Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture
© Gudjonsson et al; licensee BioMed Central Ltd. 2010
Received: 15 June 2010
Accepted: 7 July 2010
Published: 7 July 2010
Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D) co-culture assay.
Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy.
In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors.
Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.
The human breast gland is composed of two main cellular compartments, the branching epithelium, commonly referred to as the terminal duct lobular units (TDLUs) and the surrounding stroma. The TDLUs consist of an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells separated from the surrounding vascular rich stroma by a basement membrane [1, 2]. The breast stroma is composed of cellular components such as fibroblasts, immune cells and endothelial cells and the extracellular matrix (ECM) as well as entrapped growth factors within the ECM. Breast stroma accounts for roughly 80% of the total tissue volume and exerts a dominant effect on tissue morphogenesis in both the normal and malignant breast gland [3, 4]. Recent studies have underscored the dominant role of breast stroma during epithelial morphogenesis (reviewed in ). Previous studies have shown that normal and malignant breast epithelium can mimic certain aspects of the breast gland histoarchitecture - such as lumen formation and branching morphogenesis - when cultured alone or in co-culture with fibroblasts in three-dimensional matrix [5–7]. The importance of the stroma in the normal and cancerous breast is becoming increasingly appreciated. Boulanger et al. demonstrated that spermatogonial cells underwent a breast epithelial differentiation program upon interaction with the mammary gland microenvironment . Furthermore, Booth et al. showed that breast stroma can redirect neural progenitor cells to produce cellular progeny committed to breast epithelial differentiation . While the functional role of fibroblasts and various extracellular matrix components in breast morphogenesis has been extensively studied [10–12], much less is known of the role of the vascular endothelium in the process. Previously, the role of endothelial cells has been seen as a passive conducting system, transporting oxygen and nutrients to tissues. In recent years however, studies in organogenesis and stem cell research have shown that endothelial cells play a pivotal role in tissue morphogenesis and stem cell niche [13, 14]. In the prostate, vasculature expansion has been shown to precede the expansion of the epithelium following castration and androgen treatment, suggesting the importance of endothelial derived signals or epithelial growth . We have recently shown that microvessels are in close proximity with TDLUs . A detailed description of the epithelial-endothelial interactions in the human breast gland however, has until recently been largely neglected. There are, however, few reports describing in vitro the interaction between endothelial- and epithelial cells in the human breast. Shekhar et al. [17, 18] showed that interaction between endothelial cells and premalignant breast epithelial cells was necessary to allow sufficient proliferation of endothelial cells as well as to induce branching ductal-alveolar morphogenesis and hyperplasia of premalignant cells [17, 18]. In these studies, they used human umbilical vein endothelial cells (HUVEC) instead of organ-specific endothelial cells. It is becoming increasingly recognized that endothelial cells from different organs vary in terms of morphology, marker expression and metabolic properties [19–23] highlighting the need to use organotypic endothelial cells in co-cultures with breast epithelial cells. We have recently improved the isolation protocol and the culture conditions for long term culture of breast endothelial cells (BRENCs) . In this study, we describe a novel three dimensional co-culture system, where primary breast endothelial cells are seeded together with epithelial cells in three dimensional laminin rich gel. We provide evidence that BRENCs can induce proliferation of breast epithelial cells in three-dimensional culture. Furthermore, in co-culture with endothelial cells a subpopulation of luminal epithelial cells form bigger acini/ducts with larger lumens. Seeding normal and cancerous epithelial cells in rBM at clonal dilution with endothelial cells resulted in increased cloning efficacy and larger colony size. This data suggests that endothelial cells in addition to providing nutrient and oxygen to tissues, might be an important microenvironmental factor for normal morphogenesis and cancerous growth in the human breast gland.
Establishment of primary cell culture
Breast tissue specimens were obtained from reduction mammoplasties with informed consent from patients and approval from the National Bioethics Committee of Iceland, Reference number VSNa2001050056. Primary epithelial cells were processed as previously described and cultured on collagen I (Inamed, Gauting, Germany) coated culture flasks (BD Biosciences, Bedford MA) in serum free chemically defined medium (CDM3) [24, 25]. Primary breast endothelial cells were isolated from the organoid supernatant as previously described . Briefly, following centrifugation at 1000 rpm for 5 minutes, capillary organoids were isolated using CD31 coated magnetic beads (Invitrogen). Primary endothelial cells were cultured on collagen coated flasks in EGM-2 medium (Lonza, Basel, Switzerland) supplemented with 30% FBS (Invitrogen), heparin, FGF-2, EGF- VEGF, IGFR3, ascorbic acid and hydrocortisone. FBS concentration was reduced to 5% after 2 passages, this medium will be referred to as EGM5.
Isolation of luminal- and myoepithelial cells
list of antibodies usied in the study
List of cell lines used in the study
Normal like cell lines
Cancer cell lines
F: Fibrocystic disease, RM: Reduction mammoplasty, IDC: Invasive ductal carcinoma, PE: Pleural effusion, AC: Adenocarcinoma, ER: Estrogen Receptor, MES: Mesenchymal
Three-dimensional cell culture
1 × 104 primary epithelial cells were suspended in 300 μl rBM along with 2 × 105 endothelial cells and seeded in a 24-well plate. After incubation at 37°C for 30 minutes the cultures were supplemented with EGM5 medium. Co-cultures were maintained for 14 days and culture medium was changed three times per week.
The epithelial cell lines MCF10A, D382, MCF7, T47-D and MDA-MB-231 (table 2) were seeded at a clonal density (500 cells per gel) with 2 × 105 BRENCs and cultured as described above. Colony size and number was measured on days 5, 9 and 13.
To determine dose effect of endothelial cells in co-culture, BRENCs were seeded at increasing concentrations - ranging from 1,000 cells to 200,000 cells - with 250 MCF10A cells. Colony size and number was measured on day 10.
To prevent direct cell-cell contact, BRENCs were seeded on a 0.4 μm pore size Transwell (TW) filter (Corning Life Sciences, Lowell, MA) and cultured in a 12 well plate for 3 days. Epithelial cells (500 cells per well) were then seeded into 100 μl rBM in a separate plate and placed in an incubator at 37°C for 10 minutes. Confluent BRENCs on TW filters were then transferred on top of the gels. Cultures were maintained on EGM5 medium for 8 days.
Gradient co-cultures were conducted using 7 × 104 BRENCs embedded into 100 μl of rBM and seeded in a 4-well chamber slide. 3 × 103 epithelial cells were seeded in separate 100 μl rBM and placed in the same well as the BRENCs, allowing the gels to merge in the centre, achieving a gradient in the densities of the two cell types. The chamber slide was then placed in an incubator at 37°C for 20 minutes and supplemented with 1 ml EGM5. Cultures were maintained for 10 days.
Gels were frozen in n-hexane at the end of the culture period. For cryosectioning, gels were mounted in O.C.T. medium and sectioned in 9 μm slices in a cryostat. Primary tissue samples were sectioned in 9 μm slices for immunofluorescence and 5 μm slices for DAB staining. Cryostat sections were fixed in methanol at -20°C for 10 minutes and incubated with primary antibodies (table 1) mixed in PBS+10% FBS for 30 minutes. Slides were incubated with isotype specific fluorescent antibodies (Alexa fluor (AF, 488 (green), 546 (red) Invitrogen) mixed in PBS+10% FBS for 30 minutes in the dark. The specimens were then incubated with a fluorescent nuclear counterstain (TO-PRO-3, Invitrogen) and mounted with coverslips using Fluoromount-G (Southern Biotech). Co-culture gels were stained in a similar manner, with an initial blocking step using IF blocking solution  (10% goat serum (Invitrogen) and 1% Goat anti Mouse F(ab')2 Fragments (Invitrogen) in PBS) for 30 minutes. For F-actin staining sections were fixed in 3.7% formaldehyde for 10 minutes and permeabilized with 0.1% Triton-X-100 in PBS for 5 minutes. Slides were then incubated with AF488 conjugated Phalloidin (Invitrogen) for 30 minutes and counterstained with TOPRO-3.
In gel staining of endothelial cells
Endothelial cells were seeded on top or into rBM and cultured for two weeks. Visualization of CD31 was performed after 24 hours and Ac-LDL uptake after two weeks. For CD31 visualization, gels were fixed in methanol at -20°C for 10 minutes. Nonspecific binding was blocked using IF blocking solution for 30 minutes, followed by an overnight incubation with anti CD31 antibody. Secondary AF488 IgG1 antibody was incubated for 2 hours, followed by TOPRO-3 counterstaining for 15 minutes. LDL uptake of embedded endothelial cells was visualized by incubation of Alexa Fluor 488 AcLDL conjugate (Invitrogen) for 5 hours. Immunofluorescence was visualized using a Zeiss LSM 5 Pascal laser scanning microscope. See table 1 for list of antibodies used in this study.
Imaging and statistical analysis
All three-dimensional culture experiments were performed in triplicate for statistical accuracy. Imaging was performed using a Leica DMI3000 microscope and a Leica 310FX imaging system. Populations were compared using an unpaired two-tailed t test. Sample distribution was tested using an F-test. Welch correction was used for t-tests of samples with unequal variation. Graphs were created in Microsoft Excel. Error bars represent the standard error of the mean (SEM) unless stated otherwise.
Breast endothelial cells cultured in rBM are quiescent but metabolically active
BRENCs facilitate growth of primary luminal and myoepithelial cells
Clonal colony formation is enhanced by BRENCs in normal and malignant breast epithelial cell lines
Proliferative signals from BRENCs are delivered via soluble factors
In this paper we have presented a novel three dimensional co-culture system that can be used to analyze cell-cell interaction in heterotypic co-culture. We have demonstrated that isolated primary breast endothelial cells exert a density dependant proliferative effect on epithelial cells when co-cultured. These growth signals are conveyed by soluble factors that disperse from the endothelial cells.
Paracrine interactions are important in the stromal-epithelial crosstalk within the breast gland. Various stromal cells such as fibroblasts produce growth factors and extracellular matrix that influence breast morphogenesis and cancer progression but very little is known about the inductive signals from vascular endothelium. Our data supports the notion that stroma is a vital regulator of tissue morphogenesis and could have a role in cancer progression in the human breast and thus adds a new key player, endothelial cells to this scenario. Studies on epithelial-endothelial interactions in the human breast are lacking. In contrast, studies in mice have shown that angiogenesis precedes the growth of epithelium during puberty and pregnancy when mammary epithelium undergoes a dramatic growth phase . This suggests that endothelium may contribute to the breast morphogenesis. During pregnancy the mammary epithelium and its supporting intra-lobular vasculature rapidly expands to prepare for lactation, resulting in dramatic changes in the microenvironment . The vasculature of the lactating gland is composed of well-developed capillary meshwork enveloping the secretory acini with basket-like structures . During involution, apoptotic cell death returns the breast gland from active to resting state . These morphological changes are also seen during each menstruation cycle where the breast gland undergoes a miniature version of this cycle observed during pregnancy, lactation and involution . Vascular networks exist in most tissues where endothelial cell are in prime position to interact with parenchymal cells such as the epithelial cells. Indeed, recent data from various organs such as liver, pancreas, brain and bone marrow indicate that organ specific endothelial cells are important for fate control of stem cells, organogenesis and tissue maintenance (reviewed in ). Lammert et al. showed that endothelial cells are important for both pancreas and liver development before the endothelium takes up its usual roles . In the nervous system Shen et al.  demonstrated that endothelial cells were enriched in the niche occupied by neural stem cells and that these endothelial cells regulate nerve stem cell proliferation and induce these stem cells to become neurons in vitro. Lai et al.  showed that endothelial cells induced proliferation and functional differentiation in embryonic stem cell-derived neural progenitor cells. In the bone marrow, hematopoietic stem cells are regulated by the vascular niche . In vitro experiments have shown that endothelial cells can provide the right environment for growth and differentiation of megakaryocytes .
In our 3D culture model BRENCS remain proliferatively quiescent but metabolically active and generate a stimulatory microenvironment for epithelial cells. This quiescence enables visualization of proliferating cells over a long time period, as the endothelial cells themselves do not form colonies that would limit visibility in the assay. Improvement of our in vitro three-dimensional cell culture model, for example incorporating fibroblasts is important. Nonetheless, these models remain superior systems to approach the situation found in vivo. Animal models, in particular mice, have provided extensive information regarding mammary development and cancer progression. Human and mouse mammary glands, however, have distinct differences . In addition, an inherent limitation to in vivo models is the lack of information regarding cell-cell and cell-stroma interactions. Monolayer cultures (2D), due to their lack of physiological context are not suitable to study tissue morphogenesis. Breast epithelial cells cultured in 2D fail to form acinar-like structures and lose tissue specific differentiation such as apical-basal differentiation. In contrast, 3D models have proven to be highly relevant when studying the tissue morphogenesis and cancer progression where they add critical elements not found in conventional two dimensional cell culture systems .
The observation that BRENCs stimulate a subpopulation of primary luminal epithelial cells to form colonies with a larger lumen is of interest and could indicate that these epithelial cells were derived from a ductal part of the epithelium rather than the small lobuli-derived acini. Using a Transwell assay we demonstrated that the proliferative effects of BRENCs are delivered by soluble factors. However, these factors do not diffuse effectively through the gel, and are most prominent at close proximity. These factors remain to be identified. Recent studies on endothelial-epithelial interaction by Neiva et al. have identified factors produced by endothelial cells that enhance migration and survival of epithelial cells . The appearance of spindle shaped MDA-MB-231 colonies occurred most often in co-culture with complete mixing of the cell types (Figure 4), whereas in both the Transwell and gradient co-cultures the appearance rates were much lower (not shown). This suggests that even though proliferative effects are conferred, they are not as strong as in close cell-cell contact.
Our co-culture model may help define some of the key components involved in heterotypic cell-cell interactions in normal breast morphogenesis and cancer progression. This model might be relevant for hard to culture cell types such as primary breast cancer cells where one might be able to grow these cells more readily in vitro. This study strengthens the notion that to understand tissue maintenance and tumor progression it is important to gain information on stromal components interacting with the epithelial cells. It is clear from other tissues that endothelial cells play an important role in organogenesis and tissue maintenance. Our data provides important hints that this might also be true in the breast gland. Furthermore, endothelial cells and their interaction with malignant breast cells might be an important factor to take into consideration in breast cancer biology.
List of abbreviations
Breast endothelial cell
Luminal epithelial cell
Reconstituted basement membrane
Terminal duct lobular unit
Chemically defined medium
Phosphate buffered saline.
Grant support was provided by the Icelandic Research Council, Landspitali University Hospital Research Fund, University of Iceland Research Fund, Science and Technology Policy Council-Thematic program in postgenomic biomedicine. European Science Foundation (EuroCORES program, EuroSTELLS), "Göngum saman" a supporting group for breast cancer research in Iceland.
This work has been approved by the National Bioethics Committee of Iceland, Reference number VSNa2001050056.
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