Figure 2.

IL6 sustains the stem-like characteristics of bladder cancer. (A) Primary and secondary spheroid assays were performed in the spheres treated with PBS, human recombinant IL6 (50 ng/mL) or the IL6 neutralizing antibody (20 µg/mL). The total numbers of spheroids from 5,000 cells were counted. The experiments were performed in triplicate (scale bar =50 µm). (B) The expression of stem-like associated genes (CD133, CD44, ABCG2, Oct-4 and Nanog) was assessed by qRT-PCR in the RT4 or J82 cells treated with PBS, human recombinant IL6 (50 ng/mL) or the IL6 neutralizing antibody (20 µg/mL). (C) The percentage of CD133 in the RT4 or J82 cells treated with PBS, human recombinant IL6 (50 ng/mL) or human recombinant IL6 (50 ng/mL) combined with the IL6 neutralizing antibody (20 µg/mL), as assessed by flow cytometry. (D) Male, non-obese, diabetic, severe combined immunodeficient (NOD-SCID) mice (n=6 in each group) were subcutaneously injected with 10,000 cells sorted from the bladder cancer spheres. Representative subcutaneous tumors from the mice after 6 weeks of transplantation are shown, as evaluated by bioluminescent imaging. (E) Tumor volume was measured once a week when tumors were palpable. Representative results from three independent experiments are shown, and all the values are expressed as the means ± SD, *P<0.05, **P<0.01 and ***P<0.001. qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; IL6, interleukin 6.