The ACSL4 Knockout KYSE-30 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human esophageal squamous cell carcinoma line KYSE-30, in which the ACSL4 gene has been disrupted to generate a loss-of-function model. This polyclonal format avoids clonal bias and enables functional studies of ferroptosis and lipid metabolism in an esophageal cancer context.
The parental KYSE-30 cell line is derived from a well-differentiated esophageal squamous cell carcinoma resected from a 64-year-old male Japanese patient, representing a primary tumor model with epithelial characteristics. As a commonly employed cell line in cancer research, KYSE-30 retains molecular features of esophageal squamous cell carcinoma, making it suitable for investigating oncogenic signaling, metabolic reprogramming, and therapeutic vulnerabilities.
ACSL4 (acyl-CoA synthetase long-chain family member 4) catalyzes the activation of long-chain fatty acids, preferentially arachidonic acid, to acyl-CoA thioesters, thereby enriching cellular membranes with polyunsaturated fatty acid-containing phospholipids that are substrates for peroxidation. This enzymatic activity is a rate-limiting step in ferroptosis execution. ACSL4 expression is transcriptionally upregulated by SREBF1, SP1, and PPARG in response to lipogenic and stress signals, and its activity is positively influenced by MAPK/ERK signaling. The ferroptosis suppressor GPX4 directly antagonizes ACSL4-driven lipid peroxidation by reducing phospholipid hydroperoxides. Within the ferroptosis machinery, ACSL4 functions in concert with LPCAT3, ALOX12, ALOX15, and PTGS2 to generate and propagate lipid peroxides. Inhibition of system xc- or GPX4 leads to accumulation of lipid ROS, triggering ferroptotic cell death in an ACSL4-dependent manner.
Disruption of the ACSL4 gene in KYSE-30 polyclonal cells significantly attenuates ferroptotic sensitivity, providing a powerful isogenic model to investigate drug resistance mechanisms in esophageal squamous cell carcinoma. In the absence of functional ACSL4, the incorporation of arachidonic acid into membrane phospholipids is reduced, resulting in decreased lipid peroxidation and resistance to ferroptosis inducers such as erastin (system xc- inhibitor) and RSL3 (GPX4 inhibitor). This model enables the dissection of ACSL4-dependent cell death pathways and the elucidation of compensatory lipid remodeling mechanisms, including alterations in LPCAT3 activity and lipoxygenase-mediated oxidation, which may contribute to ferroptosis resistance in cancer.
This polyclonal knockout model has broad applications in ferroptosis research, including investigation of ferroptosis sensitivity in esophageal cancer, screening of novel ferroptosis-inducing compounds, and functional genomic studies of lipid metabolism. Researchers can confirm ACSL4 depletion by Western blotting and RT-qPCR, assess lipid peroxidation levels using the C11-BODIPY probe, and quantify ferroptotic cell death via cell viability assays following treatment with erastin or RSL3. Additional assays such as flow cytometry for lipid ROS and arachidonic acid incorporation assays further characterize the metabolic consequences of ACSL4 loss. This tool accelerates translational research in oncology and neurobiology. For more detailed information, please contact Ascent Research.
