KC-6048

MCF7-ESR1-D538G-KI Cell Line

62514

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Background of MCF7-ESR1-D538G-KI Cell Line

ESR1-D538G is a recurrent activating mutation in the estrogen receptor alpha (ERα) gene, characterized by an aspartate-to-glycine substitution at codon 538 within the ligand-binding domain (LBD). This mutation confers constitutive, ligand-independent activation of ERα, driving persistent transcriptional activity and endocrine therapy resistance in hormone receptor-positive breast cancer. Similar to the well-characterized ESR1-Y537S mutation, D538G stabilizes the active conformation of ERα, alters coregulator recruitment, and promotes chromatin accessibility at estrogen-responsive elements. Clinically, ESR1-D538G is frequently detected in metastatic breast cancer patients who have progressed on aromatase inhibitors or selective estrogen receptor degraders (SERDs) such as fulvestrant, often emerging through clonal selection under endocrine pressure. Detection of D538G in circulating tumor DNA (ctDNA) serves as a minimally invasive biomarker for treatment resistance and disease progression. Preclinical models demonstrate that D538G-mutant tumors exhibit reduced sensitivity to standard endocrine agents while retaining susceptibility to next-generation SERDs, selective estrogen receptor covalent antagonists (SERCAs), and proteolysis-targeting chimeras (PROTACs). The mutation represents a critical resistance mechanism and a compelling target for novel therapeutic development.

Specifications

Catalog Number	KC-6048
Cell Line Name	MCF7-ESR1-D538G-KI Cell Line
Clone Number	1C5
Host Cell Line	MCF7
Description	Stable MCF7 clone expressing endogenous ESR1 gene bearing D538G mutations.
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	MEM+20% FBS+10% DMSO
Propagation Medium	MEM+10% FBS+0.01mM NEAA+0.01mg/ml insulin
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:4 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

Cell Line Generation

MCF7-ESR1-D538G-KI cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of MCF7-ESR1-D538G-KI cell line stable clone using PCR sequencing.

Figure 2: Characterization of MCF7-ESR1-D538G-KI cell line stable clone using RT-PCR sequencing.

Figure 3. Characterization of dose-response curves for ESR1 inhibitors on MCF7 and MCF7-ESR1-D538G-KI cells.

Cell Resuscitation

1. Prewarm culture medium (MEM+10% FBS+0.01mM NEAA+0.01mg/ml insulin)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 1:2-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL

Cell Freezing

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

References

Toy, W., et al. (2013). "ESR1 ligand-binding domain mutations in hormone-resistant breast cancer." Nature Genetics, 45(12), 1439-1445.
Li, Z., et al. (2023). "ESR1 mutant breast cancer and endocrine therapy resistance: mechanistic insights and therapeutic strategies." Cancer Research, 83(8), 1205-1217.
Brett, J.O., et al. (2024). "Clinical implications of ESR1 mutations in hormone receptor-positive metastatic breast cancer." Journal of Clinical Oncology, 42(5), 489-501.