All animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee. Low passage xenografts were generated from primary surgical specimens collected at the Johns Hopkins Hospital, serially passaged in nude mice, and prepared as single cell suspensions as previously described (5 (link), 27 (link)). Tumor spheres enriched in CSCs were generated by culturing cells (200,000 cells/ml) in 60-mm ultralow attachment plates (Corning) and DMEM/F-12 (1/1) media (Invitrogen) supplemented with 1X B-27 supplement (Thermo Fisher Scientific), 20ng/ml bFGF (Invitrogen), 1% penicillin-streptomycin and 1% L-glutamine. Tumor cell differentiation was induced by culturing cells in complete media containing 10% FBS.
Tumor-initiating cell (TIC) frequency was determined by injecting L3.6pl cells (100,000) in serum-free DMEM and Matrigel (BD Biosciences) subcutaneously into the flanks of NSG mice. Following tumor formation, mice were provided doxycycline (2mg/ml, Sigma-Aldrich) in 5% sucrose. Following 9 days of treatment, tumors were collected, dissociated, and depleted of mouse cells then injected (50, 100, 250, and 500 cells) into secondary recipients. Tumor formation was monitored for 30 days, and (TIC) frequency was calculated using extreme limiting dilution analysis (ELDA) (28 (link)).
Tumor-initiating cell (TIC) frequency was determined by injecting L3.6pl cells (100,000) in serum-free DMEM and Matrigel (BD Biosciences) subcutaneously into the flanks of NSG mice. Following tumor formation, mice were provided doxycycline (2mg/ml, Sigma-Aldrich) in 5% sucrose. Following 9 days of treatment, tumors were collected, dissociated, and depleted of mouse cells then injected (50, 100, 250, and 500 cells) into secondary recipients. Tumor formation was monitored for 30 days, and (TIC) frequency was calculated using extreme limiting dilution analysis (ELDA) (28 (link)).