Transcriptomic analysis of effects of cancer cell detachment after treatment with 2-deoxy-D-glucose alone or in co-treatment with metformin

Detachment of cancer cells from tumor, anchorage-independent survival, and maintenance of proliferative capacity upon reattachment are crucial steps in metastases formation. Better understanding of the underlying molecular mechanisms could help to improve targeted cancer therapies. We show reversible detachment of cancer cells using N-glycosylation inhibitors 2-deoxy-D-glucose (2DG) and tunicamycin, with re-attached cells retaining their proliferating capacity. This phenomenon was enhanced by combining with inhibitors of the electron transport chain. Analysis of the detached MDA-MB-231 cells treated with 2DG or 2DG combined with metformin showed ER stress, upregulation of pentose phosphate pathway, stemness, and alterations in genes involved in EMT. MDA-MB-231 cells were plated on 6-well plates in RPMI-1640 medium supplemented with 25 mM glucose, 2 mM glutamine and 10 % FBS. Cells were washedand the medium was changed to complete RPMI medium with 5.6 mM glucose. Cells were treated for 72 hours with 4.8 mM 2DG or co-treated with 5 mM metformin and 0.6 mM 2DG. As additional sample, cells were grown on poly-HEMA (Poly(2-hydroxyethyl methacrylate)) coated plates were used. Medium was renewed daily, and the detached cells were collected and returned to the attached cell counterpart. After 72 hours, attached and detached cells were collected and counted separately. Both, attached and detached cells were harvested and washed twice with ice cold PBS, snap freezed in liquid nitrogen, and stored at – 80 °C. Total cell RNA was isolated from cell pellets using PureLink™ RNA Mini Kit (Invitrogen, ThermoFisher Scientific, Waltham, USA). Transcriptome sequencing of MDA-MB-231 cells was performed by Novogene (Cambridge, UK) and the sequencing was performed in triplicates in each group.