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. 2013 Aug 5;8(8):e69182.
doi: 10.1371/journal.pone.0069182. Print 2013.

Radiation therapy-induced tumor invasiveness is associated with SDF-1-regulated macrophage mobilization and vasculogenesis

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Radiation therapy-induced tumor invasiveness is associated with SDF-1-regulated macrophage mobilization and vasculogenesis

Shu-Chi Wang et al. PLoS One. .

Abstract

Radiation therapy (RT) remains the front-line treatment for high-grade gliomas; however, tumor recurrence remains the main obstacle for the clinical success of RT. Using a murine astrocytoma tumor cell line, ALTS1C1, the present study demonstrates that whole brain irradiation prolonged the survival of tumor-bearing mice, although the mice eventually died associated with increased tumor infiltration. Immunohistochemical (IHC) analysis indicated that RT decreased the microvascular density (MVD) of the primary tumor core, but increased the MVD of the tumor invasion front. RT also increased the number of tumor-associated macrophages (TAMs) and the expression of stromal cell-derived factor-1 (SDF-1) and hypoxia-inducible factor-1 (HIF-1) at the tumor invasion front. SDF-1 expression suppressed by siRNA (SDFkd tumors) showed a decrease in RT-enhanced tumor invasiveness, leading to prolonged survival of mice bearing these tumors. The invasion front in SDFkd tumors showed a lower MVD and TAM density than that in the islands of the control or irradiated ALTS1C1 tumors. Our results indicate that tumor-secreted SDF-1 is one key factor in RT-induced tumor invasiveness, and that it exerts its effect likely through macrophage mobilization and tumor revascularization.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Local brain irradiation prolonged the survival of tumor-bearing mice, but mice eventually died due to increased tumor.
(A) Kaplan-Meier survival curves of ALTS1C1 glioma-bearing mice after 8 Gy or 15 Gy single-dose irradiation. The arrow indicates the time (day 13 after brain tumor implantation) at which radiation was given. (B) The mean diameter of control and irradiated ALTS1C1 brain tumors measured at the maximum cross-section in H&E-stained tissues. (C) Low-magnification merged image showing tumor invasion into the adjacent brain tissues. Scale bar  = 1 mm. (D) Scatter plot of the invasion islands in the ALTS1C1 control and single-dose irradiated brain tumors. The invasive pattern was assessed in whole-brain serial sections by DAPI staining. The plot represents the average island number of each brain in each group, as calculated using a microscope (×100 power). * p<0.05.
Figure 2
Figure 2. Irradiation increased the MVD and TAM density in the infiltrating islands.
(A) IHC staining of the vessels by CD31 antibody (red) and of the nuclei by DAPI (blue) in the tumor core region of the control, 8 Gy, or 15 Gy single-dose irradiated ALTS1C1 tumors. Scale bar  = 100 μm. (B) Quantification of MVD in CD31-positive cells in the brain tumor core region (n = 6∼12 fields per tumor, with a total of five tumors in each group; * p<0.05). (C) Confocal imaging of IHC-stained sections for detection of CD31 (red), Iba-1 (red), CD68 (red), and nuclei staining by DAPI (blue) in infiltrating islands of control and irradiated ALTS1C1 tumors. Scale bar  = 20 μm. (D) Quantification of MVD and cells expressing Iba-1, and CD68 in the infiltrating islands of control or single-dose irradiated ALTS1C1 tumors (n = 20 islands per tumor, with a total of three tumors in each group; * p<0.05).
Figure 3
Figure 3. Peripheral myeloid cells infiltrate into the invading brain tumor.
(A) Migration of GFP-BMDCs in the ALTS1C1 brain tumor sections from adjacent normal regions to the tumor core. Cell nuclei were stained with DAPI. All scale bars: 50 μm. (B) Percentage of GFP-BMDCs in normal brain region, islands, and different areas of the tumor core in ALTS1C1 brain tumor (n = 10–15 fields per area in total 3 tumors). (C) IHC counterstaining of GFP-positive cells (green color) with CD11b (red color of top panel), CD68 (red color of middle panel), and F4/80 (red color of bottom panel) antibodies in ALTS1C1 brain tumors obtained from mice receiving GFP-BM transplantation. Low power scale bar (left 3 columns): 100 μm. High power scale bar (right column): 20 μm.
Figure 4
Figure 4. Irradiation increased SDF1 expression in infiltrating tumor islands.
(A) IHC staining of SDF-1 (red) and nuclei by DAPI (blue) in the control (CON) and in 8 Gy (RT) irradiated brain tumors. Scale bar  = 200 μm. (B) Quantification of SDF-1 expression in selected tumor island areas. * p<0.05. (C) Western blot analysis to analyze the expression of SDF-1 and HIF-1 proteins in ALTS1C1 control or single-dose 8 Gy-irradiated cells. (D) ELISA results of SDF-1 production by ALTS1C1 control or single-dose of 8 Gy-irradiated cells. * p<0.05.
Figure 5
Figure 5. In vitro and in vivo radiation response of ALTS1C1 and SDFkd tumors.
(A) The survival curve following IR was assayed by clonogenic assay. (B) Kaplan–Meier survival curves of ALTS1C1 and SDFkd tumor-bearing mice treated with various doses of irradiation. The arrow indicates the time (day 13 after brain tumor implantation) for which radiation was given. (C) The mean diameter of control and single-dose irradiated ALTS1C1 or SDFkd brain tumors measured at the maximum cross-section in H&E-stained tissues. * p<0.05. (D) Scatter plot of tumor islands in control and single-dose irradiated ALTS1C1 and SDFkd brain tumors. The invasive pattern was assessed in whole brain serial sections by DAPI staining. The plot represents the average island number of each brain in each group, as calculated using a microscope (× 100 power). * p<0.05, *** p<0.001.
Figure 6
Figure 6. Tumor MVD and TAM density in SDFkd tumor following irradiation.
(A) Confocal imaging of IHC staining for CD31 (red), Iba-1 (red), CD68 (red), and nuclei by DAPI (blue) on the infiltrating islands of SDFkd control or 15 Gy-single dose irradiated SDFkd tumors. Scale bar  = 20 μm. (B) Quantification of the change of MVD, Iba-1, and CD68 in brain tumor islands (n = 20 islands per tumor, with a total of three tumors in each group; * p<0.05).

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This study is supported by NHRI-EX101-10132BI, NSC 101-2627-N-007-001, NTHU-101N2050E1, and NHTU-101N2760E1 grants to Chiang, C. S.; NSC 100-2314-B-182A-094 grant to Tsai, C. S.; Wang S. C. was supported by the post-doctor fellowship of National Tsing Hua University, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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