Figure 3

Tumor growth in immunocompromised mice. (A) Flowchart depicting the protocol scheme for the animal study. Female athymic nude mice were inoculated with 4T1 engineered cells. On the seventh day after inoculation 4T1/EV, 4T1/IR-A, or 4T1/IR-B cells were treated or not with 10 nM insulin glargine, given s.c. for 5 days/week (n = 6 for each group). At day 25, tumor volume was measured and tumor tissue was collected. Mice were sacrificed at day 50 and pulmonary metastasis evaluated. (B) Images of explanted tumors at day 25. Scale bar: 3 cm. (C) Graph showing the tumor volume (cm³) in 4T1/IR-A, 4T1/IR-B and in control (4T1/EV) inoculated-mice. The data are the mean ± SE of the values obtained in five animals per group. N.S., p > 0.05; * p < 0.05; and ** p < 0.01, by ordinary one-way ANOVA followed by post hoc analysis of significance (Bonferroni test) for the comparison between more than two groups. (D) Enumeration of lung metastases by in vivo examination in saline-treated mice inoculated with 4T1/EV, 4T1/IR-A, or 4T1/IR-B cells. The data are the mean ± SE of the values obtained in five animals per group (left panel). Representative images of India ink-filled lungs dissected from 4T1 tumor-bearing mice on day 50 (right panel). (E) qRT-PCR measurement of IGF2 mRNA in mice tumors. NIH-3T3 and MCF7/IGF2 cells were used as positive controls. Data are mean ± SE of two independent biological replicates. (ns, not significant; * p <0.05; ** p< 0.01).