Relationship between serum IL-6 levels and survival.

<p>(<b>A</b>) A conservative threshold linked 20 or 21 pg/ml of IL-6 in serum (y-axis) with 21 to 28 days of survival time (x-axis), as determined by AIC analysis (see Methods), satisfactorily identifying the most appropriate cut-off level (z-axis, AIC = −10.395). Note that patients with serum IL-6 levels ≥21 pg/ml were predicted to have less survival time. (<b>B</b>) The probability of surviving one and three months after evaluation was lower in patients with IL-6 >21 pg/ml than those with <21 pg/ml (one month survival, 20.0% vs. 77.8%, p = 0.007; and three months survival 10.0% vs. 33.3%, p = 0.025). (<b>C</b>) The proportion of patients with IL-6 ≥21 pg/ml was 80.0% for patients surviving less than one month, 20.0% for patients surviving one to three months and 25.0% for patients surviving more than three months.</p

Patients’ characteristics and comparison between patients with or without cachexia.

<p>Plus-minus data are presented as means ± SD.</p

Effects of MR16-1 on carcass weight (a), tumor growth (b), food intake (c), and water intake (d).

<p>Transplantation of IL-6-expressing LLC cells caused cachexia in mice (groups 3 and 4), while groups 1 and 2 received only the same amount of saline solution, 0.9% sodium chloride (healthy control groups). We administered MR16-1 (groups 2 and 4) or 0.9% saline (groups 1 and 3), respectively. Body weight, tumor size and food and water intake were measured on days 0, 3, 7, 10, 14, 17 and 21 after transplantation. The measured quantity of food or water was divided by the number of mice and days to determine each intake per animal per day. Statistical significance was evaluated with Tukey’s Honest Significant Differences (HSD) after the one-way ANOVA. Data represent mean and S.D. (S.D. data of weight are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102436#pone-0102436-t004" target="_blank">Table 4</a>). Tumor growth was not significantly different between groups 3 and 4 (B), but body weight (A) and food (C) and water intake (D) were significantly improved in group 4 (†, p<0.01). No significant differences were observed in the carcass weight and food and water intakes between the untreated (group 1) and treated healthy mice (group 2).</p

Safety of MR16-1 in healthy control mice.

<p>To evaluate the safety of MR16-1, we appraised other biochemical parameters in the blood of healthy control mice. Group 2 received MR16-1 treatment, whereas group 1 received the same amount of saline solution.</p><p>Abbreviations used are: AST, aspartic acid aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase.</p

Correlation between patients’ characteristics and survival or IL-6 levels.

<p>We selected 19 patients with stage IIIB or IV and supportive care after evaluation, and analyzed the correlation between patients’ characteristics and survival time or IL-6 level. All those patients had been diagnosed with non-small cell lung cancer (NSCLC).</p

Cachectic parameters in control and treated mice at day 21.

<p>In cancer cachectic groups (groups 3 and 4), Lewis Lung Carcinoma (LLC) cells transfected with IL-6 cDNR (LLC-IL6) were implanted subcutaneously into a total of 20 C57BL/6J mice. We administered MR16-1, anti-murine IL-6 receptor monoclonal antibody, intraperitoneally at a dose of 20 mg/kg once a week into 10 mice (Group 4), while 0.9% saline into 10 mice (Group 3), respectively.</p><p>Eight mice in both the MR16-1 and control groups survived for 21 days after the tumor inoculation. After sacrifice, these mice were evaluated for cachectic parameters. Carcass weight was weight without tumor.</p><p>Data represent mean ± S.D. Statistical significance was evaluated with Turkey’s test after a one-way ANOVA.</p>*<p>Significantly different from group 1 (tumor (−), MR16-1 (−)); p<0.01.</p>**<p>Significantly different from group 1 (tumor (−), MR16-1 (−); p<0.05.</p>***<p>Significantly different from group 3 (tumor (+), MR16-1 (−)); p<0.01.</p>****<p>Significantly different from group3 (tumor (+), MR16-1 (−)); p<0.05.</p

Effect of MR16-1 on survival of LLC-IL6 transplanted mice.

<p>Each group consisted of ten LLC-IL6 transplanted mice. One group received MR16-1 (solid line), and the other group received only the same amount of saline solution (dotted line). Weekly treatment had been continued with the same interval until death was confirmed. Mean ± S.D. of the survival of LLC-IL6 transplanted mice without MR16-1 was 28.5±4.1 days. The injection of MR16-1 prolonged the survival of LLC-IL6 transplanted mice to 36.6±11.1 days (p = 0.016).</p

IGF1R was phosphorylated on hypoxic GRPs, and knockdown of IGF1 decreased the population of CD133- and Oct4-positive hypoxic GRPs.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for IGF1 in PC9 or HCC827 parental cells, GRPs, and hypoxic GRPs. Data were normalized to actin expression. **p<0.001. <b>B.</b> PC9 cells, grown on Lab-Tek chamber slides with or without 1 µM gefitinib and under normoxic or hypoxic conditions for 72 h, fixed, and incubated with the primary antibodies against phosphorylated IGF1R (pIGF1R) and then with secondary antibodies labeled with Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained using an Axioplan 2 system imaging with AxioVision software. Images used to compare PC9 parental cells, GRPs, and hypoxic GRPs were acquired with the same instrument settings and exposure times, and were processed similarly. The number of pIGF1R-positive cells was counted, and the ratio of these cells was calculated in five fields in each experiment. **p<0.001. <b>C.</b> IGF1 expression was knocked down in PC9 or HCC827 hypoxic GRPs by using small interfering RNA (siRNA) in Lab-Tek chamber slides. Immunofluorescence staining for pIGF1R, CD133, or Oct4 was then performed. Two specific siRNAs and one non-specific control were used. The numbers of pIGF1R-, CD133- and Oct4-positive cells were counted, and the ratio of these cells was calculated in five fields for each experiment. **p<0.001, *p<0.01.</p

GRPs were highly enriched for gene expression of stemness, IGF1, and IGF1R.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1, which are stemness genes in PC9 or HCC827 parental cells and GRPs. <b>B.</b> Quantitative RT-PCR was performed with primers specific for IGF1 and IGF1R in PC9 or HCC827 parental cells and GRPs. Data were normalized to actin expression. *p<0.01, **p<0.001, ***p<0.0001.</p

Sphere-forming ability of GRPs was upregulated and hypoxia increased this capacity.

<p>PC9 parental cells, GRPs, and hypoxic GRPs were prepared at densities of 2.5×10³ cells per 2 mL per well in serum-free media supplemented with growth factors and seeded into 6-well ultra-low attachment plates. PC9 parental cells and GRPs were incubated under normoxic conditions, while PC9 hypoxic GRPs were grown under hypoxic conditions. Culture medium was fed every 3 days. The number and size of spheres were recorded and immunofluorescence was performed 7 days after the start of the culture period. Spheres were fixed and incubated with primary antibodies against CD133, Oct4, or phosphorylated IGF1R (pIGF1R), and then with secondary antibody labeled with Alexa Fluor 594 goat anti-mouse IgG (red) or Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained on an Axioplan 2 imaging system with AxioVision software. <b>A.</b> The number of spheres was significantly increased in PC9 GRPs compared to in parental cells, and was further increased in PC9 hypoxic GRPs. *p<0.01, # p<0.05. <b>B.</b> Sphere size of PC9 hypoxic GRPs was significantly greater than that of parental cells. # p<0.05. <b>C.</b> Immunofluorescent images of control cells of PC9 (left) and spheres of GRPs (right) for CD133, Oct4, and pIGF1R.</p