Figure 2.

Deletion of IL-6 from KPC cells prevented muscle wasting in vitro and increased survival in mice. (A) Targeted mutagenesis of the Il6 gene was performed, and a transfection control clone (KPC) and an Il6 ablated clone (KPC IL6^KO) were selected for use in downstream experiments based on their Il6 expression relative to the untransfected parental cell line (KPC-P). Clones were cultured individually in triplicate for n = 3 per group, and error bars are mean plus SD. (B) To determine if deletion of IL-6 affected tumor cell growth, a proliferation assay was performed comparing the clones, where clones were cultured in triplicate (n = 3 per group) and measurements were taken every hour for 100 h; growth curves represent the mean proliferation of triplicate wells over time. (C) Myoblasts were differentiated into myotubes and treated in triplicate (n = 3 per group) with 30% CM from tumor clones to measure effects on myotube atrophy and the activation of STAT3. (D) Western blotting (WB) analysis using the pooled myotube protein lysates from n = 3 per group was performed to measure phosphorylation of STAT3 (p-STAT3) as an indication of STAT3 activation. (E and F) Myotubes were visualized using IF with an anti-myosin heavy chain (MHC) antibody (E), and myotube atrophy was measured from 20 random fields per well (n∼150 myotube diameter measurements per well, n = 3 wells per group). Scale bar = 50 µm. Error bars represent mean myotube diameter and SD, and significant differences (**, P < 0.001) were determined using ANOVA and Tukey’s multiple comparisons test (F). (G and H) To verify atrophy was influenced by IL-6, myotubes were treated in triplicate with KPC CM and IL-6 neutralizing antibody as well as KPC IL6^KO CM plus recombinant IL-6 with and without the presence of an anti–IL-6 neutralizing antibody. Myotubes were visualized with MHC IF, and diameter was measured as described in F. Scale bar = 50 µm. Error bars are mean and SD, and significant differences (*, P < 0.05; **, P < 0.001) were determined using ANOVA and Tukey’s multiple comparisons test. (I) KPC tumor cells were orthotopically implanted into mice, and tumors were excised, sectioned, and reacted with anti–IL-6 IHC to verify tumor cell IL-6 deletion. Insets are increased magnification to show staining; scale bar = 20 µm. (J) Survival was measured in mice orthotopically implanted with KPC and KPC IL6^KO tumor cells. The dashed line represents median survival, n = 15 per group, and statistical difference (**, P < 0.001) was determined using a Kaplan-Meier estimate. All panels represent data from single experiments. Diff. media, DM.