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MDA-MB-231 VIM RFP (ATCC® HTB-26MET™)

Organism: Homo sapiens, human  /  Tissue: mammary gland/breast; derived from metastatic site: pleural effusion  /  Disease: adenocarcinoma

Permits and Restrictions

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Organism Homo sapiens, human
Tissue mammary gland/breast; derived from metastatic site: pleural effusion
Product Format frozen 1.0 mL
Morphology fibroblast-like
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 adenocarcinoma
Age 51 years
Gender female
Ethnicity caucasian
Applications

Mesenchymal to epithelial transition, anti-EMT drug screening, metastatic breast cancer drug screening, vimentin intermediate filament dynamics. See Technical Data Sheet.

The MDA-MB-231 VIM RFP reporter cell line provides a convenient and sensitive platform for research on the mechanisms of metastasis in vitro and the development of new anti-EMT drugs for metastatic breast cancer.
Storage Conditions liquid nitrogen vapor phase
Comments

Breast cancer is the most aggressive form of all cancers, with high incidence and mortality rates. 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 (VIM) intermediate filament (IF) 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). 

The VIM RFP reporter cell line (ATCC HTB-26MET) was created using CRISPR/Cas9 gene editing and the parental MDA-MB-231 breast adenocarcinoma cell line (ATCC HTB-26). HTB-26MET harbors a C-terminal red fluorescent protein (RFP) tag on the vimentin gene. This enables the tracking of the EMT status of cells in vitro by monitoring RFP expression. The integrity of the VIM RFP knock-in has been verified at the genomic, mRNA, and protein level for sequence and expression. Functional evaluation of HTB-26MET shows sensitivity to metastatic breast cancer drugs axitinib (tyrosine kinase inhibitor) and U0126 (MEK1/2 inhibitor) via the inhibition of the inherent signaling pathways which impact EMT. 
Complete Growth Medium

The base medium for this cell line is Eagle's Minimum Essential Medium (EMEM; ATCC 30-2003). To make the complete medium, add the following components to the base medium at the indicated final concentrations:

  • 10% Fetal Bovine Serum (FBS; ATCC 30-2020)
  • 0.01 mg/mL human recombinant insulin (Thermo Fisher cat# 12585014) 
  • 10 µg/mL Blasticidin S HCl (Gibco cat# A11139-03)
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.
  6. Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended
Medium Renewal: 2 to 3 times per week
Cryopreservation Complete growth medium plus with 5% (v/v) DMSO (ATCC 4-X)
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial Approximately 2 x 106 cells
Volume 1.0 mL
STR Profile
Amelogenin: X
CSF1PO: 12,13
D13S317: 13
D16S539: 12
D5S818: 12
D7S820: 8,9
THO1: 7,9.3
TPOX: 8,9
vWA: 15, 18
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 1894 kb corresponding to Vimentin RFP junction; Sanger Sequencing – confirms junction sequence.
Sensitivity to EMT induction and anti-EMT drugs verified during product development and validation (See Technical Data Sheet).
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:

  • For-profit customers must obtain a research use license agreement from the Contributor prior to shipment.
  • 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
Product Sheet
Product Sheet
Certificate of Analysis
Certificate of Analysis
SDS
SDS
Other Documentation
Other Document
Application Data
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.

Unless the Purchaser has a separate license agreement with The University of Texas M. D. Anderson Cancer Center (“Institution”), the ATCC Material is subject to the following restrictions:

  1. The ATCC Material (and any Modifications, Unmodified Derivatives and/or Progeny thereof) may not be used (1) for commercial purposes or Commercial Use by any Purchaser, or (2) by for-profit or commercial entities for any purpose;
  2. Purchaser may not transfer ATCC Materials, Modifications, Unmodified Derivatives, or Progeny to any for-profit entity or commercial entity; and
  3. Purchaser may not use ATCC Materials (or any Modifications, Unmodified Derivatives and/or Progeny thereof) in connection with any research, collaboration or other activities involving a third party that is a commercial entity or for-profit entity.

The restrictions set forth above are in addition to the restrictions set forth in the ATCC Material Transfer Agreement. Capitalized terms have the meanings set forth in the ATCC Material Transfer Agreement unless otherwise indicated above.

For instructions on how to obtain a license from Institution, please contact the Institution Office of Technology Commercialization via email at researchtools@mdanderson.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