Orthotopic MB49 and MBT-2 challenges were 8×104 or 1×106 cells in 50 µL Dulbecco’s Phosphate Buffered Saline (DPBS, Sigma Aldrich) into bladder, respectively, via indwelling urinary catheter in female mice under isoflurane anesthesia, as described.13 Bladder tumor growth was monitored in vivo by tumor bioluminescence and bladder weight at sacrifice. Subcutaneous tumor challenge was 2×105 MB49 cells in 100 µL DPBS in each hind flank of female mice (two tumors/mouse).14 Tumor growth was measured using Vernier calipers and volume was calculated as (length x width2)/2. Orthotopic challenge of ID8agg was 4×106 cells injected intraperitoneally into females.9 Lung tumors were generated via intravenous injection of 7×105 or 2.5×105 MB49 or MBT-2 cells, respectively,15 in 200 µL DPBS into male mice. Survival was defined as moribundity, spontaneous death, tumor volume ≥ 2000 mm3 (subcutaneous challenge), or significant weight loss from baseline (≥15% for bladder and ≥20% for lung tumors). To ensure uniform tumor size across all treatment groups, mice were occasionally excluded on first tumor measurement before randomization, if determined to be an outlier by Grubbs’ test.

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