Isolation and Culture of Human Breast Cancer Cells from Primary Tumors and Metastases

Ethier, Stephen P.; Ammerman, Cheryl A.; Dziubinski, Michele

doi:10.1007/978-1-4615-4295-7_18

Cited by 5 publications

(7 citation statements)

References 21 publications

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“…Primary culture of epithelial cells derived from such tumors has been a goal of many laboratories to increase our understanding of human mammary gland biology (Speirs et al 1996;Ethier et al 2000). In contrast to immortalized cell lines that grow in the Fig.…”

Section: Discussionmentioning

confidence: 99%

“…One of the major stumbling blocks in this type of work is using a selective growth medium and methods to focus on enriching and developing cancer cell populations with a minimum number of normal epithelial and fibroblast (stromal) cells. In a medium containing many different factors, as is important for the expansion, epithelial breast cancer cells proliferate rapidly, in vitro (Speirs et al 1996;Ethier et al 2000).…”

Section: Discussionmentioning

confidence: 99%

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Isolation and characterization of the primary epithelial breast cancer cells and the adjacent normal epithelial cells from Iranian women’s breast cancer tumors

et al. 2018

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As an experimental model, most studies rely on established human cancer cell lines; however, some genetical or phenotypical differences exist between these cells and their original tumor. Therefore, primary cells isolated directly from tissue are believed to be more biologically relevant tools for studying human and animal biology. Here, we aimed to isolate primary epithelial cancer and normal cells from breast tumors of Iranian women, for the first time. Thus, we isolated the epithelial and fibroblast cells from biopsy samples of patients with breast cancer based on differential centrifugation followed by culture in selective media. Normal epithelial cells obtained from the tissue biopsy away from the core of the tumor, based on the pathological diagnosis. Flow cytometry analysis indicated the positive immunoreactivity of the isolated epithelial cells against CD24 and Epithelial Specific Antigen (ESA/EpCAM), while they displayed a concomitant low expression of CD44 and CD49f. In contrat to fibroblasts, the qPCR data indicated the expression of luminal intracellular cytokeratin (Ck18) in both normal and cancer epithelial cells, but there was no expression of myoepithelial/basal markers, CK5 and vimentin. The epithelial cancer cells were reactive to cytokeratin 19 (CK19) antibody, whereas the normal epithelial cells were not. The expression of calmodulin-like protein (CLP) was also lower in the cancer epithelial cells than in the normal ones. In conclusion, primary epithelial normal and cancer cells, in addition to the fibroblasts were isolated and characterized from breast tumor of Iranian patients; and CLP expression is suggested as a susceptibility marker for breast cancer screening.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of the primary epithelial breast cancer cells and the adjacent normal epithelial cells from Iranian women’s breast cancer tumors

et al. 2018

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“…The supplements for studied media (hormones and growth factors) and their working concentrations were chosen on the basis of studies performed by Ethier et al [ 31 , 32 ]: insulin (I; 5 μg/mL), hydrocortisone (HC; 1 μg/mL), epidermal growth factor (EGF; 10 ng/mL), estradiol (E2; 10 nM), transferrin (T; 5 μg/mL), triiodothyronine (T3; 10 μM) and cholera toxin (Ct; 1 ng/mL). The studied media were also supplemented with antioxidant sodium selenite (Se; 50 μM) and buffering agent 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 2 nM).…”

Section: Methodsmentioning

confidence: 99%

An Effect of Culture Media on Epithelial Differentiation Markers in Breast Cancer Cell Lines MCF7, MDA-MB-436 and SkBr3

et al. 2018

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Background and objectives: Cell culture is one of the mainstays in the research of breast cancer biology, although the extent to which this approach allows to preserve the original characteristics of originating tumor and implications of cell culture findings to real life situations have been widely debated in the literature. The aim of this study was to determine the role of three cell culture media on transcriptional expression of breast cancer markers in three breast cancer reference cell lines (MCF7, SkBr3 and MDA-MB-436). Materials and methods: Cell lines were conditioned in three studied media (all containing 5% fetal bovine serum (FBS) + hormones/growth factors; different composition of basal media) for four passages. Population growth was characterized by cumulative population doubling levels, average generation time, cell yield and viability at the fourth passage. Transcriptional expression of breast cancer differentiation markers and regulatory transcriptional programs was measured by qPCR. Results: Differences in the composition of growth media significantly influenced the growth of studied cell lines and the expression of mammary lineage governing transcriptional programs and luminal/basal markers. Effects of media on transcriptional expression were more pronounced in luminal cell lines (MCF7, SkBr3), than in the basal cell line (MDA-MB-436). Changes in growth media in terms of supplementation and basal medium delayed growth of cells, but improved cell yields. Conclusions: The expression of breast cancer cell differentiation phenotypic markers depends on the composition of cell growth medium, therefore cell culture as a tool in phenotypic studies should be used considering this effect. The findings of such studies should always be interpreted with caution. The formulation of cell growth media has greater effect on the expression of phenotypic markers in luminal, rather than basal cell lines. Media containing mitogens and higher vitamin content improved efficacy of cell culture in terms of cell yields, although greatly increased growth times.

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“…These procedures were adapted from 3,4 Tap the plates gently to loosen the cells. Check for detachment using a microscope.Quench the trypsin by pipetting 9 ml of complete medium on the plate and transfer the cells to a sterile 15 ml conical tube.…”

Section: Orthoptic Transplantation Of Collagen Embedded Cells In C57bmentioning

confidence: 99%

“…Expose the mouse on its back on a flat surface and immobilize the limbs using tape adhesive. 3. Make an upside down T incision between the nipples of the thoracic and inguinal mammary gland and pull the skin flap back to expose the mammary glands.…”

mentioning

confidence: 99%

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

Cheng

Lambert

2011

JoVE

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The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

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Isolation and Culture of Human Breast Cancer Cells from Primary Tumors and Metastases

Cited by 5 publications

References 21 publications

Isolation and characterization of the primary epithelial breast cancer cells and the adjacent normal epithelial cells from Iranian women’s breast cancer tumors

Isolation and characterization of the primary epithelial breast cancer cells and the adjacent normal epithelial cells from Iranian women’s breast cancer tumors

An Effect of Culture Media on Epithelial Differentiation Markers in Breast Cancer Cell Lines MCF7, MDA-MB-436 and SkBr3

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

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