<b>Defining 3-D Culture for Investigating Breast Cancer Progression</b><br/><br/>

Defining 3-D Culture for Investigating Breast Cancer Progression

by Gabriel Benton and Jay George, PhD

Introduction

Figure 1. Evaluation fo various levels of Laminin-1-dependant acinar differentiation: 1) Fully Differentiated acinar structures, b) semi-differentiated spreading structures, and c) undeveloped monolayer. Click here to enlarge.

Over the last decade a new technology has emerged that allows cells to grow in three-dimensional structures. This platform, which mimics more the natural state of cells existing in vivo and provides cells biological surfaces rather than plastics for adherence and growth, has become an invaluable tool in the study of normal cells, as well as in the study of cancer genesis and progression. When grown on biological surfaces under the correct conditions, glandular epithelial cells from breast,^(1,6,8,9,10) prostate,⁽²⁾ and salivary glands⁽⁴⁾ develop into spheroid structures (acini), containing an apoptotic-derived, hollow lumen surrounded by a monolayer of cells. Neoplastic cells, having lost cell cycle control check points, do not organize into acinar structures under identical conditions, but instead, grow in random monolayers.

More recently, the disruption of acinar architecture formation in normal epithelial cells was studied by transfection of cancer genes into MCF-10A mammary epithelial cells.⁽⁵⁾ The MCF-10A cell line was derived from the benign breast tissue from a woman with fibrocystic disease and is phenotypically normal.⁽⁶⁾ This model has been utilized, in conjunction with cell invasion assays, to propose mechanisms for neoplastic transformation of MCF-10A cells as a result of ErbB2 and transforming growth factor beta activation,⁽⁷⁾ autocrine CSF-1R activation,⁽⁸⁾ and Epidermal Growth Factor Receptor or Myc overexpression.⁽⁹⁾ In this study, a system is utilized to examine Basement Membrane Extract (BME) and Laminin-1 for their ability to promote cellular differentiation in a dose-dependent manner.

Figure 2. Cellular differentiation of MCF10A cells in assay media prepared with a) mg/ml, b) 0.4 mg/ml, c) 0.8 mg/ml , d) 1.2 mg/ml, e) 1.6 mg/ml, f) 2mg/ml, g) 2.4 mg/l, h) 2.8 mg/ml, i) 3.2 mg/ml, j) 3.6 mg/ml 3D Culture Matrix Reduced Growht Factor Basement extract. Click here to enlarge.

For this assay, a collagen I coated plate is used to promote cell attachment and growth without inducing differentiation.⁽¹⁰⁾ BME enriched medium provides a complement of basement membrane components to induce differentiation if present in adequate concentrations. Laminin-1 has been implicated as the primary factor driving differentiation,^(3,11,12) and it is evaluated for its ability to induce differentiation relative to BME concentrations. We investigated BME for its ability to induce differentiation in a dose-dependent manner, and a new assay is proposed for investigating this differentiation using a defined medium.

Materials and method

MCF-10A cells were cultured in Growth Medium, DMEM (Gibco, 11995-040), 10 μg/ml Insulin (Sigma, I1882), 20 ng/ml hEGF (Trevigen, 3443-050-01), 100 ng/ml Cholera Toxin (Sigma, C8052), 500 ng/ml Hydrocortisone (Sigma, H0888), 1× Pen/Strep (Cellgro, 30-001-CL), 5% Horse Serum (ATCC, 30-2040), to 80% confluence, and harvested using Trypsin-EDTA (Cellgro, 25-052-CI). Cells were counted with a hemocytometer and diluted to 5,000 cells/ml in the respective assay media, and 500 Μl of diluted cells were added to each well of a 48 well plate (Corning, 3548).

Figure 3. Cell differentiation of MCF10A cells in assay media prepared with a) 0 mg/ml, b) 0.4 mg/ml, c) 0.8 mg/ml, d) 1.2 mg/ml, e) 1.6 mg/ml, e) 2.0 mg/ml 3D Culture Matrix Laminin 1. Click here to enlarge.

3D Culture Matrix Collagen I (Trevigen, 3447-020-01) was diluted to 2.5 mg/ml in PBS (Gibco, 10010-031) and neutralized with 23 μl of 1N NaOH per total ml solution. Forty-eight well plates were coated with 250 μl per well collagen solution and placed at 37 C for one hour to gel.

Assay Medium was prepared with DMEM, 10 μg/ml Insulin, 20 ng/ml hEGF, 100 ng/ml Cholera Toxin, 500 ng/ml Hydrocortisone, 1× Pen/Strep, and 2% Horse Serum. 3-D Culture Matrix BME RGF (Trevigen, 3445-048-01) was added at a final concentration of 0, 0.4 mg/ml, 0.8 mg/ml, 1.2 mg/ml, 1.6 mg/ml, 2.0 mg/ml, 2.4 mg/ml, 2.8 mg/ml, 3.2 mg/ml, and 3.6 mg/ml. 3-D Culture Matrix Laminin-1 (Trevigen, 3446-005-01) was added at a final concentration of 0, 0.4 mg/ml, 0.8 mg/ml, 1.2 mg/ml, 1.6 mg/ml, and 2.0 mg/ml.

Cell suspensions were dispensed in the collagen-coated plate in 500 μl aliquots. Cells were cultured at 37 C in a CO₂ incubator for 6 days, then one well for each condition was fixed with 2% formalin in PBS, and nuclei were stained with SYBR Green (Molecular Probes, S-16158). Collagen I gels containing fixed cells were extracted from the wells and mounted on glass microscope slides for analysis. Cells were visualized with a Nikon E400 microscope using epifluorescence, and images were captured with a Kodak digital camera. To evaluate this data quantitatively, images were analyzed using SigmaPlot software. Fluorescent area (pixels) was converted to percent monolayer; acinar structures were counted for each image; and BME concentrations were converted to Laminin-1 concentration; BME is composed of 60% Laminin-1.

Results

In two independent studies, the self-organization of MCF-10A cells into acinar structures was evaluated as a function of the concentration of (1) growth factor reduced BME and (2) Laminin-1 in culture media. To better understand subsequent figures, Figure 1 distinguishes between fully differentiated acinar structures, semi-differentiated spreading structures, and undifferentiated monolayer.

Figure 4. Quantitative analysis of cellular differentiation by evaluating a) structural spreading and b) number of acini formed. By comparing the square root of acini formed with relative Laminin-1- concentration (c), a linear standard curve may be formulated. Click here to enlarge image a. Click here to enlarge image b. Click here to enlarge image c.

Increasing BME concentration induces acinar structure formation (Figure 2A). Cells cultured in assay medium without BME formed a monolayer-like morphology, void of acinar structures. With the addition of BME to a final concentration of 0.4 mg/ml (Figure 2B), the monolayer morphology is still observed, but acinar structure formation is evident within the monolayer. Increasing concentration of BME results in further differentiation with the presence of both spreading and acinar structures (figure 2c through 2I). Finally, at a concentration of 3.6 mg/ml (figure 2J), all structures are fully differentiated in the acinar conformation.

The concentration for Laminin-1-dependent differentiation differed slightly from that of BME. In the absence of Laminin-1, the MCF-10A cells formed a monolayer-like morphology (Figure 3A); however, the formation of acinar structures is not present until Laminin-1 concentrations reach 0.8 mg/ml (figure 3C). At 1.2 mg/ml, there are still artifacts of the monolayer morphology, in addition to the presence of acinar and spreading structures. As with the BME, increasing the Laminin-1 concentration results in the elimination of monolayer and spreading structures, until only fully differentiated acinar structures remain (figure 3F).

Graphic analysis of these images, as indicated in Figure 4, reveal similar endpoints for both relative area and acinar structure formation; however, multiple inflection points for the BME assay compared to a single curve for the Laminin-1 assay suggest some participation of other membrane components in the intermediate steps. In both assays, fully differentiated acinar structures are apparent at approximately 2 mg/ml Laminin-1. With regard to fluorescent area relative to monolayer formation, inflection points are evident for the BME assay at 0.24 mg/ml, 1.68 mg/ml, and 2.16 mg/ml Laminin-1 concentration while the Laminin-1 assay has one curve between 0.4 mg/ml and 1.6 mg/ml. Similarly, the BME assay has three inflection points at 0.24 mg/ml, 1.2 mg/ml and 1.68 mg/ml Laminin-1 concentration while the Laminin-1 assay has one curve between 0.4 mg/ml and 2.0 mg/ml. It is very interesting to note that both graphs exhibit parallel lines in the two curves between 1.44 mg/ml and 1.68 mg/ml, indicating dependence on Laminin-1 for transition to fully differentiated structures. Furthermore, by plotting the square root of acinar structure formation vs. Laminin-1 concentration, a linear standard curve can be constructed for the Laminin-1 assay.

Since the formation of fully differentiated structures is Laminin-1 dependent and a linear curve can be constructed based on the number of acinar structures, the Laminin-1 assay described herein provides a quantitative assay for evaluating structural disruption in MCF-10A mammary epithelial cells.

About the authors

Gabriel Benton is the Product Development Manager for Cellular Assays, and Jay George is the Chief Technology Officer, both with Trevigen, Inc..

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