Tumor progression stage and anatomical site regulate tumor-associated macrophage and bone marrow-derived monocyte polarization

Abstract

Tumor-associated macrophages (TAMs) encourage and coordinate neoplastic growth. In late stage human lung adenocarcinoma, TAMs exhibited mixed M1 (classical; argI(low)iNOS(high)) and M2 (alternative; argI(high)iNOS(low)) polarization based on arginine metabolism. In several murine cancer models including chemically and genetically-induced primary lung tumors, prostate tumors, colon xenografts, and lung metastases, TAMs expressed argI(high)iNOS(low) early during tumor formation; argI(low)iNOS(high) polarization also occurred during malignancy in some models. In a chemically-induced lung tumor model, macrophages expressed argI(high)iNOS(low) within one week after carcinogen treatment, followed by similar polarization of bone marrow-derived monocytes (BDMCs) a few days later. TAMs surrounding murine prostate tumors also expressed argI(high)iNOS(low) early during tumorigenesis, indicating that this polarization is not unique to neoplastic lungs. In a human colon cancer xenograft model, the primary tumor was surrounded by argI(high)iNOS(low)-expressing TAMs, and BDMCs also expressed argI(high)iNOS(low), but pulmonary macrophages adopted argI(high)iNOS(low) polarization only after tumors metastasized to the lungs. Persistence of tumors is required to maintain TAM polarization. Indeed, in both conditional mutant Kras- and FGF10-driven models of lung cancer, mice expressing the transgene develop lung tumors that regress rapidly when the transgene is silenced. Furthermore, pulmonary macrophages expressed argI(high)iNOS(low) on tumor induction, but then returned to argI(low) iNOS(low) (no polarization) after tumors regressed. Manipulating TAM function or depleting TAMs may provide novel therapeutic strategies for preventing and treating many types of cancer.

Figure 1

Activation state of macrophages associated with human AC. A: H&E-stained human ACs have both TAMs (circles) and TIMs (squares). B: Immunofluorescent staining for macrophages (green, CD-68) expressing both mannose receptor (blue) and iNOS (red). White circles indicate corresponding areas of macrophage localization. T indicates tumor tissue. Magnification for H&E images is ×400 and ×630 for immunofluorescent staining.

Figure 2

Time courses of macrophage activation and IL-4 and IFN-γ production in urethane-treated A/J mice. BAL fluid, pulmonary macrophages, and BDMCs were isolated from urethane treated A/J mice at the indicated times. A: BAL macrophage (filled triangle) and BDMC (open triangle) polarization were examined by arginase I and iNOS immunofluorescence and compared with control naïve macrophage expression. *P < 0.05 versus 1 day BAL macrophage, **P < 0.01 versus 1 day BDMC. B: IL-4 content in BAL from control (filled square) and urethane-treated (open square) A/J mice. *P < 0.05 versus control. C: IFN-γ expression in BAL from control (filled square) and urethane-treated (open square) A/J mice. *P < 0.05 versus control. Cytokine content was assessed by ELISA.

Figure 3

Peritoneal macrophages (indicated by white arrowheads) in urethane-treated A/J mice. Peritoneal macrophages (CD-68, green) from naïve A/J mice and mice with benign (24 weeks after urethane) and malignant (42 weeks after urethane) lung tumors express arginase I (blue) but not iNOS (red). Magnification ×630.

Figure 4

TAM activation in a primary murine model of prostate cancer. A: H&E sections show a moderately-differentiated prostate tumor (T). TAMs, indicated by white arrowheads (CD-68, green) express arginase I (blue) but not iNOS during early tumor progression. B: Tissue adjacent to a late stage, poorly-differentiated prostate tumor showing TAMs (black circles) stained by H&E. These macrophages (green) express both iNOS (red) and arginase I (blue). White circles denote corresponding areas of macrophage localization.

Figure 5

Pulmonary macrophage activation in a mutant Kras transgenic lung cancer model. Conditional overexpression of mutant Kras induces pulmonary tumors. A: Two months after DOX treatment, H&E images show the location of macrophages peripheral to, TAMs (circles), and within, TIMs (squares), pulmonary tumors (T, border indicated by white dashed line). Macrophages identified with F4/80 (green) also stain for arginase I (blue) but not iNOS (red). B: Seven days after DOX removal, H&E staining indicates lesions and associated macrophages (circles) are still present in the lungs. TAMs express arginase I (blue). C: One month after DOX removal, tumors have regressed. Pulmonary macrophages (circles) expressed neither arginase I nor iNOS. Magnification ×630 for fluorescent images, ×400 for H&E images.

Figure 6

Activation status of macrophages in a human HT-29 colon cancer xenograft model. Macrophages and BDMCs are identified with anti-F4/80 or anti–CD-68 respectively (green). A: H&E staining shows that macrophages (black circles) surrounding the xenograft (T) express arginase I (blue) but not iNOS. White circles indicate areas of macrophage localization. B: BDMCs (green) isolated from xenograft-bearing mice express arginase I (blue) but not iNOS. C: H&E section shows pulmonary macrophages (black circles) in lungs containing HT-29 metastases (black arrow). Macrophages positively stain for arginase I (blue) but not iNOS. White circles indicate areas of macrophage localization. D: Pulmonary macrophages (green, white circles) in lungs with no detectable metastases stain for neither arginase-I nor iNOS. Magnification ×630 for fluorescent images, ×400 for H&E images.

Figure 7

Schematic summarizing macrophage activation in mouse models of lung cancer, prostate cancer, tumor regression, and metastasis.