Fig. 1: Anti-tumour efficacy of combining Rova-T and CBL in vitro and in vivo.

a H82 TICs, b H526 TICs, c H82 non-TICS and d H526 non-TICs were seeded at 3000 cells per well in black-walled 96-well plates. The next day cells were treated with IgG control or Rova-T at different concentrations, or CBL, or with Rova-T + CBL. The cell viability after 72 h of treatment was determined using the CyQUANT Fluorescent Assay⁸ and normalised to controls. The experiments were repeated thrice, and each measurement was performed in triplicate. Results are represented by means ± SD. Data were analysed using Student’s t test. P values of <0.05 are considered statistically significant. *P < 0.05, and ***P < 0.001. e, f Tumour volumes in mice after treatment with the combination of Rova-T and CBL. Four PDX mice with tumours in both the flanks (N = 8) were treated with vehicle controls for CBL + IgG control, Rova-T alone (1.8 mg/kg, i.p.), CBL alone (60 mg/kg, i.v., once per week) or combinations of Rova-T with CBL. Mice were treated with vehicle controls or CBL + IgG control, Rova-T alone, CBL alone or combinations of Rova-T with CBL. e Tumour volumes were measured in all groups until they reached ~1200 mm³ in the vehicle-treated mice. f The treatment at the same doses as above was continued until the tumour volumes reached ~1200 mm³ in each group. Tumour volumes (v) were calculated using the volume for a prolate spheroid: v = 4/3 × π × a² × b, where a is the minor radius and b the major radius. Differences between groups were analysed by the Student’s t test. The results are represented as means ± SE. * indicates P < 0.05 versus the single drug treatment groups. g Survival of mice shown in f (P < 0.05 for the combination vs single drugs alone). h SOX2 protein level was determined by Western analysis in the residual tumours derived from mice after treatment with vehicle for CBL + IgG control, or with CBL, Rova-T or Rova-T + CBL. β-Actin was used as a loading control. i In vivo limiting dilution assay showing that combining Rova-T and CBL reduced the tumour-initiating frequency (TIF). Mice were scored positive for tumour growth when the tumour size exceeded 200 mm³ at 6 months after tumour cell inoculation. The TIF was calculated from N = 8 mice per group for each dilution of cells.