A Drug combination assay: different concentrations of ibrutinib and cisplatin were used in combination for calculating the combination index (CI). Normalized isobolograms demonstrated that ibrutinib and cisplatin synergistically suppressed the cell viability of OSCC spheres. CI < 1 denotes synergy. B Photo images (left) and graphical quantitative data (right) of the tumorsphere-forming ability of the ALDH+ SAS or TW2.6 cells after treatment with 5 μM cisplatin, 5 μM ibrutinib, or both compared with control cells. C Treatment of ibrutinib alone or in combination with cisplatin effectively reduces the colony-forming abilities, graphical quantitative data of the number of colonies formed by the SAS-IR or TW2.6-IR cells after treatment with 5 μM cisplatin, 5 μM ibrutinib, or both compared with control cells. D Apoptotic effect of cisplatin alone, ibrutinib alone, or cisplatin–ibrutinib combination treatment in SAS-IR or TW2.6-IR cells. Data represent experiments performed in triplicates and expressed as mean ± SD. *p < 0.05, **p < 0.01.