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. 2012 Aug 6:2:89.
doi: 10.3389/fonc.2012.00089. eCollection 2012.

Irradiation promotes an m2 macrophage phenotype in tumor hypoxia

Chi-Shiun Chiang  1 , Sheng Yung Fu, Shu-Chi Wang, Ching-Fang Yu, Fang-Hsin Chen, Chi-Min Lin, Ji-Hong Hong
Affiliations

Irradiation promotes an m2 macrophage phenotype in tumor hypoxia

Chi-Shiun Chiang et al. Front Oncol. .

Abstract

Macrophages display different phenotypes with distinct functions and can rapidly respond to environmental changes. Previous studies on TRAMP-C1 tumor model have shown that irradiation has a strong impact on tumor microenvironments. The major changes include the decrease of microvascular density, the increase of avascular hypoxia, and the aggregation of tumor-associated macrophages in avascular hypoxic regions. Similar changes were observed no matter the irradiation was given to tissue bed before tumor implantation (pre-IR tumors), or to established tumors (IR tumors). Recent results on three murine tumors, TRAMP-C1 prostate adenocarcinoma, ALTS1C1 astrocytoma, and GL261 glioma, further demonstrate that different phenotypes of inflammatory cells are spatially distributed into different microenvironments in both IR and pre-IR tumors. Regions with avascular hypoxia and central necrosis have CD11b(high)/Gr-1+ neutrophils in the center of the necrotic area. Next to them are CD11b(low)/F4/80+ macrophages that sit at the junctions between central necrotic and surrounding hypoxic regions. The majority of cells in the hypoxic regions are CD11b(low)/CD68+ macrophages. These inflammatory cell populations express different levels of Arg I. This distribution pattern, except for neutrophils, is not observed in tumors receiving chemotherapy or an anti-angiogenesis agent which also lead to avascular hypoxia. This unique distribution pattern of inflammatory cells in IR tumor sites is interfered with by targeting the expression of a chemokine protein, SDF-1α, by tumor cells, and this also increases radiation-induced tumor growth delay. This indicates that irradiated-hypoxia tissues have distinct tumor microenvironments that favor the development of M2 macrophages and that is affected by the levels of tumor-secreted SDF-1α.

Keywords: radiation; tumor microenvironment; tumor-associated macrophages.

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Figures

Figure 1
Figure 1
The association of CD68+ TAMs with hypoxia is tumor and tissue dependent. The distribution of CD68+ TAMs and PIMO+ hypoxia in ALTS1C1 astrocytoma (A,C) and GL261 glioma (B,D) grown in the thigh (i.m.) (A,B) or in the brain (i.c.) (C,D). Green: anti-PIMO stain for hypoxic region; red: anti-CD68 antibody for TAMs. Merged images: the colocalization of hypoxia and TAMs. Scale bar = 100 μm.
Figure 2
Figure 2
The gene expression profiles between ALTS1C1 and GL261 cells by (A) cDNA microarray and (B) RT-PCR analysis. The mRNA level of primary astrocyte was used as reference.
Figure 3
Figure 3
Irradiation redistributes the localization of subtypes of inflammatory cells. (A) The distribution of CD31, CD11b, Gr-1, CD68, and F4/80 staining with PIMO+ hypoxia in series 25 Gy-irradiated-TRAMP-C1 tumor sections. Green: anti-PIMO stain for hypoxic region; red: anti-CD31, anti-CD11b, anti-Gr-1, anti-CD68, or anti-F4/80 antibody. Merged images: the colocalization of hypoxia and inflammatory cells or vessels. Scale bar = 100 μm. (B) Percentage of CD11b, CD68, F4/80, or Gr-1 positive cells within control or 25 Gy-irradiated TRAMP-C1 tumors as assayed by flow cytometry.
Figure 4
Figure 4
Irradiation induces CD68+ TAMs aggregation in hypoxic regions in both ALTS1C1 and GL261 i.c. tumor models. The distribution of CD68+ TAMs and PIMO+ hypoxia in 8 Gy-irradiated ALTS1C1 astrocytoma (A) and GL261 glioma (B) grown in the brain. Green: anti-PIMO stain for hypoxic region; red: anti-CD68 antibody for TAMs. Merged images: the colocalization of hypoxia and TAMs. Scale bar = 100 μm.
Figure 5
Figure 5
Pre-irradiation induces CD68+ TAMs aggregation in hypoxic regions in ALTS1C1 tumors grown in both the brain (i.c.) and thigh (i.m.). The correlation of CD68+ TAMs with PIMO+ hypoxia in pre-irradiated ALTS1C1 astrocytoma grown in the brain (i.c.) (A) or in the thigh (i.m.) (B). Green: anti-PIMO stain for hypoxic region; red: anti-CD68 antibody for TAMs. Merged images: the colocalization of hypoxia and TAMs. Bar: 100 μm.
Figure 6
Figure 6
The nature of PIMO+ hypoxic region in control and irradiated tissues is different. The IHC staining for the distribution of hypoxia and vascular in control (A,C), irradiated (B), or pre-irradiated (D) ALTS1C1 tumors. The hypoxic regions in control tumors were vascularized. There was almost no vasculature in the hypoxic regions of irradiated (B) or pre-irradiated tumors (D). Green: anti-PIMO stain for hypoxic region; red: anti-CD31 antibody for vessels. Merged images: the colocalization of hypoxia and vessels. Scale bar = 100 μm.
Figure 7
Figure 7
IHC staining for CD31,Gr-1, and CD68 in series sutent-treated tumor sections. Administration of sutent decreased vascular density and accumulated Gr-1+ cells at central necrosis in chronic hypoxia. However, CD68+ TAM does not aggregate at chronic hypoxia. Scale bar = 100 μm.
Figure 8
Figure 8
Aggregated TAMs express higher level of Arg-1 than random TAMs. (A) The IHC staining for the expression of Arg-1 by random or aggregated CD68+ TAMs in control, irradiated (IR) or pre-irradiated (pre-IR) ALTS1C1 tumor Green: anti-Arg-1 antibody; red: anti-CD68 antibody. Merged images: the colocalization of Arg-1 and TAMs. Scale bar = 100 μm. (B) The percentage of Arg-1+ TAMs (left graph) and the mean intensity of Arg-1 staining (right graph) by random or aggregated CD68+ TAMs in control (rectangular dot region), irradiated (IR) (white bar) or pre-irradiated (pre-IR) (dark dot bar) ALTS1C1 tumors. The rectangular dot region represents the average value ± SD in control ALTS1C1 tumor for the purpose of clarity.
Figure 9
Figure 9
Suppression of SDF-1 expression by ALTS1C1 tumor disrupts pre-IR-induced TAM association with hypoxia in both i.c. (A) and i.m. (B) models and prolong pre-IR-induced growth delay in both i.c. (C) and i.m. (D) models. Green: anti-PIMO stain for hypoxic region; red: anti-CD68 antibody for TAMs. Merged images: the colocalization of hypoxia and TAMs. Scale bar = 100 μm.
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