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BT-474 ECAD-EmGFP (ATCC® HTB-20EMT)

Organism: Homo sapiens, human  /  Tissue: mammary gland, breast, duct; derived from a solid, invasive ductal carcinoma  /  Disease: ductal carcinoma of the breast

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Organism Homo sapiens, human
Tissue mammary gland, breast, duct; derived from a solid, invasive ductal carcinoma
Product Format frozen 1.0 mL
Morphology epithelial
Culture Properties adherent
Biosafety Level 2

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Disease ductal carcinoma of the breast
Age 60 years
Gender female
Ethnicity Caucasian
Applications Epithelial to mesenchymal transition (EMT), anti-EMT drug screening, metastatic breast cancer drug screening. See Technical Data Sheet for data validating protein expression and transition to mesenchymal status.
Images
Comments

Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the incidence of cancer metastasis and drug resistance, their impact in cancer progression and patient survival is not fully understood (Nieto et al. 2016). During EMT, epithelial cells lose their polarity, as well as their cell-cell adhesions, and acquire the motile and invasive characteristics of mesenchymal cells (Hay 1995). Proteins such as vimentin intermediate filament (IF) proteins are generally upregulated when the cell is in the mesenchymal relative to the epithelial status (Gilles et al. 1999; Thiery and Sleeman 2006; Richardson et al. 2012; Lamouille et al. 2014). Here, we created an ECAD-EmGFP reporter cell line (HTB-20EMT) using the CRISPR/Cas9 gene editing platform and the parental BT-474 breast ductal carcinoma cell line (ATCC HTB-20). The HTB-20EMT cell line harbors a C-terminal green fluorescent protein (EmGFP) tag on the E-cadherin gene. This will enable the tracking of the EMT status of cells in vitro by monitoring GFP expression. The integrity of the ECAD-EmGFP knock-in has been verified at the genomic, mRNA and protein level for sequence and expression. The BT-474 ECAD-EmGFP reporter cell line provides a convenient and sensitive platform for research on the mechanisms of metastasis in vitro and the development of new antitumor drugs for metastatic breast cancer.

Complete Growth Medium

The base medium for this cell line is Dulbecco's Modified Eagle's Medium (DMEM; ATCC 30-2002). To make the complete medium add the following component:

  • 10% Fetal Bovine Serum (FBS; ATCC 30-2020)
Subculturing
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
    Cultures can be established between 2 x 104 and 2 x 105 viable cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 2 X 104 and 2 X 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Note: HTB-20EMT recovers slowly from cryopreservation. It may take two to four weeks for the cells to reach 70-80% confluence in a T-75 flask after thaw. Cells form adherent patches of epithelial-like cells. The patches are compact multilayered colonies that rarely become confluent.
Cryopreservation Complete growth medium plus 5% (v/v) DMSO (ATCC 4-X)
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial Approximately 5 x 106 cells
Volume 1.0 mL
STR Profile
Amelogenin: X
CSF1PO: 10,11
D13S317: 11
D16S539: 9,11
D5S818: 11,13
D7S820: 9,12
TH01: 7
TPOX: 8
vWA: 15,16
Sterility Tests Bacteria and yeast: No growth
Mycoplasma: No growth
Viral Testing Hepatitis B: None detected
Cytomegalovirus: None detected
Human immunodeficiency virus: None detected
Epstein-Barr virus: None detected
Human papillomavirus: None detected
Viability ≥ 70%
Functional Tests Genotype Testing for KI mutation: PCR band at 1702 bp corresponding to ECAD-EmGFP junction; Sanger Sequencing confirms junction sequence
Name of Depositor ATCC
Year of Origin 2018
References

Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7(2): 131-142, 2006. PubMed: 16493418

Richardson F, et al. The evaluation of E-Cadherin and vimentin as biomarkers of clinical outcomes among patients with non-small cell lung cancer treated with erlotinib as second- or third-line therapy. Anticancer Res 32(2): 537-552, 2012. PubMed: 22287743

Gilles C, et al. Vimentin contributes to human mammary epithelial cell migration. J Cell Sci 112 (Pt 24): 4615-4625, 1999. PubMed: 10574710

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
Restrictions

For commercial accounts, this cell line is only distributed under the terms of a fully signed and executed ATCC® Material Transfer Agreement and Addendum. If the commercial account is screening per completed Addendum, the recipient will be required to pay a Screening Fee (ATCC® ACS-2103F™).

Screening Use is defined as use of Biological Material in small molecule and biologic drug discovery, including initial target identification and validation, assay development, high throughput screening, hit identification, lead optimization, and selection of candidates for clinical development.

If the commercial account is not screening per the completed Addendum, the recipient will not be required to pay a Screening Fee.

In addition to the foregoing, this product's use is governed by the CRISPR Label License Agreement. For information on purchasing a license to use this product for purposes other than those permitted in the CRISPR Label License Agreement, please contact The Broad Institute at partnering@broadinstitute.org.

References

Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7(2): 131-142, 2006. PubMed: 16493418

Richardson F, et al. The evaluation of E-Cadherin and vimentin as biomarkers of clinical outcomes among patients with non-small cell lung cancer treated with erlotinib as second- or third-line therapy. Anticancer Res 32(2): 537-552, 2012. PubMed: 22287743

Gilles C, et al. Vimentin contributes to human mammary epithelial cell migration. J Cell Sci 112 (Pt 24): 4615-4625, 1999. PubMed: 10574710