Fig. 4

In vivo validation of β-sitosterol pre-treatment. a Experimental overview: Treatment started one week prior to intracardiac injections of 5 × 10⁵ H1_DL2 cells in NOD/SCID mice. Quantification of tumor cell exposure in the mouse brains was carried out 24 h after injections for group homogenization (Additional file 6: Figure S5a). Test groups received daily i.p. injections of 0.1 mL vehicle (olive oil; n = 10) or β-sitosterol diluted in olive oil (5 mg/kg; n = 9). Vehicle treatment was continued until euthanization and β-sitosterol was given for ten weeks. See Additional file 6: Figure S5 for more details on this experiment. b Development of brain metastases assessed by MRI at four, six, seven and eight weeks. Scale bar MRIs (T1-weighted images with contrast), = 0.25 cm. c Number of brain metastases assessed by T1-weighted MRI with contrast (Student’s t-test). The mean number of brain metastases in the vehicle group decreased slightly from seven to eight weeks as four mice with the greatest number of brain metastases were sacrificed between these observation points. d Brain BLI at seven and eight weeks (total photon count = dorsal + ventral region of interest (ROI); Student’s t-test). e Kaplan-Meier survival plot (Mantel-Cox log-rank test). The experiment was terminated at 100 days, and three mice in the β-sitosterol group were still alive and healthy. * P < 0.05; ** P < 0.01; **** P < 0.0001. All values are given as the mean ± s.e.m