Three-Dimensional Cultures of Normal and Malignant Human Breast Epithelial Cells to Achieve in vivo-like Architecture and Function
Apicobasal polarity, properly positioned cell-cell contacts, and attachment to basement membrane are fundamental characteristics of simple glandular epithelia, such as the mammary gland. The development and maintenance of this polarized structure are essential for the formation of tissue architecture, control of proliferation, and the differentiated function of epithelial cells (Roskelley et al., 1995). Loss of architectural intactness and polarity is one of the pathological hallmarks of epithelial carcinoma (Bissell and Radisky, 2001). Although traditional studies of epithelial cells grown as a monolayer on tissue culture plastic remain a powerful tool to dissect and understand the molecular events of signaling machineries, tissue culture plastic does not recapitulate the microenvironment or morphology of glandular epithelium in vivo. Animal models have provided us with invaluable insights, which have led to a greater understanding of the events involved in mammary morphogenesis and tumorigensis in vivo, and aid in the translation from basic cellular research into clinical application. However, the complexity of animal model systems prevents us from precisely pinning down the specific biochemical and cell biological pathways involved in mammary morphogenesis and tumor formation. Therefore an in vitro cell-based model system that provides epithelial cells with an in vivo-like microenvironment, recapitulates both the three-dimensional (3D) organization and multicellular complexity, and is conducive to systematic experimental pertubation is optimal to bridge the gaps between epithelial monolayer cultures and animal models.
We have developed an assay in which primary or phenotypically normal human breast epithelial cells cultured in a laminin-rich basement membrane (lrBM) undergo a three-dimensional reorganization to form structures that mimic in vivo acinar structures in culture (Petersen et al., 1992). LrBM, available commercially as Matrigel, is a mixture of basement membrane proteins that include ~80% laminin, ~10% type IV collagen, entactin, and ~10% heparin sulfate proteoglycan, derived from Engelbreth-Holm-Swarm mouse tumor (Orkin et al., 1977; Kleinman et al., 1986, 1987). The malleability of this specialized gel and its ability to signal through integrins, as well as other ECM receptors, induce changes in the cells, which allow them to withdraw from the cell cycle, organize, and form a central lumen. The entire acinar structures achieve apical basal polarity, and cells within the acini express tissue-specific genes, processes that are not easily accomplished by the same cells cultured on conventional tissue-culture plastic. We refer to the assay as 3D BM assay. The following are advantages of 3D BM assays in studying normal and malignant cells. (1) Because the cells are able to form tissue-like structures, mechanisms of tissue specificity can be studied ex vivo. These cultures are amenable to a variety of perturbations and manipulations that can be used to understand how cells may signal in vivo. (2) The assay allows breast tumor cells to be distinguished easily from their nonmalignant counterparts because the tumor cells fail to stop proliferating and do not become organized into tissue-like structures (Petersen et al., 1992; Weaver et al., 1996). (3) Addition of specific signaling inhibitors or blocking antibodies or delivery of genes to the tumorgenic cells can be used to analyze signaling pathways involved in the acquisition of the nontumorgenic phenotype (Weaver et al., 1997; Wang et al., 1998, 2002). (4) The 3D BM cultures can be used to understand the novel roles of oncogenes and tumor supressors genes (Howlett et al., 1994; Spancake et al., 1999; Muthuswamy et al., 2001; Debnath et al., 2002). (5) These assays can assess the response of tumorigenic vs nontumorigenic cells to potential therapeutics and unravel novel mechanisms (Weaver et al., 2002). (6) The assays (3-10 days) can be performed rapidly and in a high throughput manner relative to costly animal studies. (7) Last but not least, these assays allow us to study and manipulate human cellular responses in physiological context. For a brief review of the results obtained with these models and studies in rodents cells in 3D cultures and in vivo, see Bissell et al. (1999). For a history of development of 3D cultures in general see Schmeichel and Bissell (2003). For a review of more complex organotypic cultures, see Gudjonsson et al. (2003).
II. MATERIALS AND INSTRUMENTATION
Primary breast luminal cells from human reduction mammoplasty (Petersen et al., 1992) as well as human mammary epithelial cell lines have been successfully cultured utilizing the 3D BM assay. These include the following.
The HMT3522 progression series (Briand et al., 1987) consists of immortal human mammary epithelial cells originally isolated from fibrocystic breast tissue and includes the phenotypically normal S1 cells, as well as their tumorigenic derivative T4-2 cells, which were selected for their ability to grow in the absence of EGF (Briand et al., 1996; Weaver et al., 1996).
This article focuses mainly on the HMT3522 series but provides examples of the morphology of a few other nonmalignant cell lines, such as 184B5 (Walen and Stampfer, 1989); for a description of these series, see http://www.lbl.gov/~mrgs/review.html and MCF10A (Soule et al., 1990). For further information and culture conditions, see http://www.atcc.org /ATCC Number: CRL-10317.
A. Culture Media Composition
Media for the HMT3522 progression series of human mammary epithelial cells are composed of DMEM/F12 media (GibcoBRL Cat. No. 12400-024) supplemented with insulin (Sigma Cat. No. 1-6634), human transferrin (Sigma Cat. No. T-2252), sodium selenite (Collaborative Research Cat. No. 40201), estradiol (Sigma Cat. No. E-2758), hydrocortisone (Sigma Cat. No. H-0888), prolactin (Sigma Cat. No. L-6520), and epidermal growth factor (EGF) (Roche Cat. No. 855731) as described in Briand et al. (1987) and Blaschke et al. (1994). Concentrations and stability are outlined in Table I.
B. Additional Reagents Required for Culture and Manipulation in Three-Dimensional Culture
Vitrogen (collagen I, Cohesion Technologies) can be made from rat tail collagen, phosphate-buffered saline (PBS) (any vendor), 0.25% trypsin-EDTA (any vendor), soybean trypsin inhibitor (Sigma Cat. No. T6522), trypan blue (Sigma Cat. No. T8154), growth factor reduced Matrigel (Becton-Dickenson-Collaborative Research Cat. No. 354230), AIIB2, blocking antibody against 131 integrin (Sierra BioSource Inc. Cat. No. SB959), LY294002 (Cell Signaling Technologies Cat. No. 9901), Mab225 (Oncogene Research Cat. No. GR13L), PP2 (Calbiochem Cat. No. 529573), PD98059 (Calbiochem Cat. No. 513000) and tyrphostin AG1478 (Calbiochem Cat. No. 658552). All other equipment and supplies can be purchased from a variety of vendors.
C. Minimal Equipment Required for Cell Culture
Hemacytometer, low-speed centrifuge, inverted light microscope equipped with 4, 10, 20, and 40× objectives, pipetman p20, p200, pl000, pipette aid, biological safety cabinet, 37°C, 5% CO2 humidified culture incubator, air-tight plastic container, 37~ water bath.
D. General Tissue Culture Supplies
Tissue culture plasticware including T75 flasks, 4-, 6-, 24-, 48-, and 96-well dishes, 5-, 10-, and 25-ml individually wrapped sterile pipettes, 15- and 50-ml sterile conical centrifuge tubes, sterile 1- to 200- and 500- to 1000-µl pipette tips, and sterile 9-in. Pasteur pipettes and cell lifters.
Successful growth of these cells requires special attention to the media, confluency, trypsinization, and feeding regime in order to maintain healthy cells capable of undergoing differentiation. Insufficient attention to any of the aforementioned parameters will quickly result in cells acquiring a fibroblast-like morphology and concomitant failure to undergo 3D organization when cultured in IrBM.
A. Cell Maintenance
1. Preparation of Collagen-Coated Tissue Culture Flasks
2. Passage of HMT3522 Cells
Change media every 2-3 days; it is important to ensure that fresh media be added 1 day before splitting. HMT3522 S1 cells should be passaged once the 2D colonies form rounded islands with the flask being approximately 75% confluent, as shown in Fig. 1A. This is usually occurs 8 to 10 days after the initial plating.
Passage HMT3522 T4-2 cells when the flask reaches 75% confluency, as shown in Fig. lB. This usually occurs 3 to 5 days after initial plating. Maintain T4-2 cells on Vitrogen (collagen I) or rat tail collagen-coated flasks.
Perform the following steps in a tissue culture hood.
3. Three-Dimensional BM Assay Embedded in I rBM/EHS/(Ma trige 1)
Single phenotypically normal mammary epithelial cells embedded into lrBM will undergo several rounds of cell division, withdraw from cell cycle between 6 and 8 days (depending on the cell type), organize into polarized structures, and form acini-like structures, including a central lumen. Malignant cells, however, continue to proliferate and form disorganized, tumorlike structures (Fig. 2). Starting on day 4 but clearly by day 10, one can distinguish nonmalignant from malignant cells. Immunohistochemistry analysis for the Ki67 antigen on day 10 shows that ~60% of the malignant cells are still proliferating, whereas only <10% of the nonmalignant remain in the cell cycle.
The following volumes and concentrations given are appropriate for a 35-mm tissue culture dish (9.6- cm2 surface area). The volumes and concentrations must be adjusted to correct for the area of the plate used in the analysis. IrBM should be stored at -80°C. Thaw immersed in ice at 4°C overnight before use. Once thawed it should not be refrozen but can be kept in ice at 4°C for more than 1 month. Matrigel lots are tested before use and batches are purchased for experimental use. Alternatively, the basement membrane mixture (EHS) can be made from the Engelbreth-Holm-Swarm mouse tumor described in Kleinman et al. (1982); see Section III,B details.
Note: EHS or Matrigel should always be kept on ice as it will polymerize quickly at room temperature.
4. Three-Dimensional BM Assay on Top of EHS/ Matrigel
The culture of mammary epithelial cells, developed previously in our laboratory for functional studies of mouse cells (Barcellos-Hoff et al., 1989; Roskelley et al., 1994), has now been adopted to human cells as well. Making 3D BM cultures on top of EHS as opposed to inside the gel has some advantages and a few drawbacks. The proliferation and morphological differences between nonmalignant and malignant cells can be distinguished by 4 days in culture as opposed to the 8-10 days required for embedded cultures. The cell colonies can readily be imaged utilizing a live cell imager, allowing one to follow a single cell to an acini-like structure. Furthermore, cell colonies can be harvested easily by scraping the culture gently with a pipette tip and depositing the isolate on a glass slide for analysis by immunohistochemistry. The advantage of this method of harvest is that the remaining culture can be harvested for DNA, RNA, or protein. However, neither the morphology nor the growth rate of the acini is as tightly controlled as the embedded cultures. An additional disadvantage of the on-top culture is that the amount of material collected per plate is less than the embedded cultures, as plating of the cells is restricted to one plane. Therefore, there are significantly less cells per millimeter dish to be harvested compared to the embedded assay. The following volumes and concentrations are appropriate for a 35-mm tissue culture dish (9.6-cm2 surface area). The volumes and concentrations must be adjusted to correct for the area of the plate used in the analysis.
Before splitting the cells, prepare the EHS-coated plates.
5. Reversion Assay
The reversion assay can determine the ability of tumor cells to become phenotypically normal or to undergo apoptosis as a function of treatments. In addition, the differential response of tumorigenic and nonmalignant cells to a wide variety of perturbations can be analyzed. Analyses can be performed both on cells cultured embedded in or on top of EHS. This assay can be used as a screen for potential therapeutics by manipulating various genes, signaling pathways, and/or protein modifications and for determining their role in the establishment or maintenance of the tumorigenic phenotype. Cancer cells, cultured as described earlier, can be treated with a wide variety of agents, including inhibitory or stimulatory antibodies to integrins or growth factor receptors, small molecule inhibitors to different signaling pathways, gene delivery via transfection with expression vectors or transduction with virus overexpressing genes of interest, and RNAi for specific gene silencing.
Quantitative end points include morphology, proliferation index, degree of polarity, and level of expression of genes of interest using RT-PCR, Northern, or Western analysis, as well as immunofluorescence. Table II details a variety of molecules that have been shown to revert T4-2 cells. A typical reversion morphology of T4-2 cells is illustrated in Fig. 4.
6. Release of Cellular Structures from 3D BM
For molecular analysis of cell extracts, it is important to remove them from the gel. Apart from scraping the colonies as described earlier, which will still have minor EHS contamination, intact cellular structures can be released from EHS utilizing chelating agents, a procedure described in Weaver et al. (1997). A modified version is described here. Note that the solutions must be cold and the cells kept on ice for the entire harvest period. The maximum harvest should be 1 h as the cell-cell junctions will begin to come apart if left in these solutions for too long.
Perform the following steps on the day of harvest.
Note: Trizol or Tripure can be used to isolate RNA and DNA directly from the cultures without release of the cells from the gel.
B. Additional Procedures
1. Criteria for Purchase of Appropriate Matrigel Lots
Test lots of Matrigel and choose those that are low in epidermal growth factor (EGF), low endotoxin, which have a protein concentration close to 10mg/ ml.
Test the cells of interest by always comparing old and new lots side by side using the 3D BM-embedded and on top culture conditions as described earlier. Also test the ability of malignant cells of interest to revert in the presence of tyrphostin AG1478 or known reverting agents.
2. Preparation of EHS Matrix from EHS Tumors
The EHS tumor is grown in C57 black mice. Typical propagation of the tumor starts with one-third of a fresh tumor, which is enough to inject 10 mice. The tumor sample is minced with a scalpel blade sussuspended into 2 ml PBS and is then further disassociated by pushing it through an 18-gauge needle followed by a 20-gauge needle. This tumor slurry is then injected intramuscularly (IM) into the hind limb (0.2ml/limb) of mice. Tumors are allowed to grow for 4 weeks. For the last 2 weeks of tumor growth, 0.1% β-aminopropionitrile fumarate (BAPN) is added to the drinking water to prevent the collagen from cross-linking. The tumors should be harvested, weighed, frozen in liquid nitrogen, and stored at-80°C until use. For further information, see Kleinman et al. (1986).
The following buffers are needed for isolation.
Buffer 1: 3.4M NaCl, 50mM Tris-Cl, pH 7.4, 4mM EDTA, and 2mM N-ethylmalenmide (NEM) (make up 100× stock in H2O fresh for each other preparation)
Buffer 2: 0.2M NaCl, 50mM Tris-Cl, pH 7.4, 4rmM EDTA, 2mM NEM (make up 100× stock in H2O fresh for each preparation), and 2M urea, ultrapure or deionized
Buffer 3: 0.15M NaCl, 50mM Tris-Cl, pH 7.4, 4mM EDTA, and 2 mM NEM (make up 100× stock in H2O fresh for each preparation). Sterilize
All solutions should be at 4°C and kept on ice
Using this assay system, one can screen a wide variety of breast tumor cell lines and assess their ability to revert or die when treated with various signaling inhibitors and/or potential therapeutics, as outlined in Wang et al. (2002).
Always include a cell line with a known positive response when performing the reversion assay.
Work from the authors' laboratory was supported by the United States Department of Energy, Office of Biological and Environmental Research (DE-AC03 SF0098 to M.J.B.). Additional funding was contributed by the Department of Defense Breast Cancer Research Program (#DAMD17-02-1-0438 to M.J.B.) and the National Cancer Institute (CA64786-02 to M.J.B., and CA57621 to Zena Werb and M.J.B). The authors thank Paraic Kenny for editorial comments.
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