Figure 5. Macrophage-mediated proliferation of NBT2 cells is dependent on STAT3 phosphorylation.

(A) Mean fold change in percent of BrdU-positive NBT2 cells treated with solvent or JAK1/2 inhibitors AZD1480 (AZD) or ruxolitinib at indicated concentrations for NBT2 cells either alone or in co-culture with macrophages. Data were compiled from at least 3 independent experiments in triplicates; (B) Effects of AZD1480 and ruxolinitib on pSTAT3, STAT3, and MYC protein levels in lysates of NBT2 cells without or with a 6 and 24 hour co-culture with syngeneic macrophages in presence of AZD1480 (5 μM) or ruxolitinib (1 μM) as assessed by immunoblotting; (C) Immunoblot analysis of pSTAT3, STAT3, and MYC levels in protein lysates of three low MYC-expressing human NBL cell lines (LAN-6, CHLA-172, and CHLA-79) after 24 hour co-cultures with macrophages without or with ruxolitinib (1 μM); (D) Immunoblot analysis of STAT3 and pSTAT3 levels in protein lysates of tumors from NB-Tag mice or of subcutaneous tumors (Sub-Q) growing in NSG mice treated with ruxolitinib (60 mg/kg) by oral gavage twice daily for one week. (E) Tumor growth in NSG mice injected subcutaneously with 1X10⁶ NBT2 tumor cells alone in one shoulder or co-injected in the opposite shoulder with equal number of macrophages that were conditioned for 24-36 hours with NBT2 cells in the transwell system. Animals were administered ruxolitinib (60 mg/kg) or drug vehicle by oral gavage twice daily for 3 weeks [ANOVA p < 0.005 between the untreated group (n = 12) and the treated group (n = 6); no significant difference was observed between treatment groups].