Citation
- Authors: Cumin C. et al.
- Year: 2022
- Journal: Cell Rep 40 111181
- Applications: in vitro / DNA / jetPEI
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Method
For preparation of lentiviral particles, HEK293T cells were seeded at 50% confluency in a T75 flask one day prior to transfection. 4 μg of lentiCRISPR v2, 2 μg of pMD2.G (addgene #12259) and 2 μg of pCMVR8.74 (addgene #22036) were co-transfected using 24 μL of jetPEI reagent in 1 mL of 150 mM NaCl solution (Polyplus-transfection, Chemie Brunschwig AG, Switzerland). Medium was changed 24h after transfection. Virus supernatant was collected 48h later and filtered with a 0.45 μm polyvinylidene fluoride filter (Sartorius AG, Germany). Transduced cells were kept under selection with a concentration of 1 μg/μL puromycin.
Abstract
The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.