Three-dimensional culture models of normal and malignant breast epithelial cells

Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype1. Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of laminin-rich extracellular matrix (lrECM)2. These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form disorganized, proliferative and nonpolar colonies3. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane–like gels4–7. Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype ex vivo. We describe here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for molecular analyses. The procedure below describes the 3D ‘embedded’ assay, in which cells are cultured embedded in an lrECM gel8 (Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells9. For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D ‘on-top’ assay, in which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 (Fig. 1 and Fig. 2). Figure 1 Breast epithelial cell morphology in different culture conditions. (a) Schematic of nonmalignant breast epithelial cells grown as a monolayer on tissue-culture plastic (left), in the 3D embedded assay (middle) and in the 3D on-top assay (right). (b) Phase-contrast ... Figure 2 Breast cancer cell lines in 3D culture. (a–c) Phase-contrast images (left) and confocal cross-sections of Phalloidin staining of F-actin (right) of BT-474 (a), SK-BR-3 (b) and MDA-MB-231 (c) cell lines grown for 4 d in the 3D on-top assay. In ... Materials Reagents Engelbreth-Holm-Swarm extracellular matrix extract (EHS), growth factor–reduced (Matrigel, BD Biosciences or Cultrex BME, Trevigen) Diaminophenylindole (DAPI) Hard-set mounting medium (VECTASHIELD HardSet, Vector Laboratories or ProLong Gold, Invitrogen) Immunofluorescence (IF) buffer: 0.2% Triton X-100, 0.1% BSA (radioimmunoassay grade), 0.05% Tween 20 in PBS (pH 7.4; sterilized, 0.22 μm filter); for long-term storage, add 7.7 mM NaN3 IF blocking solution: 10% goat serum, 1% goat F(ab')2 anti-mouse immunoglobulin G (IgG; Caltag) in IF buffer PBS: 130 mM NaCl, 13 mM Na2HPO4, 3.5 mM NaH2PO4 (pH 7.4) PBS-EDTA: 5 mM EDTA, 1 mM NaVO4, 1.5 mM NaF in PBS PBS-glycine: 100 mM glycine in PBS Procedure Culturing cells in 3D 1 Thaw EHS at 4 °C overnight. 2 Coat prechilled culture surface (for example, dish or well) with a thin layer of EHS. Slowly pipette the appropriate volume of “EHS coat” (Table 1) directly onto surface and spread evenly with a pipette tip or plunger of a 1-ml syringe for smaller areas, or cell lifter for larger areas. Incubate for 15–30 min at 37 °C to allow the EHS to gel (but do not let it overdry). Table 1 Suggested volumes for 3D culture 3 Trypsinize cells from a monolayer to a single-cell suspension. Use cells that are healthy and not more than 75% confluent. 4 Aliquot cells to be plated into a 1.5 ml microcentrifuge tube. To perform drug response assays in 3D cultures7,10,11 (Fig. 3 and Supplementary Videos 1 and 2 online), compounds may be added to the culture in one of two manners: Figure 3 3D drug response assay. (a–c) HMT-3522 S1 (a), HMT-3522 T4-2 (b) and HMT-3522 T4-2 treated with an EGFR inhibitor, AG1478 (c) were cultured in the 3D on-top assay for 4 d. Colonies were then extracted and immunostained against α6 integrin ... Small-molecule inhibitors: add to medium when cells are plated in Step 6 of the main protocol and Step b of the 3D on-top protocol (Box 2). Include the compound in all media changes for the duration of the culture. Blocking antibodies: mix with cells before plating in Step 5 of the main protocol and Step b of the 3D on-top protocol (Box 2) to ensure complete exposure of the cells to antibodies. Include the antibody in all media changes for the duration of the culture. 5 Gently pellet the cells by centrifugation at ∼115g and, on ice, resuspend the cells into the appropriate volume of EHS (Table 1). The number of cells to be plated per milliliter of EHS depends on the growth properties of the cell line and may need some optimization, but we recommend the following ranges: for nonmalignant cells, 0.85 × 106 cells/ml; for malignant cells, 0.50–0.60 × 106 cells/ml. 6 Pipette the mixture of cells and EHS onto the precoated surface. Incubate 30 min at 37 °C to allow EHS to gel. Add the appropriate volume of medium (Table 1). 7 Maintain the culture for 10 d, changing medium every 2 d. BOX 1 EHS USER'S GUIDE EHS is available commercially from several sources, including BD Biosciences (Matrigel) and Trevigen (Cultrex Basement Membrane Extract). EHS can also be prepared directly from EHS tumors grown as xenografts22. As EHS is a biological product, its components and properties, including ECM protein and growth factor concentrations, endotoxin levels and stiffness, vary between lots. It is therefore desirable to perform a series of experiments using the same lot number to minimize variation introduced by slight differences in the properties of the EHS. It is also important, when a new lot is obtained, to test whether it is appropriate for culture by performing a side-by-side comparison with cells grown in EHS from a previous lot. We routinely evaluate new lots for the typical and appropriate morphogenesis of nonmalignant and malignant cells along with the expression of several markers of interest. We also exclusively use growth factor–reduced EHS as much of our work is done in the absence of serum and under defined medium conditions. Depending on the nature of the cell type and parameters to be measured, you may develop your own strategy for validation and arrive at your own EHS preferences.