In vivo Xenograft studies and drug treatments

All animals were housed and handled in accordance with the approved guidelines of the Canadian Council on Animal Care (CCAC) “Guide to the Care and Use of Experimental Animals”. All experiments were performed under the approved McGill University Animal Care protocol (AUP # 7497 to JJL). All transplantation procedures were undertaken using isoflurane anesthesia. SUM159 cells infected with GECKO library were prepared in PBS (Phosphate Buffered Saline 1X, WISENT INC.) and transplanted into NSG mice by means of subcutaneous injection. For single KO cell transplantation, 1 million SUM159 cells were initially diluted in 20 µl PBS and 20 µl Matrigel (BD Bioscience) and then transplanted into mammary glands of NSG mice. When the tumors became palpable, after 3–4 weeks, paclitaxel (Sigma) and vehicle (control) were intraperitoneally administered twice per week. Paclitaxel was dissolved in 10% DMSO (Sigma), 40% PEG300 (Sigma), 5% Tween-80 (Sigma) and 45% saline. The mice were treated for 2–3 weeks before tumors reached maximum volume of 1000 mm³ and then were euthanized. Tumor volumes were documented. For tail vein injection, 1 million cells were prepared in 100 µl PBS and injected into the median tail vein. The mice were euthanized after ~4 weeks and the lung tissues were collected and stained in Bouin’s solution (Sigma) for at least 48 h. Lung metastatic nodules were counted under a microscope.