KC-4045

LLC-Luc2-EPCAM-Low Cell Line

40548

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Background of LLC-Luc2-EPCAM-Low Cell Line

EPCAM (epithelial cell adhesion molecule) is a type I transmembrane glycoprotein, which is also known as CO171A, EGP, EGP40, GA7332, KSA, M4S, MIC18, MK1, TROP1, hEGP2. EPCAM is highly expressed in a variety of epithelial tumor tissues and promotes tumor proliferation. EPCAM contributes to various biological processes including cell adhesion, signaling, migration and proliferation. Intact EPCAM and its proteolytic fragments interact with claudins, CD44, E-cadherin, epidermal growth factor receptor (EGFR), and intracellular signaling components of the WNT and Ras/Raf pathways, respectively. EPCAM is a prognostic marker and target for primary and systemic tumor cells. Oportuzumab Monatox, an ADC targeting epithelial cell adhesion molecule (EPCAM) developed by Sesen Bio, has completed phase III clinical trials.

Specifications

Catalog Number	KC-4045
Cell Line Name	LLC-Luc2-EPCAM-Low Cell Line
Host Cell Line	LLC-Luc2
Description	Stable LLC-Luc2 clone expressing exogenous human EPCAM gene in low level
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin + 500 μg/mL Hygromycin B
Selection Marker	Puromycin, Hygromycin B
Morphology	Epithelial-like cells growing as a monolayer
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

LLC-Luc2-EPCAM-Low cell line was generated using a lentiviral vector expressing the human EPCAM sequence.

Characterization

Figure 1: Characterization of EPCAM overexpression in the LLC-Luc2-EPCAM-Low stable clone using FACS.

Figure 2: Characterization of human EPCAM in the LLC-Luc2-EPCAM-Low stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS, 1μg/mL puromycin and 500 μg/mL Hygromycin B)in a 37°C water bath. 2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes. 3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol. 4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium. 5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet. 6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask. 7. Incubate the flask at 37°C, 5% CO₂ incubator. 8 .Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use. 2. Keep the freezing medium on ice and label cryovials. 3. Transfer cells to a sterile, conical centrifuge tube, and count the cells. 4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium. 5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium. 6. Aliquot 1 mL of the cell suspension into each cryovial. 7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer. 8. Transfer vials to liquid nitrogen for long-term storage.

References

1. Huang L, Yang Y, Yang F, Liu S, Zhu Z, Lei Z, Guo J. Functions of EpCAM in physiological processes and diseases (Review). Int J Mol Med. 2018 Oct;42(4):1771-1785. doi: 10.3892/ijmm.2018.3764. Epub 2018 Jul 11. PMID: 30015855; PMCID: PMC6108866. 2. Gires O, Pan M, Schinke H, Canis M, Baeuerle PA. Expression and function of epithelial cell adhesion molecule EpCAM: where are we after 40 years? Cancer Metastasis Rev. 2020 Sep;39(3):969-987. doi: 10.1007/s10555-020-09898-3. PMID: 32507912; PMCID: PMC7497325. 3. Fagotto F. EpCAM as Modulator of Tissue Plasticity. Cells. 2020 Sep 19;9(9):2128. doi: 10.3390/cells9092128. PMID: 32961790; PMCID: PMC7563481.