ATCC Gold Seal HCT116 VIM RFP (ATCC® CCL-247EMT)

Organism: Homo sapiens, human  /  Tissue: colon/rectum  /  Disease: colorectal carcinoma

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Organism Homo sapiens, human
Tissue colon/rectum
Verified By ATCC Cell Line Authentication Service
Functional Testing
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 colorectal carcinoma
Age adult
Gender male
Applications Epithelial to mesenchymal transition (EMT), anti-EMT drug screening, colon cancer drug screening, vimentin intermediate filament dynamics.
Storage Conditions liquid nitrogen vapor phase
Images
Comments

The HCT116 VIM RFP reporter cell line, derived from the parental HCT116 (ATCC® CCL-247™) colorectal carcinoma cell line, was created using CRISPR/Cas9 gene editing technology. The HCT116 VIM RFP reporter cell line carries a knock-in red fluorescent protein (RFP) reporter, which was integrated before the stop codon at the last exon of the endogenous vimentin (VIM) gene.

Epithelial to mesenchymal transition (EMT) has been recognized to play an important role in cancer cell metastasis and drug resistance. The EMT pathway is of increasing interest as a novel therapeutic avenue in the treatment of cancer. HCT116 VIM RFP cell line (ATCC® CCL-247EMT™) was created using the CRISPR-Cas9 platform, in which a red fluorescent protein (RFP) reporter was integrated before the stop codon at the last exon of the endogenous vimentin gene, a widely used mesenchymal cell marker. The integrity of the VIM RFP knock-in allele has been verified at genomic, transcriptional, and translational levels. In HCT116 VIM RFP cells, vimentin-RFP expression can be robustly turned on in response to miR-200 family inhibitor treatment, as previously reported (Park et al, Genes Dev, 22: 894-907, 2008). In contrast, the expression of E-cadherin, a marker of epithelial cells, is significantly reduced upon miR-200 inhibitor treatment. In addition, hypomethylating agent 5-Aza-2’-Deoxycytidine treatment can induce an increase in VIM RFP expression effectively. The HCT116 VIM RFP cell line could be a useful in vitro cell model for dissecting the molecular switches underlying EMT and for identifying compounds that target EMT in colorectal cancer.

Complete Growth Medium

Base growth media for this cell line is McCoy’s 5A Medium (ATCC 30-2007). To make the complete medium add the following component to the base medium:

  • 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 4 x 104 viable cells/cm2.
  6. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 2 X 104 and 8 X 104 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Cryopreservation Complete growth medium plus 5% (v/v) DMSO
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Cells per Vial Approximately 4 x 106 cells
Volume 1.0 mL
STR Profile
Amelogenin: x,y
CSF1PO: 7,10
D13S317: 10,13
D16S539: 11,13,14
D5S818: 10,11
D7S820: 11,12
THO1: 8,9
TPOX: 8
vWA: 20,22


Note:

The STR profile of the HCT-116 VIM RFP reporter cell line is approximately an 80% match to the parental HCT-116 cell line indicating that the cell lines are related (derived from common ancestry). The STR differences are likely attributed to microsatellite instability in the HT-116 cell line, which is a common feature of this cell line.1,2,3

  1. Ahmed DP, et al. Epigenetic and genetic features of 24 colon cancer cell lines. Oncogenesis Sep 16;2:e71. doi: 10.1038/oncsis.2013.35, 2013. PubMed: 34042735
  2. Gu C, et al. Inositol pyrophosphate profiling of two Hct116 cell lines uncovers variation in Insp8 levels. PLoS One 11(10):e0165286. doi: 10.1371/journal.pone.0165286, 2016. PubMed: 27788189
  3. Eshleman JR, et al. Increased mutation rate at the hprt locus accompanies microsatellite instability in colon cancer. Oncogene 10(1):33-7, 1995. PubMed: 7824277
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
Functional Tests Genotype Testing for KI mutation: PCR- Band at 1127 kb corresponding to Vimentin-RFP LHA junction and band at 918 bp corresponding to RHA junction; Sanger Sequencing – confirms junction sequence
Population Doubling Time 22 hours
Name of Depositor MG Brattain
Year of Origin 2018
References

Park SM, et al. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 22(7): 894-907, 2008. PubMed: 18381893

Schroy PC, et al. Detection of p21ras mutations in colorectal adenomas and carcinomas by enzyme-linked immunosorbent assay. Cancer 76: 201-209, 1995. PubMed: 8625092

Brattain MG, et al. Heterogeneity of malignant cells from a human colonic carcinoma. Cancer Res. 41: 1751-1756, 1981. PubMed: 7214343

Sun L, et al. Autocrine transforming growth factor-beta 1 and beta 2 expression is increased by cell crowding and quiescence in colon carcinoma cells. Exp. Cell Res. 214: 215-224, 1994. PubMed: 8082724

Santoro IM, Groden J. Alternative splicing of the APC gene and its association with terminal differentiation. Cancer Res. 57: 488-494, 1997. PubMed: 9012479

Brattain MG, et al. Enhancement of growth of human colon tumor cell lines by feeder layers of murine fibroblasts. J. Natl. Cancer Inst. 69: 767-771, 1982. PubMed: 6956756

Bender CM, et al. Inhibition of DNA methylation by 5-Aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res. 58: 95-101, 1998. PubMed: 9426064

Landers JE, et al. Translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild-type p53 protein. Cancer Res. 57: 3562-3568, 1997. PubMed: 9270029

Kutchera W, et al. Protaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect. Proc. Natl. Acad. Sci. USA 93: 4816-4820, 1996. PubMed: 8643486

Wang R, et al. Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor. Proc. Natl. Acad. Sci. USA 93: 8425-8430, 1996. PubMed: 8710887

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

Park SM, et al. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 22(7): 894-907, 2008. PubMed: 18381893

Schroy PC, et al. Detection of p21ras mutations in colorectal adenomas and carcinomas by enzyme-linked immunosorbent assay. Cancer 76: 201-209, 1995. PubMed: 8625092

Brattain MG, et al. Heterogeneity of malignant cells from a human colonic carcinoma. Cancer Res. 41: 1751-1756, 1981. PubMed: 7214343

Sun L, et al. Autocrine transforming growth factor-beta 1 and beta 2 expression is increased by cell crowding and quiescence in colon carcinoma cells. Exp. Cell Res. 214: 215-224, 1994. PubMed: 8082724

Santoro IM, Groden J. Alternative splicing of the APC gene and its association with terminal differentiation. Cancer Res. 57: 488-494, 1997. PubMed: 9012479

Brattain MG, et al. Enhancement of growth of human colon tumor cell lines by feeder layers of murine fibroblasts. J. Natl. Cancer Inst. 69: 767-771, 1982. PubMed: 6956756

Bender CM, et al. Inhibition of DNA methylation by 5-Aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res. 58: 95-101, 1998. PubMed: 9426064

Landers JE, et al. Translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild-type p53 protein. Cancer Res. 57: 3562-3568, 1997. PubMed: 9270029

Kutchera W, et al. Protaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect. Proc. Natl. Acad. Sci. USA 93: 4816-4820, 1996. PubMed: 8643486

Wang R, et al. Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor. Proc. Natl. Acad. Sci. USA 93: 8425-8430, 1996. PubMed: 8710887