3D Outgrowth and Bioluminescence Imaging

3D-outgrowth quantification and in vivo bioluminescence imaging were carried out as described (Gooding et al, 2017 (link)). Cells were cultured in appropriate media supplemented with 5% Cultrex, as well as 5-FdU or 5-fluorouracil (5-FU; Sigma-Aldrich), the ATR inhibitors AZ20 (4-{4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}-1H-indole; MedChem Express) or VE-821 (3-amino-6-[4-(methlsulfonyl)phenyl]-N-phenyl-2-pyrazinecarboxamide; Sigma-Aldrich), the BLM inhibitor ML216 (1-(4-fluoro-3-(trifluoromethyl)phenyl)-3-(5-(pyridine-4-yl)-1,3,4-thiadiazol-2-yl)urea; MedChem Express), or the telomerase inhibitor BIBR1532 (Selleckchem) as indicated. For U2OS cells, Cultrex cushions were supplemented with type I collagen (3 mg/ml; BD Biosciences). In mice, 5-FdU and 5-FU were administered by slow intravenous injection (0.1 ml at a rate of 0.4 ml/min). Mice were randomly assigned to receive cell lines or treatments. Endpoints for 3D-outgrowth and pulmonary tumor assays were determined prospectively. Growth was normalized to an initial reading taken 24 h post-plating (in vitro) or immediately after inoculation (in vivo).