A Western blot indicated the expression levels of BTK, pBTK, stemness marker CD133, and EMT marker vimentin in SAS-IR and TW2.6-IR cell lines. GAPDH was used as a loading control. B Representative data showing the effect of pre-exposing SAS-IR and TW2.6-IR cells to 0 μM (control) or 10 μM (treated) ibrutinib for 48 h, on their ALDH activity as detected by flow cytometry-based ALDEFLUOR assay and the graphical quantitative analysis of treated ALDH+ compared with control cells. C The reducing effect of ibrutinib pretreatment on OSCC tumorsphere formation from SAS and TW2.6 (left), and quantitative representation of the effect of ibrutinib treatment on primary and secondary tumorsphere formation from SAS and TW2.6 cells (right). D, E Representative images illustrating that treatment with ibrutinib significantly reduced the migration and invasion ability of SAS-IR and TW2.6-IR OSCC cells in a dose-dependent manner. Graphical quantitative data showing the relative number of cells in ibrutinib-treated cells compared with control cells. All experiments performed in triplicates and expressed as mean ± SD. *p < 0.05, **p < 0.01.