In Vivo Tumor Growth Assay

Xenograft tumor growth assays were performed as previously described.⁹ Briefly, subcutaneous tumor xenografts were established by injecting EAC cells (4 × 10⁶ cells per flank), suspended in 50% Geltrex, bilaterally into the flanks of 4- to 5-week old female athymic Crl:NU(NCr)-Foxn1^nu mice. For shRNA xenograft studies, mice were switched to a 625-mg/kg Dox diet 24 hours after inoculation for the duration of the study. For MEK inhibitor studies, after the xenograft tumors reached a minimum size of 50 to 60 mm³, mice were randomized and treated via oral gavage with vehicle (0.5% methylcellulose) or with vehicle containing 10 mg/kg of cobimetinib, once daily. Tumor volumes were estimated 2 to 3 times weekly. A 2-sided Student t test, assuming unequal variances, was used to determine significant differences in tumor volumes across comparisons. All animal procedures were approved by the Case Western Reserve University Institutional Animal Care and Use Committee and followed National Institutes of Health guidelines.