A Critical Role for Macrophages in Promotion of Urethane-Induced Lung Carcinogenesis

Characterization of macrophage phenotype in lungs after urethane treatment. A, Representative FACS plots demonstrating identification of M2 polarized macrophages in whole lungs of a naive (untreated) mouse using anti-CD204 (scavenger receptor) and anti-CD206 (mannose receptor) Abs. B, Analysis of the percentage of CD204 and CD206 double-positive cells within CD45⁺CD68⁺ macrophages in lungs of naive mice (N) and mice at weeks 1–6 after urethane treatment. C–H, mRNA expression for M1 markers IL-12p35, CCL3, and IL-1β and M2 markers mannose receptor, Ym1, and IL-10 in total CD11b⁺ cells isolated from lungs of naive mice and 1–6 wk after a single injection of urethane. n = 4 mice per time point. *p < 0.05 compared with naive mice. I, Percentage of CD204 and CD206 double-positive cells within lung CD45⁺CD68⁺ macrophages at 4 mo after urethane injection. n = 4 mice/group. J–M, Representative photomicrographs of lung sections stained with anti–arginase-1 Abs at different time points after injection of urethane. Scale bars, 100 μM.

Depletion of lung macrophages during “early” and “late” phases of lung tumorigenesis reduces tumor number and size. A, Schematic representation of early- and late-stage macrophage depletion experiment. B, The number of total BAL cells and (C) alveolar macrophages in BAL of urethane-injected mice treated with clodronate or PBS liposomes. D, The number of lung surface tumors, (E) surface tumor diameter, (F) tumor number per lung section, and (G) AAH lesions per H&E-stained lung section. Data are presented as mean ± SEM of 12 mice for the PBS group (early and late PBS control groups are combined) and 11 mice for each clodronate group. *p < 0.05 compared with PBS control group.

Depletion of alveolar macrophages reduces tumor angiogenesis. A, VEGF concentration in BAL from mice treated with weekly IT clodronate beginning at 4 wk posturethane (late stage) or throughout the 4-mo period compared with control mice treated with PBS liposomes. B, Density of blood vessels in tumors and (C) blood vessel area in tumors. The blood vessel density (or area) was assessed as the number (or area) of CD34⁺ endothelial cells per square millimeter of tumor. D, Representative photomicrographs of lung sections from tumor-bearing mice immunostained for CD34. Scale bars, 100 μM. n = 10–12 mice/group. *p < 0.05 compared with PBS liposome controls.

Depletion of macrophages with liposomal clodronate attenuates activity of NF-κB and alters inflammatory mediator expression in the lungs after urethane. A, Time course of NF-κB–dependent lung bioluminescence in NGL mice treated weekly with PBS or clodronate liposomes. n = 6 for PBS liposome; n = 10 for clodronate groups. *p < 0.05 compared with PBS liposome group at the same time point. B, Representative images for NF-κB–dependent lung bioluminescence, (C) NF-κB–dependent luciferase activity (relative light units) in lung homogenates, and (D) representative photomicrographs of lung sections immunostained for GFP (scale bars, 100 μM; arrows point to GFP⁺ cells) at day 14 after urethane injection in mice treated with PBS or clodronate liposomes. n = 5 per group. E–I, Expression of mRNA for IL-12p35, iNOS, IL-6, TNF-α, and IL-10 in lung tissue at day 11 after urethane injection in mice treated with PBS or clodronate liposomes (n = 5/group; *p < 0.05). Animals were given PBS or clodronate liposomes on days 0 and 7 after injection of urethane.