Female severe combined immunodeficiency scid mice

Female severe combined immunodeficiency (SCID) mice (18–20 g; 5 weeks of age; 5 mice/group) were purchased from Charles River Laboratories. All experimental procedures were conducted following a protocol approved by the Institutional Animal Care and Use Committee of Asan Institute for Life Sciences (2013–01–066). Tumors were grown by implanting cells (1 × 10⁶ HCC827/GR cells or 5 × 10⁶ HCC827/ER cells) in Matrigel (BD Biosciences) and subsequently into the mouse flanks. Treatment with the vehicle control commenced when the tumors reached a volume of 50–100 mm³ (20 mg/kg AUY922 via intra-peritoneal injection; 5 days/week). Treatment was stopped on the indicated day, and mice received follow-up examinations to document tumor recurrence. To measure tumor size, the length (L) and width (W) of each tumor was measured using calipers, and tumor volume (TV) was calculated as TV = (L×W²)/2. Immunohistochemical staining was performed using a specific primary antibody (Ki-67; DakoCytomation, Los Angeles, CA), the EnVision Plus staining kit (DakoCytomation), and the APO-Direct terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit (Millipore) as directed by the supplier’s instructions. Quantitative analysis of each stained section was performed by counting all immunopositive cells in 5 arbitrarily selected fields (×400 magnification).

To establish the xenograft model, female severe combined immunodeficiency (SCID) mice (18–20 g, 6 weeks of age) were purchased from Charles River Laboratories. All experimental procedures were conducted following a protocol approved by the Institutional Animal Care and Use Committee of Asan Institute for Life Sciences (2015-02-062). Tumors were grown by implanting cells (1–5 × 10⁶ cells/0.1 mL) in 50% Matrigel (BD Biosciences), and subcutaneously injected into the right flank of the animals. Drug treatment was started in 5 mice per group when the tumor volume reached 50–100 mm³ with control (10% 1-methyl-2-pyrrolidinone: 90% PEG-300, oral gavage), WZ4002 (30 mg/kg, oral gavage, 5 days a week), BI836845 (100 mg/kg, intraperitoneal, 2 days a week), or WZ4002 plus BI836845. To estimate the tumor size, the length (L) and width (W) of each tumor were measured using calipers, and the tumor volume (TV) was calculated as TV = (L× W²)/2. To evaluate the p-IGF1R in tumors from xeongrafts, p-IGF1R was measured by immunohistochemistry (IHC) with p-IGF1R antibody (phosphor Y1161, Abcam). IHC staining was performed using the EnVision Plus staining kit (DakoCytomation).

All in vivo experiments were performed according to the institutional guidelines and approved by the Institutional Animal Care and Use Committee. shRNA expression was induced in the +DOX group 24 h prior to implantation by addition of 1 μg/ml doxycycline to the culture media, while cells in the −DOX group were left untreated. Female Severe combined immunodeficiency (SCID) mice (Charles River laboratory) in the +DOX group were switched to 400 ppm doxycycline diet (Harlan) 3 days prior to implant, while animals in the −DOX group were maintained on standard mouse diet. On the day of implant, the SKHEP1 reporter cell lines were suspended in PBS at a concentration of 1 × 10⁷ cells per milliliter, and 500 μl of the suspension was injected into the peritoneal cavity of each mouse. Luciferase imaging was performed on a Xenogen IVIS imaging system (Perkin Elmer) following intraperitoneal injection of 150 mg/kg of D-luciferin (Perkin Elmer) prepared in PBS. Anti-miR compounds were formulated in PBS and administered intraperitoneal injection at 25 mg/kg.