Fig. 9.

IGF-1 is required for polarization and the proliferation and migration of lung cancer cells. A–H PBMC-derived macrophages were transfected with the indicated plasmid by lentivirus. A The relative expression of IGF-1 in macrophages was measured by qPCR. The mRNA expression was normalized to that of GAPDH, and the experiments were performed in triplicate. N = 5. B The knockdown efficiency of IGF-1 was further verified by western blotting, and β-actin was used as the loading control. N = 5. C–E After shRNA transduction, PBMC-derived macrophages were polarized to M1 or M2 macrophages for 48 h. C, D The relative expression of the M1 markers IL-1β, IL-12 and iNOS (C) and the M2 markers IL-10, Arg-1 and Mrc-1 (D) was determined by qPCR. The mRNA expression was normalized to that of GAPDH, and the experiments were performed in triplicate. N = 5. E After shRNA transduction, PBMC-derived macrophages were polarized to M2 macrophages for 48 h, which was then analysed by FACS and statistical analysis of the proportions of M2 macrophages. N = 5. F–H Forty-eight hours later, macrophage CM was collected to culture A549 and HCC827 cells for the indicated assays. F The proliferation of A549 and HCC827 cells was assessed by MTT assay at the indicated times. N = 5. G The survival and proliferation of A549 and HCC827 cells were determined by colony formation assay and statistical analysis of the colony numbers. N = 5. H The migration of A549 and HCC827 cells was measured by Transwell assay (left panel). Scale bar, 50 μm. The statistical analysis of the migrated cells is shown in the right panel. I–K Six-week-old nude mice were subcutaneously injected with 2 × 10⁶ A549 cells, a mixture of 2 × 10⁶ A549 cells plus 1 × 10⁶ shNC-transfected M2 macrophages, or a mixture of 2 × 10⁶ A549 cells and 1 × 10⁶ shGRP78-transfected M2 macrophages. N = 5. I Four weeks later, the mice were euthanized, and the tumour tissues were dissected for analysis. J The tumour growth kinetics were monitored by measuring the tumour volume every 7 days. K The expression of GRP78 in xenografted NSCLC tumour samples and in tumour-associated macrophages was measured by IHC. Scale bar, 100 μm. A–E, G–H The results are representative of three independent experiments. F, I, J The results are representative of five independent experiments. A, C, D, F, G, H, J, K, M Error bars represent the mean ± SD. p values were determined by one-way analysis of variance (ANOVA) (A, C, D, G, H), two-way ANOVA (F, J) or by unpaired two-tailed t test (E). ***p < 0.001, **p < 0.01, *p < 0.05