To organize lung sections, mouse lungs had been perfused with PBS to reduce circulatory blood. The many representative pictures have been obtained under 200x magnification. CD31 blood vessels or CD11b myeloid cells had been counted in 6 random fields. Actual time quantitative PCR RNA extraction, cDNA synthesis and true time PCR have been described previously. The primers for mouse VEGF, IL 1B, MMP9, FGF 2, S100A8, S100A9 and GAPDH were bought from SuperArray Bioscience. Western blot Harvested cells had been lysed and tissue samples had been homogenized with a homogenizer with modified RIPA buffer. Equal amounts of proteins have been subjected to SDS Page and immunoblotted with indicated antibodies. Evaluation of lung metastasis Mouse lungs had been perfused by intra tracheal injection of India ink and fixed in Feketes remedy for 10 min.
Metastatic nodules have been counted. For histological evaluation of lung micro metastases, five sections of lung from each mouse have been stained with H&E and examined under light microscope. The number of micro metastatic foci per field was counted in all 5 sections. Intravital multiphoton microscopy Implantation of 786 O pRC vector or 786 selelck kinase inhibitor O pRC STAT3C tumor cells into nude mice and treatment with AZD1480 or vehicle had been described previously. Tumor vasculature, apoptosis and extracellular matrix have been visualized by dextran rhodamine, Annexin V and Hoechst 33342. Statistics Two tailed students t test was used for statistical analysis. Differences had been considered statistically significant when p 0. 05. , p 0. 001; , p 0. 01 and, p 0. 05.
Results AZD1480 inhibits Renca tumor growth in vivo by using a reduction in tumor myeloid cell infiltration Our previous studies indicated that although AZD1480 could induce tumor growth inhibition and tumor cell apoptosis in vivo, in certain tumor cell lines it did not effectively inhibit tumor cell proliferation and induce apoptosis in vitro. Consistent with this observation, we found LY2811376 that AZD1480 treatment of 786 O human renal cancer cells and mouse Renca cells in vitro had only limited reduction in cell viability, although phosphorylated JAK2 and p STAT3 had been inhibited. These findings prompted us to investigate the in vivo antitumor effects of AZD1480 on Renca, a syngeneic murine renal carcinoma model. Renca tumor cells have been subcutaneously injected into BALB/c mice and treated with AZD1480 or vehicle for 21 days.
We observed a significant inhibition of tumor growth in AZD1480 treated group compared with vehicle treated group. Western blot analyses of the whole tumor lysates revealed a dramatic inhibition of p STAT3 by AZD1480 treatment.