In vivo mouse xenograft tumor model

In vitro cultured CD133⁺ GBM cells (2-5x 10⁶ in 0.1 ml) were injected subcutaneously into the flanks of 3-5 week old Balb/c nude mice (n=8/group; 18-22g). After the tumor was established (about 50-75 mm³), as determined by caliper measurements, the mice were randomly distributed into the following 4 experimental groups: (a) DMSO control: 100μL DMSO/PBS (1:1) ; (b) TMZ-only: 20mg/kg TMZ in 100μL DMSO/PBS; (C) DMC-only: 10 or 30mg/kg DMC in 100μL DMSO/PBS; and (iv) 20mg/kg TMZ plus 10 or 30mg/kg DMC in 100μL DMSO/PBS. The treatments were given daily for 24 days. Then, tumor volumes were calculated using the following formula: tumor volume (TV) = (length × width²) × 0.5. Based on the absolute tumor volume measurements, we calculated the relative tumor volume (RTV) as RTV = V_t/V₀ (V₀, the measured tumor volume at day 0; Vt, the measured tumor volume at each time point). Further, the relative tumor proliferation rates (T/C %) were calculated using the following formula: T/C % =(T_RTV/C_RTV) x 100%, (T_RTV: the treatment group RTV; C_RTV: model control group RTV). Also, the tumor growth inhibition rates (TGI %) were calculated using the following formula: Tumor growth inhibition rate =(Average tumor weight of experimental group-Average tumor weight of model group)/Average tumor weight of model group x 100%. All mice procedures were conducted in accordance with the Animal Care guidelines by the First People's Hospital of Kunshan affiliated with Jiangsu University. After 30 days, tumor samples were snap-frozen for further experiments.