P7045

NT2197 mammary tumor cells (5×10⁴) were injected into the right side of the fourth mammary fat pads of Nude mice (Charles River Laboratories). 16 days post-injection, mice were given vehicle control (PBS;methylcellulose/Tween-80/DMSO), phenformin (50mg/kg) (Sigma-Aldrich P7045) via intraperitoneal injection, and/or lapatinib (50mg/kg) (lapatinib: LC Laboratories L-4804) via gavage every 24hr, for 11 days. On day 11, 3h post-treatment, tumors were rapidly dissected, washed with saline, flash frozen in liquid nitrogen and kept at −80 °C. All animal experiments were performed in accordance with the guidelines of the McGill University animal ethics committee and the Canadian Council on Animal Care as approved by the facility animal care committee (protocol #5518). Tumors were finely grounded in a mortar under liquid nitrogen, weighed and processed for GC/MS extraction (see section “Metabolite extraction and GC/MS analysis of tumor tissues”).

NT2197 mammary tumor cells (5×10⁴) were injected into both sides of the fourth mammary fat pads of Nude mice (Charles River Laboratories). The mammary tumor growth (mm³ ± SEM) was obtained by caliper measurements (n=10 tumors/treatment group) and were calculated according to following formulae: 4/3π (w² × l) as previously described, where l and w are diameter measurements of the length and width of the tumor, respectively (Ursini-Siegel et al., 2007 (link)). Once tumor volume reached 100~200 mm³, mice were given vehicle control (PBS;methylcellulose/Tween-80/DMSO), phenformin (50mg/kg) (Sigma-Aldrich P7045) via intraperitoneal injection, and/or lapatinib (50mg/kg) (lapatinib: LC Laboratories L-4804) via gavage every 24hr. All treatment conditions contained 12.5% DMSO and were resuspended in 0.5% methylcellulose-0.1% Tween-80 solution (Tween-80: Sigma-Aldrich P4780, methyl cellulose: 274429). On the day of necropsy mice were sacrificed 4h post-treatment, the mammary tumors were placed in 10% formalin to be fixed and later embedded in paraffin for immunohistochemistry. All animal experiments were performed in accordance with the guidelines of the McGill University animal ethics committee and the Canadian Council on Animal Care as approved by the facility animal care committee (protocol #5951).