New drugcombination may effectively treat often fatal childhood brain tumor CUAnschutz
in UW228-3 cells and stable expression of NF-κB reporter construct pHAGE NF-κB-TA-LUCUBC-GFP-W in D425 MED and MED8A cells were achieved by lentiviral transduction. The NF-κB reporter plasmid contains four copies of a consensus NF-κB binding sequence in addition to a minimal promoter . Pure populations of each stable cell line were sorted by flow cytometry for stable GFP expression using the Moflo XDP .
Statistical gene set analysis was performed using the non-parametric Kruskal-Wallis test to determine differential expression at the gene level using anti-CD14 microbeads , then plated at 1×106/ml in 150×25 mm tissue culture plates in RPMI 1640 with 10% FBS, penicillin/streptomycin, glutamine, and HEPES -1-piperazineethanesulfonic acid). To generate monocyte-derived macrophages, isolated monocytes were treated for 7 days with human recombinant macrophage colony-stimulating factor .
Tumor formation was followed by bioluminescence imaging on an IVIS spectrum instrument and quantified with Live Image V.4.0 software . MB-engrafted mice were given a daily treatment of CI-994 or vehicle control per oral gavage until they reached morbidity. The drug solution of CI-994 was prepared according to the manufacturer’s instructions. Briefly, CI-994 powder was first dissolved in DMSO and stored at −20°C. On the day of administration, a final solution was prepared using diH0 and administered at a concentration of 30 mg/kg per oral gavage. Control mice received DMSO alone. For combination studies with anti-CD47 mAb, anti-CD47 was administered as before at 10 mg/kg every 48 hours.
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