER and Flk-1/KDR) polyacrylamide gels (33) , and subjected to Western blot analysis. The following antibodies to human proteins were used: rabbit polyclonal anti-VEGF antibody (recognizes the COOH terminus present in VEGF165, VEGF189, and VEGF206; Oncogene Science), mouse monoclonal anti-ER antibody (Clone 1D5; reacts with the NH2 terminal domain or A/B region of the receptor; Dako Corp., Carpenteria, CA), mouse monoclonal anti-Flk-1/KDR antibody (Chemicon International, Inc.), and goat polyclonal anti-IL-8 antibody (R&D Systems). Immunoreactive bands were visualized by chemiluminescence, and band intensities were quantitated with a Model 300A densitometer (Molecular Dynamics, Sunnyvale, CA).

Effects of Estrogen on Three-dimensional Growth.
To assess growth effects of E2 on homotypic (EIII8) or heterotypic (EIII8-HUVEC) three-dimensional cultures, 50 x 103 EIII8 cells were seeded alone or mixed with an equivalent number of HUVEC cells in the appropriate medium in eight-well chamber slides coated with Matrigel as described above. Slides were incubated overnight to allow attachment of cells to surface and treated with vehicle (0.01% ethanol, v/v), pure antiestrogen ICI 182,780 alone (100 or 1000 nM; a gift from Dr. A. Wakeling, Zeneca Pharmaceuticals, Cheshire, United Kingdom), E2 (0.1, 1, or 10 nM; Sigma Chemical Co., St. Louis, MO), or a combination of 1 nM E2 and 100- or 1000-fold molar excess of ICI 182,780. Cultures were incubated at 37°C for 5 days, after which cell viability was measured with the MTS kit according to the manufacturer’s directions (Promega Corp., Madison, WI). Measurements were made from triplicate sets of wells for each treatment. Background absorbance was corrected by preparing triplicate sets of wells containing only Matrigel ("no cell") and same volumes of culture medium and MTS reagent as in experimental wells. Average absorbances from "no cell" wells were subtracted from sample absorbance values containing cells to yield corrected absorbance, and the results represent the average ± SE of triplicate samples. Each experiment was repeated at least three times. Because the MTS assays performed here differ from routine MTS assays in that they are done on three-dimensional cultures growing in Matrigel rather than monolayers, results of MTS assays were validated by performing direct cell counts of viable cells in the three-dimensional cultures. For this, the medium was removed, wells were rinsed with PBS, and Matrigel was digested with dispase for 2 h at 37°C. The digested material was centrifuged at 4000 x g for 10 min, and the pellet was treated with trypsin to recover single cells from the three-dimensional tubular structures. The number of viable cells was determined by trypan blue exclusion in a hemocytometer. Results were expressed as mean ± SE from three independent experiments.

Morphological Evaluation.
For histological evaluation, three-dimensional cocultures were fixed in buffered formalin and embedded in paraffin, and 4-µm sections were stained with H&E. For immunohistochemical evaluation of mammary epithelial and endothelial cell functional markers, sections were incubated with monoclonal antibodies directed against the following human proteins: pan-cytokeratin 5/6/8/18 is a cocktail of monoclonal antibodies designed to recognize epithelial cells and their tumors (Novocastra Laboratories, Ltd., Newcastle upon Tyne, United Kingdom); muc-1 glycoprotein is a mammary type apomucin also known as milk fat globule membrane antigen (Novocastra Laboratories, Ltd.); cd31 or PECAM-1 (Dako Corp.); factor VIII-related antigen (Dako Corp.); and rabbit polyclonal antibody against PCNA (Dako Corp.). In each instance, negative controls were overlaid with appropriate mouse or rabbit IgG isotype. The slides were overlaid with avidin-biotin conjugated goat antimouse or antirabbit IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA), incubated in peroxidase substrate solution (3,3'-diaminobenzidine), and counterstained by Mayer’s hematoxylin (Sigma Chemical Co.).

Gelatin Zymography.
The activity of gelatinolytic enzymes in conditioned media of MCF10A, EIII8, HUVEC, MCF10A-HUVEC, or EIII8-HUVEC three-dimensional cultures was detected by electrophoresis in 7.5% (w/v) polyacrylamide gels containing gelatin at a final concentration of 0.6 mg/ml. Aliquots of conditioned media containing equivalent amounts of protein were mixed with SDS-sample buffer (33) and electrophoresed under nonreducing conditions. After electrophoresis, the gel was soaked for 10 min in 2.5% Triton X-100/10 mM Tris-HCl (pH 8.0) at room temperature, rinsed, and incubated at 37°C for 16 h in 5 mM CaCl2/50 mM Tris-HCl (pH 8.0). Gels were stained with 0.1% Coomassie Brilliant Blue R250 and destained. The purified precursor form of MMP-2 (pro-MMP-2; gift from Dr. Rafael Fridman, Wayne State University, Detroit, MI) was activated with 1 mM 4-aminophenylmercuric acetate and used as a positive control for activated MMP-2 form (34) .

Statistical Analysis.
Data were analyzed with an ANOVA. Specific differences among treatments were examined using the Student’s t test. Statistical significance was determined using the Student’s t test with P < 0.02 considered as statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Three-Dimensional Basement Membrane Coculture of EIII8 Cells with HUVEC Cells Recapitulate the Phenotypic Characteristics of Preneoplastic Breast Tissue in Vivo: Effects of Estrogen on Three-dimensional Growth of Homotypic Cultures.
When MCF10A cells or ras-transformed MCF10AT1 or EIII8 cells are seeded on Matrigel, within 24 h all of the lines organize into a three-dimensional tubular network of cells that are arranged in a duct-like pattern around a central space (Fig. 1, a–dCitation ). However, profound differences between the duct-like structures of EIII8 cells and MCF10A cells become evident after about 4 days in culture. Tubes produced by EIII8 cells appear multicellular in contrast to the unicellular structures formed by MCF10A cells. The lack of tubular thickening is not attributable to loss of viability of MCF10A cells because these tubular structures are stable and persist indefinitely as those formed by EIII8 cells. Rather, this difference is attributable to the difference in proliferative capacities of EIII8 and MCF10A cells on Matrigel (Fig. 1Citation , compare a and b with c and d), although the two cell lines have similar doubling times (~19 h) on tissue culture plastic (data not shown). Because MCF10AT1 cells exhibit growth characteristics intermediate to those of MCF10A and EIII8 cells, most of our work used EIII8 cells. A major difference between MCF10A and EIII8 cells was the growth-stimulatory effects of E2 on EIII8 three-dimensional structures. Treatment with 1 or 10 nM E2 resulted not only in marked thickening of tubes over those of control cultures but also in the formation of several new "central spaces" and "connecting bridges" (Fig. 2Citation , compare b and d with a). These effects could be blocked by a 100-fold molar excess of the pure antiestrogen, ICI 182,780, indicating the specificity of E2-induced growth effects (Fig. 2cCitation ).



View larger version (130K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Phase contrast morphology of cells in three-dimensional Matrigel culture. a and b, MCF10A cells at 24 h and 10 days, respectively; c and d, EIII8 cells at 24 h and 10 days, respectively; e and f, HUVEC cells at 24 h and 5 days, respectively. Note the difference in contrast on day 10 between MCF10A (b) and EIII8 (d) cultures that is produced by multilayering of epithelium. Magnification, x100.

 


View larger version (114K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Phase contrast morphology of EIII8 cells in three-dimensional Matrigel culture after treatment with estrogen. a, cultures treated with 100 nM ICI 182,780; b and d, cultures treated with 1 or 10 nM E2, respectively; c, cultures treated with a combination of 1 nM E2 and a 100-fold molar excess of ICI 182,780. All cultures represent morphologies at 5 days of culture. Note the remarkable differences in contrast between cultures exposed to ICI 182,780 (a and c) and E2 (b and d). Also, note that addition of ICI 182,780 significantly inhibits epithelial multilayering induced by E2. Treatment with E2 also induces formation of several new central spaces and connecting bridges (b and d). Magnification, x100.

 
In contrast to stable tubular networks formed by the breast epithelial cell lines, similar cultures of HUVECs in Matrigel resulted in formation of tubes within 24 h that remained stable only for an additional 48–72 h and disintegrated by day 5 of culture (Fig. 1, e and fCitation ).

Heterotypic Cultures and Effects of E2 on Growth.
We compared the abilities of normal MCF10A and preneoplastic EIII8 cells to support and maintain endothelial cell growth. When heterotypic cocultures of MCF10A or EIII8 cells were set up with HUVEC cells, the tubular networks observed with the homotypic cultures (Figs. 1Citation and 2Citation ) were preserved. However, although equal numbers of epithelial and endothelial cells were seeded, the tubular frameworks were comprised mainly of epithelial cells, whereas endothelial cells demonstrated preferential affinity to attach and proliferate at certain sites of the tubular framework. These regions, referred to as "endothelial cell enriched spots," became prominent at ~2–3 days of culture (Fig. 3, a, b, d, and eCitation ). This distribution pattern of the two cell types in the three-dimensional structures was confirmed in cocultures of EIII8 and HUVEC cells that were prelabeled with DiI and DiO, respectively (Fig. 4, a and bCitation ). It is interesting to note that although both MCF10A and EIII8 cells provided "soil" for endothelial cell attachment, only EIII8 cells sustained active proliferation of endothelial cells for >3 weeks. This is evident from the remarkable difference in sizes and capillary outgrowths of "endothelial enriched spots" formed between EIII8-HUVEC and MCF10A-HUVEC cocultures (Fig. 3Citation , compare a, b and d, e). The "endothelial cell enriched spots" present on MCF10A-induced tubular networks remained viable for only ~1 week, although the epithelial framework persisted (data not shown). These results suggest major differences in the angiotropic response between normal and transformed human breast epithelial cells, i.e., whereas MCF10A cells switch from a "pro-angiogenic" to an "anti-angiogenic" phenotype, preneoplastic EIII8 cells remained turned-on in the "pro-angiogenic" state. Another interesting feature unique to EIII8-HUVEC three-dimensional cultures and not observed in MCF10A-HUVEC cocultures is the development within 2–3 days of coculture of "branching end buds" or "ductular-alveolar outgrowths" in close proximity with "endothelial enriched spots"(Fig. 3, d and eCitation ; Fig. 4Citation ). Treatment with 1 nM E2 enhanced both growth of "endothelial cell enriched spots" and "ductal-alveolar outgrowths" over those of control cultures, as seen by an increase in size of both "spots" and ductal branches in 5-day-old cocultures (Fig. 3Citation , compare d and e). Although no sera and only phenol red-free media were used, the magnitude and specificity of E2-mediated stimulatory effects on angiotropic response and ductal-alveolar morphogenesis/growth became more obvious when cultures were treated with a combination of 1 nM E2 and a 100-fold molar excess of 4(OH)-tamoxifen (data not shown) or the pure antiestrogen ICI 182,780. By day 5, these cultures showed dramatic inhibition of both endothelial sprouting and associated ductal-alveolar morphogenesis (Fig. 3fCitation ) that disintegrated by days 10–14 of culture (Fig. 3fCitation , inset). These data suggest that the presence of contaminating estrogen in the culture media probably contributed to endothelial cell growth and ductal-alveolar morphogenesis observed in control cultures (Fig. 3dCitation ). It is interesting to note that although addition of E2 to MCF10A-HUVEC cocultures had no significant influence on growth and proliferation of endothelial or epithelial cells (Fig. 3, a and bCitation ), addition of ICI 182,780 abolished formation of "endothelial enriched spots" while maintaining the MCF10A epithelium (Fig. 3cCitation ).



View larger version (82K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Phase contrast morphology of heterotypic EIII8-HUVEC or MCF10A-HUVEC cocultures in Matrigel at day 5. MCF10A-HUVEC or EIII8-HUVEC cultures were treated with vehicle (0.01% ethanol, v/v; a and d, respectively), 1 nM E2, (b and e, respectively), or a combination of 1 nM E2 and a 100-fold molar excess of ICI 182,780 (c and f, respectively). Note the difference in the sizes of "endothelial cell enriched spots" (open arrows) formed on EIII8 and MCF10A epithelia. Also, note the specific association of "ductal-alveolar outgrowths" (arrows) with "endothelial cell enriched spots" in EIII8-HUVEC cultures (d and e), and the specific inhibition by ICI 182,780 of ductal-alveolar growth and endothelial cell enriched spots in EIII8-HUVEC (f) and endothelial cell growth in MCF10A-HUVEC cultures (c). Inset in f, results of exposure to ICI 182,780 for 14 days. Bar, 100 µm.

 


View larger version (124K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Phase contrast micrographs of three-dimensional EIII8-HUVEC cocultures. a and b, cocultures established with DiI- and DiO-prelabeled EIII8 and HUVEC cells, respectively. Note that ductal-alveolar outgrowths are comprised of epithelial cells (a), whereas DiO-labeled endothelial cells are concentrated at this region as a spot (b). Note the presence of immature buds at day 5 of culture (c and d) that have developed into distinct and well-formed buds by day 10 (e and f). The dark areas in close association with alveolar structures represent the endothelial enriched sites. Bar, 100 µm (a, b, c, and e) and 50 µm (d and f).

 
Quantitative Assessment of Estrogen Effects on 3-Dimensional Growth of Heterotypic (EIII8-HUVEC) Cultures.
Because exposure to E2 confers pronounced growth enhancement of EIII8 cells both in homotypic and heterotypic cultures (Figs. 2Citation and 3Citation ), we measured the effects of E2 on cell proliferation in heterotypic three-dimensional cocultures by both MTS and trypan blue dye exclusion assays of dispase-treated cultures. By both assays, E2 elicited a dose-dependent induction of growth at concentrations >1 nM; a 2-fold increase in growth over control cultures was observed with 10 nM E2 (P < 0.01; Fig. 5Citation ). This induction of growth by E2 occurs through the ER-dependent pathway, because the proliferation effects of E2 were abolished by inclusion of 100-fold molar excess of the pure antiestrogen, ICI 182,780 (P < 0.001; Fig. 5Citation ). Only the results of MTS assay are shown in Fig. 5Citation , because results of both assays were in good agreement.



View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Regulation of three-dimensional growth of EIII8-HUVEC cultures by estrogen. Growth was quantitated by MTS assay on day 5 of culture. Control wells received additions of vehicle (0.01% ethanol (v/v). Treatments included E2 at 0.1, 1, or 10 nM; ICI 182,780 at 100 or 1000 nM; and combinations of 1 nM E2 with 100-fold or 1000-fold excess of ICI 182,780. Results obtained from three independent experiments performed in triplicate are expressed as mean; bars, SE. *, doses of compounds that increased cell growth significantly over nonhormone-treated control (P < 0.01). **, doses of ICI 182,780 that significantly decreased cell growth induced by E2 (P < 0.001).

 
Only Conditioned Media from Heterotypic Cocultures Have Endothelial Cell Growth Stimulatory Activity.
To determine whether the stimulatory effects on endothelial cell proliferation observed in EIII8-HUVEC cell cocultures are attributable to soluble factor(s) secreted by EIII8 cells, we examined the effects of conditioned media prepared from homotypic (EIII8 or MCF10A) or heterotypic (EIII8-HUVEC or MCF10A-HUVEC) three-dimensional cultures on HUVEC cell proliferation. Addition of up to 50 µl of conditioned media from homotypic EIII8 (Fig. 6Citation ) MCF10A (data not shown), or heterotypic MCF10A-HUVEC (Fig. 6Citation ) cultures were ineffective in stimulating growth of HUVEC cells over that of control cultures. In contrast, conditioned media from heterotypic EIII8-HUVEC cocultures elicited a significant dose-dependent induction of growth at volumes >5 µl (P = 0.02), and addition of 20 µl of conditioned medium elicited a 3-fold increase in HUVEC cell proliferation over that of control cultures (P < 0.001; Fig. 6Citation ). These results indicate that secretion of growth-stimulatory activity into the culture media requires intimate interaction between epithelial and endothelial cells. Because the cytokine, IL-8 (35, 36, 37, 38) , and the endothelium-specific mitogen, VEGF, have been demonstrated to function as survival and anti-apoptotic factors for endothelial cells, we tested the effects of antibodies to VEGF, its receptor, Flk-1/KDR, and IL-8 on conditioned media-induced growth of HUVEC cells. Because expression of Flk-1/KDR is confined to endothelial cells, effects of neutralization of Flk-1/KDR with its antibody would indicate the selective nature of VEGF-induced mitogenesis. The addition of 10 µg/ml of antibodies to either VEGF or its receptor, Flk-1/KDR, abolished conditioned media-induced HUVEC cell proliferation (P < 0.001), whereas the addition of similar amounts of polyclonal anti-human IL-8 antibody evoked only 40% inhibition of growth (P < 0.02; Fig. 6Citation ). Inclusion of equivalent amounts of the corresponding normal IgG had no effect on conditioned media-stimulated growth (Fig. 6Citation ). This inability of anti-IL-8 antibody to evoke a greater degree of endothelial cell growth inhibition is not attributable to incomplete neutralization of IL-8 in the conditioned media because the addition of higher amounts (up to 25 µg/ml) of IL-8-specific antibody failed to increase the magnitude of growth inhibition (data not shown). Our results suggest that although IL-8 is an important endothelial cell survival factor in our assays, it is not as potent as VEGF. Support for this assumption is also provided by the equally potent inhibition of growth by anti-Flk-1/KDR antibody as by VEGF antibody.



View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Effect of conditioned medium (CM) from MCF10A-HUVEC, EIII8-HUVEC, or EIII8 cultures on proliferation of HUVEC cells. HUVEC cells were treated with 50 or 1–20 µl of unconcentrated CM from EIII8, MCF10A-HUVEC, or EIII8-HUVEC cultures, respectively. Effects of antibodies on CM-induced HUVEC cell proliferation were tested in cultures treated with a combination of 20 µl of CM from EIII8-HUVEC cultures and 10 µg/ml of anti-VEGF, anti-Flk-1, or anti-IL-8 antibodies or an equivalent amount of mouse or rabbit normal IgG. Because no differences in cell numbers were observed between cultures treated with mouse or rabbit normal IgG, the results are grouped together. Results obtained from three independent experiments performed in triplicate are expressed as means; bars, SE. *, cell proliferation that is significantly increased by CM from EIII8-HUVEC cocultures over untreated control (P < 0.001). **, significant decrease in cell proliferation caused by addition of anti-VEGF or anti-Flk-1 antibodies relative to cultures treated with normal IgG (P < 0.001). ***, significant decrease in cell proliferation induced by anti-IL-8 antibody as compared with cultures treated with normal IgG (P < 0.02).

 
Heterotypic EIII8-HUVEC 3-Dimensional Structures Express Epithelial and Endothelial Cell Function Markers.
Results of Figs. 3Citation , 4Citation , and 6Citation demonstrate clearly that in heterotypic EIII8-HUVEC cocultures, not only do both endothelial and epithelial cell populations remain proliferative but that there is an intimate interaction that is mutually beneficial. This is evident from the colocalization of branching "ductal-alveolar outgrowths" with "endothelial cell enriched spots" (Figs. 3Citation and 4Citation ). Histological evaluation of H&E-stained paraffin-embedded sections of three-dimensional cocultures revealed the presence of multilayered epithelium at several regions of the tubular framework with branching end buds (resembling finger-like projections) invading into the surrounding ECM with coincident ECM degradation (Fig. 7ACitation ). To confirm the proliferative potential of the three-dimensional structures and to provide biochemical evidence for breast epithelial and endothelial cell growth and function, we used immunochemistry to examine the distribution of epithelial (cytokeratins, muc-1), endothelial (cd31, factor VIII-related antigen), and proliferation (PCNA) markers in 10-day-old cocultures. Although positive cytoplasmic immunoreactivity to pan-cytokeratins was observed in the majority of cells as expected (Fig. 7CCitation ), muc-1 glycoprotein expression was predominantly localized in the lumen or lumen-forming areas of epithelium (Fig. 7DCitation ). These data confirm that the main tubular network is indeed comprised of breast epithelial cells that in three-dimensional cultures synthesize and secrete mucin, a characteristic feature of epithelial glandular differentiation (39) . In contrast to widespread distribution of cytokeratin immunoreactivity, reactivity to the endothelial cell marker cd31 is restricted to areas on the tubular framework that probably correspond with "endothelial cell enriched spots" (Fig. 7ECitation ). Expression of factor VIII-related antigen is localized to "endothelial cell enriched spots," whereas the epithelial branching end buds in its immediate vicinity and tubular framework are negative (Fig. 7FCitation ). It is interesting to note that the majority of nuclei, particularly in the branching end buds, demonstrate positive nuclear immunoreactivity to anti-PCNA antibody, corroborating the high proli-ferative capacity of the cells in the three-dimensional structures (Fig. 7BCitation ).



View larger version (152K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Formalin-fixed, paraffin-embedded sections of EIII8-HUVEC three-dimensional cultures were either stained with H&E (A) or with antibodies to PCNA (B), cytokeratins (C), muc-1 (D), cd31 (E), or factor VIII (F). Although staining was done on serial sections, the sections look different. This inherent difficulty in obtaining identical serial sections is contributed by the small size and irregular growth of invading branching buds in Matrigel. Note the presence of numerous proliferating cells (B) in the finger-like projections or branching end buds that are invading into the surrounding ECM with coincident ECM degradation (A and B). Arrows and double-headed arrows, degraded ECM and intact ECM, respectively. Note the widespread immunoreactivity to cytokeratins (C) as compared with the localized distribution and membrane staining of cd31-positive (E) and factor VIII-expressing endothelial cells (F) that are denoted by arrows. MUC-1 staining (D) is predominantly localized in the lumen or lumen-forming areas of epithelium (arrows). A, C, E, and F, x10; D, x4; B, x25.

 
Expression of VEGF, IL-8, ER, and Flk-1 Proteins Is Up-Regulated in EIII8-HUVEC Cell Cocultures.
We have shown that unlike normal MCF10A cells, preneoplastic EIII8 cells provide a "good soil" for optimal survival, proliferation, and functioning of endothelial cells. This ability appears to be facilitated, at least in part, by secretion of endothelial cell growth-stimulatory factors, VEGF and IL-8, into the culture media (Fig. 6Citation ). Densitometric analysis of the steady-state levels of VEGF protein in the culture media of homotypic (EIII8, MCF10A, or HUVEC) and heterotypic (EIII8-HUVEC or MCF10A-HUVEC) three-dimensional cultures showed the presence of ~20-fold higher levels of VEGF165 in culture media of EIII8-HUVEC cultures when compared with corresponding fractions from homotypic EIII8 or MCF10A, or MCF10A-HUVEC cultures (Fig. 8ACitation ). Because the antibody used for Western analysis recognizes the COOH terminal epitope that is present in VEGF165, VEGF189, and VEGF206 but not in VEGF121 (11) , alterations in relative levels of VEGF121 have not been determined. Densitometric analysis of VEGF165 in culture media of EIII8-HUVEC three-dimensional cultures at various times of culture revealed that although control samples maintain a constant level of VEGF165 from days 1 to 10 of culture, treatment with 1 nM E2 induces 4–8-fold increase in VEGF165 levels over those of control cultures by days 5 and 10 of culture, respectively (Fig. 8BCitation ). The role of estrogen in regulation of VEGF expression became more apparent when addition of pure antiestrogen, ICI 182,780, at 100-fold molar excess of E2 abolished the E2-stimulated increase in VEGF165 levels, returning them to control levels (Fig. 8BCitation ). Analysis of expression of the cytokine, IL-8, in culture media of HUVEC (Fig. 8CCitation , Lane 1) or MCF10A-HUVEC three-dimensional cultures (Fig. 8CCitation , Lanes 2 and 3) showed the presence of only trace amounts of IL-8. In contrast, levels of IL-8 are significantly up-regulated (>10-fold) in culture media of corresponding 5- and 10-day-old EIII8-HUVEC cocultures (Fig. 8CCitation , Lanes 4 and 7). Interestingly, loss of IL-8 expression in 10-day-old MCF10A-HUVEC cocultures (Fig. 8CCitation , Lane 3) is coincident with loss of viability and stability of endothelial-enriched sites on MCF10A epithelium. In contrast to the inductory effects of estrogen on VEGF expression, E2 does not appear to regulate IL-8 expression (Fig. 8CCitation , Lanes 5 and 8), and addition of 100-fold molar excess of the pure antiestrogen, ICI 182,780, had no effect on IL-8 levels (Fig. 8CCitation , Lanes 6 and 9).



View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. 3-D EIII8-HUVEC cocultures show up-regulated expression of VEGF165, Flk-1, ER, IL-8, and MMP-2. Twenty or 40 µg of protein present in culture media and corresponding matrix fractions, respectively, were analyzed by Western blotting. A, VEGF165 levels were analyzed in culture media of 5-day-old homotypic (EIII8 or MCF10A) or heterotypic (EIII8-HUVEC or MCF10A-HUVEC) cultures or in HUVEC cells at 48 h of culture. B, levels of VEGF165 secreted into culture media from EIII8-HUVEC cocultures were analyzed on days 1, 5, and 10 of culture. Culture media were collected at indicated times from cultures treated with vehicle (0.01% ethanol, v/v), 1 nM E2, or a combination of 1 nM E2 and a 100-fold molar excess of ICI 182,780. C, steady-state levels of Flk-1 and ER or IL-8 were analyzed in matrix and corresponding culture media, respectively, from HUVEC cultures on day 2 (Lane 1), MCF10A-HUVEC (Lanes 2 and 3), or EIII8-HUVEC (Lanes 4–9) cultures on days 5 (Lane 2 and Lanes 4–6) and 10 (Lane 3 and Lanes 7–9). Cultures were treated with vehicle (Lanes 1- 4 and 7), 1 nM E2 (Lanes 5 and 8), or with a combination of 1 nM E2 and a 100-fold molar excess of ICI 182,780 (Lanes 6 and 9). Positions of Flk-1, wild-type ER, Mr 42,000 ER-reactive band, and IL-8 are indicated. D, zymographic analysis of gelatinases secreted from HUVEC (Lane 2), EIII8 (Lane 3), MCF10A (Lane 4), EIII8-HUVEC (Lane 5), and MCF10A-HUVEC (Lane 6) cultures. Lane 1, activated MMP-2 used as positive control. Conditioned media prepared from 5-day-old EIII8, MCF10A, EIII8-HUVEC, or MCF10A-HUVEC cultures or at 2 days of HUVEC cell cultures were analyzed on gelatin-embedded substrate gels. Arrow, position of the active form of MMP-2. The gels are representative of three independent experiments.

 
Analysis of levels of Flk-1/KDR and ER proteins in the corresponding matrix fractions of homotypic (HUVEC) and heterotypic (MCF10A-HUVEC or EIII8-HUVEC) three-dimensional cultures revealed the presence of the endothelial cell-specific receptor, Flk-1/KDR, in HUVEC cells as well as in MCF10A-HUVEC and EIII8-HUVEC cocultures (Fig. 8CCitation ). However, levels of Flk-1/KDR protein are significantly up-regulated (~12-fold) in 5-day-old EIII8-HUVEC cocultures (Fig. 8CCitation , Lane 4) over those expressed in corresponding MCF10A-HUVEC cocultures (Fig. 8CCitation , Lane 2) or homotypic HUVEC cultures (Fig. 8CCitation , Lane 1). Although the levels of Flk-1/KDR protein in control and E2-treated 5-day-old EIII8-HUVEC cultures appear to be equally up-regulated (Fig. 8CCitation , Lanes 4 and 5), addition of ICI 182,780 at 100-fold molar excess of E2 caused ~80% reduction in Flk-1/KDR protein levels (Fig. 8C,Citation compare Lanes 5 and 6). Although the levels of VEGF165 (Fig. 8BCitation ) and IL-8 (Fig. 8CCitation ) proteins remained elevated in 5- and 10-day-old EIII8-HUVEC cultures, Flk-1/KDR protein levels in 10-day-old EIII8-HUVEC cocultures declined by ~75% and were no longer regulated by E2 or ICI 182,780 (Fig. 8CCitation , Lanes 7–9). It is interesting to note that the pattern of expression of Flk-1/KDR protein parallels the expression pattern of ER in EIII8-HUVEC cocultures. HUVEC cells are ER positive (40) , whereas MCF10A cells are ER negative (41) , and as expected, a constant level of wild-type ER protein (Mr 67,000), probably originating from HUVEC cells, is detected both in homotypic HUVECs (Fig. 8CCitation , Lane 1) and heterotypic MCF10A-HUVEC three-dimensional cultures (Fig. 8CCitation , Lanes 2 and 3). As observed in the case of Flk-1/KDR protein expression, levels of wild-type ER protein are enhanced ~25-fold in heterotypic EIII8-HUVEC cocultures relative to those in HUVEC or MCF10A-HUVEC cultures (Fig. 8CCitation , Lanes 4 and 5). Treatment with ICI 182,780 at 100-fold molar excess of E2, resulted in significant reduction in amounts of wild-type ER with concomitant appearance of a Mr 42,000 protein that is immunoreactive with the anti-ER antibody (Fig. 8C,Citation compare Lanes 5 and 6). It is not yet clear whether the Mr 42,000 (Fig. 8CCitation , Lanes 6–9) and the Mr 26,000 bands (Fig. 8CCitation , Lanes 4 and 5) represent variant or truncated forms of ER (42) . In 10-day-old EIII8-HUVEC cocultures, the Mr 67,000 wild-type ER band is either undetectable or present in only trace amounts, whereas the Mr 42,000 band represents the major immunoreactive ER band (Fig. 8CCitation , Lanes 7–9). It is not yet clear whether this shift in ER protein expression from Mr 67,000 to Mr 42,000 species reflects an alteration in hormonal sensitivity or responsiveness of the EIII8-HUVEC three-dimensional cultures.

Expression of an Active Form of MMP-2 Is Enhanced in EIII8-HUVEC Cocultures.
Degradation and remodeling of ECM are essential processes for angiogenesis and involve the MMP/tissue inhibitor of metalloproteinases family of proteases (43, 44, 45) . MMP-mediated matrix remodeling also appears to promote the growth of tumor cells, possibly by facilitating the proliferation and migration of endothelial cells and the neovasculature of tumor tissue (46) . Results of histological evaluation of three-dimensional cultures have shown that in the presence of endothelial cells, EIII8 cells have acquired the ability to invade and degrade the surrounding ECM (Fig. 7ACitation ). MMPs, including MMP-2 (47 , 48) and MMP-9 (49 , 50) , have been shown to play major roles in degradation of ECM in tumor invasion. To identify the gelatinolytic activity of three-dimensional cultures, we performed gelatin zymography of conditioned media from homotypic (HUVEC, EIII8, or MCF10A) and heterotypic (EIII8-HUVEC or MCF10A-HUVEC) cultures. Although the latent or pro-form of MMP-2 (Mr 72,000) was detected in all samples at low levels, the active form of MMP-2 (Mr 62,000) was present at significantly elevated levels only in the conditioned media of EIII8-HUVEC cocultures (Fig. 8DCitation , Lane 5). No difference in the amounts of proteins corresponding to Mr 92,000 (MMP-9) and Mr 96,000 bands was observed (Fig. 8DCitation ). This lack of regulation of MMP-9 levels suggests either the presence of contaminating MMP-9 that is secreted from Matrigel during culture (51) or a less important role for MMP-9 in interactions between EIII8 and HUVEC cells in our system.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Despite wide agreement about the involvement of estrogen in the etiology of breast cancer, there is uncertainty as to its precise role(s) in the biology of breast cancer development. In this report, we describe an in vitro assay system that allows exploration of the interactions between HBECs and endothelial cells on reconstituted basement membrane and show that distinct patterns of angiogenesis permit discrimination between normal (or benign) and premalignant mammary epithelial cells. Results from this study show that estrogen exerts a direct and early effect on mammary carcinogenesis by stimulating proliferation of both endothelial cells (an important stromal component) and premalignant epithelial cells. This assay system is unique from those reported by others in that morphogenesis of ductal-alveolar units resembling terminal ductal lobular units occurs de novo (from single cells) rather than from organoids of primary cultures or simple organization from single cells into spherical structures with acini. When normal MCF10A or preneoplastic EIII8 cells are cocultured with HUVEC cells on a layer of Matrigel, all cell types in both cocultures rapidly organize into interconnected tubes, with the endothelial cells preferring to grow on the epithelium. However, although further differentiation of endothelial cells into complex three-dimensional networks is observed in both MCF10A- and EIII8-HUVEC cocultures, stable and sustained proliferation of endothelial networks is observed only in EIII8-HUVEC cocultures. Although the endothelial cells grow on top of epithelial cells, stable and functional three-dimensional vascular networks (established by factor VIII expression) develop only at specific sites on the EIII8 epithelium. It is not yet clear whether the occurrence of three-dimensional vascular networks at specific sites is induced by a subpopulation of EIII8 cells (with morphological, genetic, or functional alterations) that are committed progenitors of cancers. This is possible because like its parental line, MCF10AT1 (20) , lesions arising from EIII8 cells in immune-deficient mice exhibit a heterogeneous spectrum of advanced histological grades of progression (atypical hyperplasia, carcinoma in situ, and invasive carcinoma) besides simple ducts, with prominent angiogenesis.4 The inability of normal MCF10A cells to sustain stable three-dimensional vascular networks in vitro is consistent with its inability to produce persistent lesions in immune-deficient mice (30) .

The onset of malignancy is an active process that requires reciprocal paracrine interactions between endothelial and tumor cells (52) . Data from our in vitro assay exemplify the need for close and cooperative paracrine interactions at a very early stage between preneoplastic mammary epithelial cells and endothelial cells in the generation of both an angiogenic response and ductal-alveolar morphogenesis. We show that preneoplastic breast epithelial cells facilitate active proliferation and development of stable endothelial cell-derived three-dimensional vascular networks via soluble angiogenic factors such as VEGF and IL-8, and the three-dimensional vascular networks or "sustained angiogenesis" in turn assist in generation of local "ductal-alveolar outgrowths." It must be noted that although "sustained angiogenesis" induces formation of alveolar outgrowths, an indicator of mammary glandular differentiation (established by morphology and muc-1 expression), these outgrowths are not normal because they have tremendous potential to proliferate and invade/degrade the surrounding ECM. Because no productive interactions occur between MCF10A and endothelial cells, our data indicate that the epithelial cell background plays a major role in expression and maintenance of the angiogenic phenotype.

One of the factors that may contribute to the observed differences in angiogenic response between normal MCF10A and preneoplastic EIII8 cells is the presence of activated Ha-ras in the latter (31) . Previous studies have indicated that the angiogenic switch in ras-transformed cells may be physiologically promoted by the tumor microenvironment through induction of the angiogenic mitogen, VEGF (53 , 54) . Although our data support the role of activated ras in sustaining angiogenesis, it is clearly not sufficient because all MCF10AT cells stably express activated ras, yet the development of stable three-dimensional vascular networks occurs only at specific sites on the EIII8 epithelium.

Another important difference between MCF10A and MCF10AT cells that may contribute to observed differences in angiogenic response is the expression of functional wild-type ER in MCF10AT cells (55 , 56) , whereas MCF10A cells are ER negative (41 , 57) . We have shown previously that estrogen enhances preneoplastic progression of MCF10AT1 cells in vivo as lesions of MCF10AT1 (20) , or EIII8 cells4 harvested from estrogen-supplemented animals exhibit rapid growth and advanced histological grades of progression with prominent angiogenesis as compared with simple or moderate hyperplasia without atypia or angiogenesis in control unsupplemented animals (20) . The importance of estrogen in regulation of the angiogenic response in vivo is recapitulated in our in vitro assays, because estrogen specifically stimulates growth of both three-dimensional vascular networks and ductal-alveolar outgrowths, and these processes are blocked or significantly inhibited by the pure antiestrogen ICI 182,780.

Endothelial cells have been found to possess ER (40) , and estrogen has been reported to increase endothelial cell proliferation (58) . In our system, much of the E2/ER-mediated effects on angiogenesis appear to emanate from its stimulatory effects on expression of angiogenesis-regulating factors, VEGF and its receptor, Flk-1/KDR (VEGFR-2). These data are consistent with previous reports that estrogens influence VEGF/PF mRNA expression in the uterus (18) and in the well-vascularized, DMBA-induced, hormone-dependent rat mammary tumors (59) . Fukeda et al. (60) observed that the growth of capillary endothelial cells in the DMBA-induced tumors is estrogen-dependent, and that treatment of DMBA-exposed rats with E2 after ovariectomy prevents tumor necrosis and maintains high rates of endothelial cell proliferation. Similarly, estrogen-induced angiogenesis in rat pituitary tumors is associated with E2-mediated increases in the expression of both ligand and its receptor, VEGF and Flk-1/KDR, respectively, suggesting an important role for estrogen in the initial step of angiogenesis regulation (61) . In contrast to effects of estrogen on VEGF and Flk-1/KDR expression in our system, steady-state levels of the cytokine, IL-8, an important endothelial cell survival factor are unaffected by estrogen and remain elevated throughout the culture period.

It is interesting to note that in 10-day-old cultures, although high levels of IL-8 and VEGF165 proteins are maintained, Flk-1/KDR protein levels are significantly reduced but maintained at a steady level that is unaffected by exposure to either estrogen or ICI 182,780. Although the significance of this is not yet clear, this alteration in regulation of Flk-1/KDR protein is correlated with a dramatic down-regulation in levels of the Mr 67,000 wild-type ER and concomitant appearance of a prominent Mr 42,000 anti-ER immunoreactive protein. It remains to be established whether the Mr 42,000 protein represents a form of variant ER or is simply a protein that is cross-reactive with an NH2 terminus-specific anti-ER antibody. A plethora of studies have reported the detection by reverse transcription-PCR of ER mRNA splice variants in normal and cancerous human breast tissues, the biological and clinical significance of which might be significant but remain to be established because of a lack of evidence for their existence at the protein level (62 , 63) . However, it is intriguing to speculate that such a shift in ER status, coupled with alterations in sensitivity of Flk-1/KDR expression to estrogen and ICI 182,780, may signify a switch from an estrogen-responsive to an estrogen-insensitive phase of angiogenesis and mark the beginning of "new autonomous growth."

Expression of angiogenic activity is a predictable property of many preneoplastic cells and may represent one of the earliest indication that a cell population has become committed to malignancy (64) . Results from our novel in vitro assay system reinforce the concept that like tumor cells, preneoplastic breast epithelial cells actively produce diffusible angiogenic factors and cytokines that directly activate endothelial cells, stimulating them to sprout and initiate development of three-dimensional vascular networks, which in turn induce development of ductal-alveolar outgrowths with capacity to invade and degrade the surrounding ECM. The characteristic expression of activated MMP-2 observed only in preneoplastic EIII8-HUVEC cocultures fortifies the importance of proteolytic enzymes in the release of angiogenic factors sequestered in ECM (65 , 66) . The direct correlation observed between growth and development of vascular networks and ductal-alveolar outgrowths with coincident ECM remodeling suggests that increased gelatinolytic activity secreted by epithelial and/or endothelial cells may facilitate release of angiogenic growth factors locally from ECM.

In summary, we have established a novel, physiologically relevant in vitro model system that not only recapitulates several important aspects of estrogen-induced growth and preneoplastic progression of MCF10AT1 cells in vivo but also demonstrates for the first time the integral role endothelial cells play in ductal-alveolar morphogenesis and proliferation of preneoplastic HBECs. This assay system will provide a unique tool to explore systematically the expression of growth-regulatory molecules that determine epithelium-specific and endothelium-specific requirements for angiogenesis and progression of preneoplastic breast disease.

ACKNOWLEDGMENTS
We thank Dr. Rafi Fridman for purified pro-MMP2, and Dr. A. Wakeling, Zeneca Pharmaceuticals, for generously providing ICI 182,780. We also thank Drs. Gloria Heppner and Michael Tainsky for critical reading of the manuscript and helpful suggestions.


   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 DAMD17-94-J-4427 (to P. V. M. S.) from the United States Army Medical Research and Materiel Command and Grants CA60881 (to P. V. M. S.) and CA22453 (Core grant to the Karmanos Cancer Institute) from the NIH. Back

2 To whom requests for reprints should be addressed, at Breast Cancer Program, Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, MI 48201. Phone: (313) 833-0715, extension 2326/2259; Fax: (313) 831-7518; E-mail: shekharm{at}kci.wayne.edu Back

3 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; PF, permeability factor; DMBA, 7,12-dimethylbenz(a)anthracene; E2, 17ß-estradiol; ECM, extracellular matrix; MMP, matrix metalloproteinase; HBEC, human breast epithelial cell; EGF, epidermal growth factor; HUVEC, human umbilical vein endothelial cell; bFGF, basic fibroblast growth factor; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiO, 3,3'-dilinoleyloxacarbocyanine perchlorate; IL, interleukin; ER, estrogen receptor; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PCNA, proliferating cell nuclear antigen; HUVEC, human umbilical vein endothelial cell. Back

4 P. V. M. Shekhar and D. W. Visscher, manuscript in preparation. Back

Received 6/16/99. Accepted 11/10/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Folkman J., Cotran R. Relation of vascular proliferation to tumor growth. Int. Rev. Exp. Pathol., 16: 207-248, 1976.[Medline]
  2. Gimbrone M. A., Jr., Cotran R. S., Leapman S. B., Folkman J. Tumor growth and neovascularization: an experimental model using the rabbit cornea. J. Natl. Cancer Inst., 52: 413-427, 1974.
  3. Folkman J. What is the evidence that tumors are angiogenesis dependent?. J. Natl. Cancer Inst., 82: 4-6, 1990.[Free Full Text]
  4. Auerbach R. Angiogenesis-inducing factors: a review Pick E. eds. . Lymphokines, 4: 69-84, Academic Press New York 1981.
  5. Klagsbrun M., D’Amore P. A. Regulation of angiogenesis. Annu. Rev. Physiol., 53: 217-239, 1991.[Medline]
  6. Ferrara N., Henzel W. J. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem. Biophys. Res. Commun., 161: 851-858, 1989.[Medline]
  7. Connolly D. T., Olander J. V., Heuvelman D., Nelson R., Monsell R., Siegel N., Haymore B. L., Leimgruber R., Feder J. Human vascular permeability factor. Isolation from U937 cells. J. Biol. Chem., 264: 20017-20024, 1989.[Abstract/Free Full Text]
  8. Leung D. W., Cachianes G., Kuang W. J., Goeddel D. V., Ferrara N. Vascular endothelial growth is a secreted angiogenic mitogen. Science (Washington DC), 246: 1306-1309, 1989.[Abstract/Free Full Text]
  9. Alon T., Hemo I., Itin A., Pe’er J., Stone J., Kesher E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat. Med., 1: 1024-1028, 1995.[Medline]
  10. Watanabe Y., Dvorak H. F. Vascular permeability factor/vascular endothelial growth factor inhibits anchorage disruption-induced apoptosis in microvessel endothelial cells by inducing scaffold formation. Exp. Cell. Res., 233: 340-349, 1997.[Medline]
  11. Neufeld G., Cohen T., Gengrinovitch S., Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J., 13: 9-22, 1999.[Abstract/Free Full Text]
  12. Berse B., Brown L. F., Van de Water L., Dvorak H. F., Senger D. R. Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol. Biol. Cell, 3: 211-220, 1992.[Abstract]
  13. Connolly D. T., Heuvelman D. M., Nelson R., Olander J. V., Eppley B. L., Delfino J. J., Siegel N. R., Leimgruber R. M., Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J. Clin. Investig., 84: 1470-1477, 1989.
  14. Kim K. J., Li B., Winer J., Armanini M., Gillett N., Philips H., Ferrara N. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature (Lond.), 362: 841-844, 1993.[Medline]
  15. Plate K. H., Breier G., Weich H. A., Risau W. Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo. Nature (Lond.), 359: 845-848, 1992.[Medline]
  16. Senge D. R., Galli S. J., Dvorak A. M., Perruzzi C. A., Harvey V. S., Dvorak H. F. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science (Washington DC), 219: 983-985, 1983.[Abstract/Free Full Text]
  17. Yeo K. T., Wang H. H., Nagy J. A., Sioussat T. M., Ledbetter S. R., Hoogewerf A. J., Zhou Y., Masse E. M., Senger D. R., Dvorak H. F. Vascular permeability factor (vascular endothelial growth factor) in guinea pig and human tumor and inflammatory effusions. Cancer Res., 53: 2912-2918, 1983.[Abstract/Free Full Text]
  18. Cullinan-Bove K., Koos R. Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology, 133: 829-837, 1993.[Abstract/Free Full Text]
  19. Nakamura J., Savinov A., Lu Q., Brodie A. Estrogen regulates vascular endothelial growth/permeability factor expression in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. Endocrinology, 137: 5589-5596, 1996.[Abstract]
  20. Shekhar P. V. M., Nangia-Makker P., Wolman S., Tait L., Heppner G. H., Visscher D. W. Direct action of estrogen on sequence of progression of human preneoplastic breast disease. Am. J. Pathol., 152: 1129-1132, 1998.[Abstract]
  21. Polverini P. J. How the extracellular matrix and macrophages contribute to angiogenesis-dependent diseases. Eur. J. Cancer, 32A: 2430-2437, 1996.
  22. Barcellos-Hoff M. H., Aggeler J., Ram T. G., Bissell M. J. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development (Camb.), 105: 223-235, 1989.[Abstract]
  23. Boudreau N., Sympson C. J., Werb Z., Bissell M. J. Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science (Washington DC), 267: 891-893, 1995.[Abstract/Free Full Text]
  24. Liotta L. A., Steeg P. S., Stetler-Stevenson W. G. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell, 64: 327-336, 1991.[Medline]
  25. Liotta L. A., Tryggvason K., Garbisa S., Hart I., Foltz C. M., Shafie S. Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature (Lond.), 284: 67-68, 1980.[Medline]
  26. Liotta L. A., Tryggvason K., Garbisa S., Robey P. G., Abe S. Partial purification and characterization of a neutral protease which cleaves type IV collagen. Biochemistry, 20: 100-104, 1981.[Medline]
  27. Wilhelm S. M., Collier I. E., Marmer B. L., Eisen A. Z., Grant G. A., Goldberg G. I. SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. J. Biol. Chem., 264: 17213-17221, 1989.[Abstract/Free Full Text]
  28. Stetler-Stevenson W. G., Krutzsch H. C., Wacher M. P., Margulies I. M., Liotta L. A. The activation of human type IV collagenase proenzyme. Sequence identification of the major conversion product following organomercurial activation. J. Biol. Chem., 264: 1353-1356, 1989.[Abstract/Free Full Text]
  29. Collier I. E., Wilhelm S. M., Eisen A. Z., Marmer B. L., Grant G. A., Seltzer J. L., Kronberger A., He C. S., Bauer E. A., Goldberg G. I. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. J. Biol. Chem., 263: 6579-6587, 1988.[Abstract/Free Full Text]
  30. Miller F. R., Soule H. D., Tait L., Pauley R. J., Wolman S. R., Dawson P. J., Heppner G. H. Xenograft model of human proliferative breast disease. J. Natl. Cancer Inst., 85: 1725-1732, 1993.[Abstract/Free Full Text]
  31. Basolo F., Elliot J., Tait L., Chen X. Q., Maloney T., Russo I. H., Pauley R., Momiki S., Caamano J., Klein-Szanto A. J., Russo J. Transformation of human breast epithelial cells by c-Ha-ras oncogene. Mol. Carcinog., 4: 25-35, 1991.[Medline]
  32. Dawson P. J., Wolman S. R., Tait L., Heppner G. H., Miller F. R. MCF10AT: a model for the evolution of cancer from proliferative breast disease. Am. J. Pathol., 148: 313-319, 1996.[Abstract]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.[Medline]
  34. Ishibashi M., Ito A., Sakyo K., Mori Y. Procollagenase activator produced by rabbit uterine cervical fibroblasts. Biochem. J., 241: 527-534, 1987.[Medline]
  35. Polverini P. J. The pathophysiology of angiogenesis. Crit. Rev. Oral Biol. Med., 6: 230-247, 1995.[Abstract/Free Full Text]
  36. Bar-Eli M. Role of interleukin-8 in tumor growth and metastasis of human melanoma. Pathobiology, 67: 12-18, 1999.[Medline]
  37. Kumar R., Yoneda J., Bucana C. D., Fidler I. J. 1998. Regulation of distinct steps of angiogenesis by different angiogenic molecules. Int. J. Oncol., 12: 749-757, 1998.[Medline]
  38. Miller L. J., Kurtzman S. H., Wang Y., Anderson K. H., Lindquist R. R., Kreutzer D. L. Expression of interleukin-8 receptors on tumor cells and vascular endothelial cells in human breast cancer tissue. Anticancer Res., 18: 77-81, 1998.[Medline]
  39. Kufe D., Inghirami G., Abe M., Hayes D., Justi-Wheeler H., Schlom J. Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma, 3: 223-232, 1984.[Medline]
  40. Kim-Schulze S., McGowan K. A., Hubchak S. C., Cid M. C., Martin M. B., Kleinman H. K., Greene G. L., Schnaper H. W. Expression of an estrogen receptor by human coronary artery and umbilical vein endothelial cells. Circulation, 94: 1402-1407, 1996.[Abstract/Free Full Text]
  41. Soule H. D., Maloney T. M., Wolman S. R., Peterson W. D., Brenz R., McGrath C. M., Russo J., Pauley R. J., Jones R. F., Brooks S. C. Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res., 50: 6075-6086, 1990.[Abstract/Free Full Text]
  42. Murphy L. C., Dotzlaw H., Leygue E., Douglas D., Coutts A., Watson P. H. Estrogen receptor variants and mutations. J. Steroid Biochem. Mol. Biol., 62: 363-372, 1997.[Medline]
  43. Moses M. A., Sudhalter J., Langer R. Identification of an inhibitor of neovascularization from cartilage. Science (Washington DC), 248: 1408-1410, 1990.[Abstract/Free Full Text]
  44. Mignatti P., Tusboi R., Robbins E., Rifkin D. B. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases. J. Cell Biol., 108: 671-682, 1988.[Abstract/Free Full Text]
  45. Johnson M. D., Kim H-R. C., Chesler L., Tsao-Wu G., Bouck N., Polverini P. J. Inhibition of angiogenesis by tissue inhibitor of metalloproteinase. J. Cell Physiol., 160: 194-202, 1994.[Medline]
  46. Ingher D. E. Extracellular matrix as a solid-state regulator of angiogenesis: identification of new targets for anti-cancer therapy. Cancer Biol., 3: 57-63, 1992.
  47. Gabrisa S., Pozzatti R., Muschel R. J., Saffiotti U., Ballian M., Goldfarb R. H., Khoury G., Liotta L. A. Secretion of type IV collagenolytic protease and metastatic phenotype induction by transfection with c-Ha-ras but not c-Ha-ras plus Ad2–E1a. Cancer Res., 47: 1523-1528, 1987.[Abstract/Free Full Text]
  48. Liotta L. A., Stetler-Stevenson W. G. Metalloproteinases and malignant conversion: does correlation imply causality?. J. Natl. Cancer Inst., 81: 556-557, 1989.[Free Full Text]
  49. Bernhard E. J., Muschel R. J., Hughes E. N. Mr 92,000 gelatinase release correlates with the metastatic phenotype in transformed rat embryo cells. Cancer Res., 50: 3872-3877, 1990.[Abstract/Free Full Text]
  50. Okada Y., Tsuchiya H., Shimizu H., Tomita K., Nakanishi I., Sata H., Seiki M., Yamashita K., Hayakawa T. Induction and stimulation of 92 kDa gelatinase type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumor necrosis factor {alpha}. Biochem. Biophys. Res. Commun., 171: 610-617, 1990.[Medline]
  51. Schnaper H. W., Grant D. S., Stetler-Stevenson W. G., Fridman R., D’Orazi G., Murphy A. N., Bird R. E., Hoythya M., Fuerst T. R., French D. L., Quigley J. P., Kleinman H. K. Type IV collagenase(s) and TIMPs modulate endothelial cell morphologies in vitro. J. Cell Physiol., 166: 235-246, 1993.
  52. Rak J., Filmus J., Kerbel R. S. Reciprocal paracrine interactions between tumor cells and endothelial cells: the "angiogenesis progression" hypothesis. Eur. J. Cancer, 32A: 2438-2450, 1996.
  53. Bouck N. Tumor angiogenesis: role of oncogenes and tumor suppressor genes. Cancer Cells, 2: 179-185, 1990.[Medline]
  54. Bouck, N. Angiogenesis: a mechanism by which oncogenes and tumor suppressor genes regulate tumorigenesis. In: C. C. Benz and E. T. Liu (eds.), Oncogenes and Tumor Suppressor Genes in Human Malignancy, pp. 359–371. Boston: Kluwer Academic, 1993.
  55. Shekhar P. V. M., Werdell J., Basrur V. S. Environmental estrogen stimulation of growth and estrogen receptor function in preneoplastic and cancerous human breast cells. J. Natl. Cancer Inst., 89: 1774-1782, 1997.[Abstract]
  56. Shekhar P. V. M., Chen M. L., Werdell J., Heppner G. H., Miller F. R., Christman J. K. Activation of expression of functional endogenous estrogen receptor in MCF10AT xenografts, a model for early human breast cancer. Int. J. Oncol., 13: 907-915, 1998.[Medline]
  57. Pilat M. J., Christman J. K., Brooks S. C. Characterization of the estrogen receptor transfected MCF10A breast cell line 139B6. Breast Cancer Res. Treat., 37: 253-266, 1996.[Medline]
  58. Johannisson E. Effects of oestradiol and progesterone on the synthesis of DNA and the anti-hemophilic factor VIII antigen in human endometrial endothelial cells in vitro: a pilot study. Hum. Reprod., 1: 207-212, 1986.[Abstract/Free Full Text]
  59. Kaidoh T., Yasugi T., Uehara Y. The microvasculature of the 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor I vascular patterns as visualized by scanning electron microscopy of corrosion casts. Virchows Arch. A. Pathol. Anal. Histopathol., 418: 111-117, 1991.
  60. Fukeda M., Maekawa J., Hosokawa Y., Urata Y., Sugihara H., Hattori T., Miyoshi N., Nakanishi K., Fujita S. Hormone-dependent changes of blood vessels in DMBA-induced rat mammary carcinoma and its regression studied by 3H-thymidine autoradiography. Basic Appl. Histochem., 29: 21-43, 1985.[Medline]
  61. Banerjee S. K., Sarkar D. K., Weston A. P., De A., Campbell D. R. Over expression of vascular endothelial growth factor and its receptor during the development of estrogen-induced rat pituitary tumors may mediate estrogen-initiated tumor angiogenesis. Carcinogenesis (Lond.), 18: 1155-1161, 1997.[Abstract/Free Full Text]
  62. Dowsett M., Daffada A., Chan C. M., Johnston S. R. Oestrogen receptor mutants and variants in breast cancer. Eur. J. Cancer, 33: 1177-1183, 1997.
  63. Tonetti D. A., Jordan V. C. The role of estrogen receptor mutations in tamoxifen-stimulated breast cancer. J. Steroid Biochem. Mol. Biol., 62: 119-128, 1997.[Medline]
  64. Folkman J. Tumor angiogenesis. Adv. Cancer Res., 43: 175-203, 1985.[Medline]
  65. Vlodavsky I., Fuks Z., Ishai-Michaeli R., Bashkin P., Levi E., Korner G., Bar-Shavit R., Klagsbrun M. Extracellular matrix-resident basic fibroblast growth factor: implications for control of angiogenesis. J. Cell Biochem., 47: 167-176, 1991.
  66. Vlodavsky I., Bar-Shavit R., Ishai-Michaeli R., Bashkin P., Fuks Z. Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism?. Trends Biochem. Sci., 16: 268-271, 1991.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
M. P.V. Shekhar, S. Santner, K. A. Carolin, and L. Tait
Direct Involvement of Breast Tumor Fibroblasts in the Modulation of Tamoxifen Sensitivity
Am. J. Pathol., May 1, 2007; 170(5): 1546 - 1560.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
E. Rubin, X. Wu, T. Zhu, J. C.Y. Cheung, H. Chen, A. Lorincz, R. K. Pandita, G. G. Sharma, H. C. Ha, J. Gasson, et al.
A Role for the HOXB7 Homeodomain Protein in DNA Repair
Cancer Res., February 15, 2007; 67(4): 1527 - 1535.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Qiao, P. Shapiro, M. Fosbrink, H. Rus, R. Kumar, and A. Passaniti
Cell Cycle-dependent Phosphorylation of the RUNX2 Transcription Factor by cdc2 Regulates Endothelial Cell Proliferation
J. Biol. Chem., March 17, 2006; 281(11): 7118 - 7128.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
S. E. Bulun, Z. Lin, G. Imir, S. Amin, M. Demura, B. Yilmaz, R. Martin, H. Utsunomiya, S. Thung, B. Gurates, et al.
Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Disease: From Bench to Treatment
Pharmacol. Rev., September 1, 2005; 57(3): 359 - 383.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
M. P.V. Shekhar, P. Nangia-Makker, L. Tait, F. Miller, and A. Raz
Alterations in Galectin-3 Expression and Distribution Correlate with Breast Cancer Progression: Functional Analysis of Galectin-3 in Breast Epithelial-Endothelial Interactions
Am. J. Pathol., December 1, 2004; 165(6): 1931 - 1941.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
J. E. Maglione, E. T. McGoldrick, L. J.T. Young, R. Namba, J. P. Gregg, L. Liu, D. Moghanaki, L. G. Ellies, A. D. Borowsky, R. D. Cardiff, et al.
Polyomavirus middle T-induced mammary intraepithelial neoplasia outgrowths: Single origin, divergent evolution, and multiple outcomes
Mol. Cancer Ther., August 1, 2004; 3(8): 941 - 953.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Tei, N. Kawakami-Kimura, O. Taguchi, K. Kumamoto, S. Higashiyama, N. Taniguchi, K.'i. Toda, R. Kawata, Y. Hisa, and R. Kannagi
Roles of Cell Adhesion Molecules in Tumor Angiogenesis Induced by Cotransplantation of Cancer and Endothelial Cells to Nude Rats
Cancer Res., November 1, 2002; 62(21): 6289 - 6296.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. A. Porter, I. E. Krop, S. Nasser, D. Sgroi, C. M. Kaelin, J. R. Marks, G. Riggins, and K. Polyak
A SAGE (Serial Analysis of Gene Expression) View of Breast Tumor Progression
Cancer Res., August 1, 2001; 61(15): 5697 - 5702.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. P. V. Shekhar, J. Werdell, S. J. Santner, R. J. Pauley, and L. Tait
Breast Stroma Plays a Dominant Regulatory Role in Breast Epithelial Growth and Differentiation: Implications for Tumor Development and Progression
Cancer Res., February 1, 2001; 61(4): 1320 - 1326.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shekhar, M. P. V.
Right arrow Articles by Tait, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shekhar, M. P. V.
Right arrow Articles by Tait, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online