macrophages tropism to glioma

Journal of Neuroimmunology 232 (2011) 75–82

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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Glioma-initiating cells: A predominant role in microglia/macrophages tropism to glioma Liang Yi a,1, Hualiang Xiao b,1, Minhui Xu c, Xianzong Ye b, Jun Hu a, Fei Li a, Mei Li a, Chunxia Luo a, Shicang Yu b, Xiuwu Bian b, Hua Feng a,⁎ a b c

Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing, China Institute of Pathology, Southwest Hospital, Third Military Medical University, Chongqing, China Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China

a r t i c l e

i n f o

Article history: Received 23 July 2010 Received in revised form 7 October 2010 Accepted 11 October 2010 Keywords: Cancer stem cell Brain macrophages Microglia Gliomas

a b s t r a c t The relationship between cancer-initiating cells and cancer-related inflammation is unclear. Exploring the interaction between glioma-initiating cells (GICs) and tumor-associated microglia/macrophages (TAM/Ms) may offer us an opportunity to further understand the inflammatory response in glioma and the cellular/molecular features of the GIC niche. Here we reported a positive correlation between the infiltration of TAM/Ms and the density of GICs. The capacity of GICs to recruit TAM/Ms was stronger than that of adhesive glioma cells (AGCs) in vitro. In vivo experiments suggested that implantations formed by GICs had a higher level of TAM/M infiltration than those formed by AGCs. Our studies indicate a predominant role of GICs in microglia/macrophages tropism to glioma and a close positive correlation between the distribution of GICs and TAM/Ms. As an important part of cancer-related inflammation, TAM/Ms may participate in the architecture of the GIC niche. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Glioma-initiating cells (GICs) have been proven to play key roles in growth, invasion, angiogenesis and immune evasion of glioma (Bao et al., 2006b; Stiles and Rowitch, 2008; Wei et al., 2010). They have also been identified as the sources of chemo- and radio-resistance (Bao et al., 2006a; McCord et al., 2009; Dey et al., 2010), which offers a new perspective and target to explore and treat glioma. The GIC niche determines the characteristics of GICs and controls the malignant behaviors of glioma (Calabrese et al., 2007). However, there is only limited knowledge about the composition of the GIC niche. Inflammatory mediators and inflammatory cells are indispensable components of the neoplastic microenvironment (Mantovani et al., 2008). Cancer-related inflammation promotes tumorigenesis and progression. Ginestier et al. found that chronic inflammation can

⁎ Corresponding author. Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. Tel.: +86 23 6876 5762; fax: +86 23 6531 8230. E-mail addresses: [email protected] (L. Yi), [email protected] (H. Xiao), [email protected] (M. Xu), [email protected] (X. Ye), feifl[email protected] (F. Li), [email protected] (M. Li), [email protected] (C. Luo), [email protected] (S. Yu), [email protected] (X. Bian), [email protected] (H. Feng). 1 Liang Yi and Hualiang Xiao contributed equally to this work. 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.10.011

stimulate the proliferation of human breast cancer stem cells and contribute to the maintenance of their stemness (Ginestier et al., 2010). Veeravagu et al. hypothesized that inflammatory cells may be an important component of the GIC niche and modulate characteristics of GICs (Veeravagu et al., 2008). But, the relationship between GICs and inflammatory cells is unclear. Tumor-associated microglia/macrophages (TAM/Ms), which represent the largest population of infiltrating inflammatory cells in malignant glioma (Graeber et al., 2002), promote the growth, invasion and immune evasion of glioma cells (Badie et al., 2002; Nakano et al., 2008; Hong et al., 2009). A number of tumor-derived chemoattractants are thought to ensure the TAM/Ms recruitment, including colony-stimulating factor-1 (CSF-1) (Imai and Kohsaka, 2002), the CC and CX3C chemokines (Platten et al., 2003; Okada et al., 2009; Held-Feindt et al., 2010), vascular endothelial growth factor (VEGF) (Forstreuter et al., 2002)and neurotensin (NTS) (Martin et al., 2003). The levels of many of these proteins in glioma have a positive correlation with the numbers of TAM/Ms present in those tumors. In this study, we found that TAM/M infiltration was positively correlated to the histological grade of the malignancies and the number of GICs. TAM/Ms always distributed around the GICs. GICs from human glioma tissues had a stronger potential to recruit human primary microglia than adhesive glioma cells (AGCs) and the implantations derived from GICs had a higher level of TAM/M infiltration than those derived from AGCs. Our results indicate that GICs play a predominant role in inducing the infiltration of TAM/Ms, which may then participate in the architecture of the GIC niche.

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2. Materials and methods 2.1. Case selection Our study was approved by the Ethics Committee of Southwest Hospital, Third Military Medical University, Chongqing, PR China. 32 consecutive surgically resected astrocytomas were identified from the surgical pathology database of the Pathology Department of Daping Hospital, Third Military Medical University (Supplemental Table 1). None of the patients had undergone chemotherapy or radiotherapy before surgery except for two cases of recurrent glioblastoma. All tumors were selected and classified based on WHO Grade criteria. 2.2. Cell culture Tumor tissues from three cases of human primary glioblastoma were obtained with the informed consent of the patients as approved by the institutional Research Ethics Board at Southwest Hospital, Chongqing, China. GICs from human glioma tissues and AGCs were isolated and identified as we previously described (Yi et al., 2007). Briefly, the primary tumor cells were detached with trypsin (Gibco, USA) and suspended in defined stem cell medium, that is, Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12, Hyclone, USA) supplemented with penicillin/streptomycin (Sigma, USA), B-27 (1×, Gibco), 20 ng/mL basic fibroblast growth factor (bFGF, Peprotech, USA) and 20 ng/mL epidermal growth factor (EGF, Peprotech). After 24 h, most of tumor cells became adherent, with about 10% to 20% of primary cells floating and forming spheres composed of two to three cells. After an additional 7 days, the spheres further expanded, with cell numbers in each sphere ranging from 200 to 300. Primary human microglia (HM) were purchased from ScienCell Research Laboratories (Carlsbad, USA) and cultured in DMEM/F12 (Hyclone) supplemented with 10% fetal bovine serum (FBS, Sigma) and 10 ng/ml Macrophage-Colony Stimulation Factor (M-CSF, Sigma). Primary HM were isolated from primary human brain cell culture and the purity was N95% (Supplemental Figure 1C). These cells were characterized by immunocytochemistry with antibodies to CD11b and CD68 and were negative for astrocytic and neuronal cell markers (Supplemental Figure 1D-G). The murine glioma cell line GL261 (defined as GL261 adherent cells, GL261-AC) was obtained from ATCC, USA and cultured in DMEM/F12 (Hyclone) supplemented with 10% FBS (Sigma), penicillin and streptomycin (Sigma). GICs from glioma cell line GL261 were obtained and cultured as we previously described (Yu et al., 2008). In summary, GL261 neurospheres (GL261-NS) which had been confirmed to be enriched in GICs were obtained by seeding GL261-AC into defined stem cell medium described previously for primary GICs at a density of 1000 cells/mL. Under these conditions, only GICs and early progenitor cells survived and proliferated, whereas differentiated cells died. About 8% of these single cells could develop into spheres containing 10–20 cells after 5 days. In 2 weeks, the size of spheres expanded by 10- to 30-fold. Adult primary human astrocytes were isolated from postmortem brain specimens obtained at autopsy through Southwest Hospital, Chongqing, China, and cultured as described (Nielsen et al., 2009).

primary antibodies were used overnight, followed by the appropriate secondary antibodies and detection using the DouSPTM Detection System (MaiXin Bio, China). CD133+ cells were indicated by a brownish black using BCIP/NBT, and CD68+ cells were indicated by a scarlet using AEC. Immunofluorescence of CD68 and CD133 in orthotopic implantations and immunofluorescence of stem cell markers and differentiation markers in primary cultured GICs and AGCs were detected as previously described (Yi et al., 2007). Briefly, the cryostat-prepared sections of orthotopic implantations were incubated with rabbit anti-CD133 polyclonal antibody (1:200 dilution, Abcam) and rat anti-CD68 monoclonal antibody (1:200 dilution, Abcam), primary cultured GICs from human glioblastoma were incubated with rabbit anti-CD133 polyclonal antibody (1:200 dilution, Abcam), mouse anti-Nestin monoclonal antibody (1:200 dilution, Millipore, USA) and mouse antiOct-4 monoclonal antibody (1:100 dilution, Millipore), primary cultured AGCs from human glioblastoma were incubated with rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:100 dilution, Zhongshan Jinqiao Biotech, China), rabbit anti-microtubuleassociated protein 2 (MAP2) polyclonal antibody (1:100 dilution, Millipore) and rabbit anti-myelin basic protein (MBP) polyclonal antibody (1:100 dilution, Millipore). Immunohistochemical quantification was performed by two observers who were completely blinded to any clinical information. Following a brief scan of the maximum cross sections at low power, the number of TAM/Ms was determined in the three areas of densest cell infiltration using a square micrometer eyepiece at 200× magnification. The GICs were counted at 200× magnification in a manner similar to the TAM/Ms. The mean count of three areas was calculated as the TAM/M index (MI), which represented the total number of CD68+ cells divided by the total number of nuclei in each field, and the GIC index (GI), which represented the total number of CD133+ cells divided by the total number of nuclei in each field. The distributional features of GICs and TAM/Ms were assessed by calculating the percentages of CD133+ cells and CD68+ cells in 20 random fields at 200× magnification in representative samples (G10, G26 and G61) and the locational correlation of CD133 and CD68 expression was then analyzed. The percentages of CD133+ and CD68+ cells that were located in incremental distances of 20 μm from the nearest microvessel in surgical biopsy specimens were also calculated. 2.4. Real-time PCR Total RNA was prepared using the TRIzol reagent (Invitrogen, USA), A reverse transcription reaction using 1 μg of RNA template was performed using Taqman reverse transcription reagents (Takara, Japan) as per the manufacturer's instructions. The sequences of the primer pairs used in this analysis are indicated in Supplemental Table 2. Real-time PCR was performed using SyBr Green PCR Master Mix (Takara, Japan) and detected with an ABI-Prism 5700 Sequence Detector (Applied Biosystems, USA). Data were processed using GeneAmp software (Applied Biosystems) and were normalized against GAPDH gene expression levels. The data are from three independent experiments done in triplicate.

2.3. Staining and quantification

2.5. Chemotaxis assay

Immunohistochemistry (IHC) was performed on paraffin-embedded sections in order to investigate GICs and TAM/Ms in surgical biopsy specimens. The tumor sections were incubated with rabbit anti-human CD133 polyclonal antibody (1:100 dilution, Abcam, USA) to detect GICs, goat anti-human Iba1 polyclonal antibody (1:200 dilution, Abcam) and mouse anti-human CD68 monoclonal antibody (1:200 dilution, Abcam) to detect TAM/Ms, respectively, followed by detection using a ChemMate Detection kit (Dako, Denmark). A positive reaction was indicated by brown color using DAB and was counterstained with hematoxylin. For double labeling of CD133 and CD68, both of the

To obtain conditioned medium (CM), the primary human astrocytes, primary cultured GICs and AGCs were seeded at 1 × 106 per 25 cm2 overnight. Thereafter, the media were replaced with DMEM (Hyclone) without FBS and the supernatants were collected after 24 h of additional incubation. CM taken from astrocytes, primary cultured GICs and AGCs are referred to as astrocyte-CM, GICs-CM and AGCs-CM, respectively. To verify effects of tumor-derived chemoattractants effects, several antibody blocking assays were done. In detail, before starting chemotaxis assays, preincubation of GICs-CM with 20 μg/ml respective neutralizing antibodies (mouse anti-human CCL2,

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eBioscience; mouse anti-human CCL5, Santa Cruz; mouse anti-human CCL7, Santa Cruz; mouse anti-human VEGF-A, Santa Cruz; mouse antihuman NTS, Sigma;) or an appropriate IgG1/2 isotype control (20 μg/ml mouse IgG1-control antibody plus 20 μg/ml mouse IgG2control antibody; eBioscience;) was performed for 1 h. Chemotaxis assays were performed by using 8 μm pore size cell culture inserts within a 24-well plate (BD falcon, USA). HM (5 × 104 cells/100 μl) within DMEM/F12 medium (Hyclone) were seeded into the upper well of the insert, whereas the lower well was filled 600 μl of various conditioned media. Chambers were incubated at 37 °C in a CO2 incubator for 5 h, and cells that migrated through and adhered to the bottom of the filter were stained with crystal violet. The number of cells on the lower surface was assessed at 100× magnification using an inverted bright-field microscope. Nine fields at 100× magnification per insert were counted. Migration is expressed as a percentage (of cells per field) of migration toward astrocyte-CM. Each experiment was performed in triplicate respectively.

astrocytomas (WHO grade II) and 16.7% (2 of 12) of anaplastic astrocytomas (WHO grade III). To evaluate whether there was a correlation between marker expression and the pathological grade of the gliomas, a bivariate correlation analysis (Pearson correlation coefficients and Spearman correlation coefficients) was conducted. Statistically significant correlations between the percentages of immunostained cells and the grades of the gliomas were found for Iba1 (cp = 0.830, p b 0.01), CD68 (cp = 0.742, p b 0.01) and CD133 (rs = 0.712, p b 0.01), indicating that higher expression levels were found in higher malignant grades of gliomas. Furthermore, the Spearman correlations (rs) were 0.634 (p b 0.01) and 0.730 (p b 0.01), indicating that the expression levels of Iba1 and CD68 were each positively correlated with that of CD133. Representative images of immunohistochemistry staining and the correlation of marker expression with the clinical grading are shown in Fig. 1.

2.6. Syngeneic orthotopic glioma implantation

The locational features of TAM/Ms and GICs were evaluated by double labeling sections from the representative samples (G10, G26 and G61) with an antibody to CD133 and an antibody to CD68. Coimmunofluorescence indicated that a large number of GICs and TAM/Ms, which were both arranged in nests or cords, were distributed in tumor cores and margins. More TAM/Ms were localized in areas with a higher number of GICs and close contacts between the two cell types were obvious (Fig. 2D). Conversely, fewer TAM/Ms could be found in areas lacking GICs (Fig. 2D). Furthermore, the correlation coefficient (cp) was 0.819 (p b 0.01), confirming that the distribution of TAM/Ms was positively correlated with that of GICs (Fig. 2E). We also found that both GICs and TAM/Ms were mainly located around microvessels regardless of histologic diagnosis (Fig. 1C, L, M and Fig. 2F). The quantitative analyses indicated that most of the GICs were located within 50 μm of the nearest microvessels and most of the TAM/Ms were located within 70 μm of the nearest microvessels (Fig. 2G). In summary, these results indicated that both GICs and TAM/Ms are mainly localized in the perivascular region.

All procedures involving mice were conducted in accordance with the Guidelines of Animal Experiments of Third Military Medical University. GL261-AC (1× 104), and GL261-NS (1× 104) cells were injected orthotopically into the brains of four-week-old female C57BL/6 mice, a syngeneic glioma implantation model (n= 5 each, Center of Experimental Animals, Third Military Medical University, Chongqing, China). Mice were maintained for four weeks. The brains of euthanized mice were collected and frozen rapidly in liquid nitrogen for the subsequent preparation of sections. 2.7. Statistical analysis Parametric data are expressed as the mean value ± standard deviation (s.d.). Data were analyzed with SPSS10.0 statistical software. When two groups were compared, the unpaired dependent-samples t-test or exact Scheffe test was used. One-way ANOVA was used to determine the statistical significance of the differences among multiple groups. Bivariate correlation analyses (Pearson correlation coefficients and Spearman correlation coefficients) were calculated to evaluate the correlation between two groups. A value of P b 0.05 was considered statistically significant. 3. Results 3.1. TAM/M infiltration and the density of GICs in astrocytomas with different histological grades Expression of Iba1, CD68 and CD133 was assessed by immunohistochemistry in paraffin sections of 32 gliomas with different WHO grades. With increasing malignancy, the mean proportion of Iba1+ cells in gliomas increased. The percentages of Iba1+ cells in gliomas were 9.2% in WHO grade II (Fig. 1A), 21.0% in WHO grade III (Fig. 1B) and 33.7% in WHO grade IV (Fig. 1C and D). Similarly, the expression levels of CD68, a marker well known to be expressed on TAM/Ms, also increased with malignant progression. The mean proportions of CD68+ cells were 7.9% in WHO grade II (Fig. 1H), 19.7% in WHO grade III (Fig. 1I) and 29.2% in WHO grade IV (Fig. 1J and K). Substantial differences in CD133 expression were observed between tumors of different WHO grades. The proportions of CD133+ cells were considerably variable among gliomas ranging from complete lack of immunoreactivity (Fig. 1K) to expression in single cells (Fig. 1L) or staining of cell clusters (Fig. 1M and N). The mean proportions of CD133+ cells in WHO grade II (Fig. 1K), III (Fig. 1L) and IV (Fig. 1M and N) gliomas were 0.9%, 7.2% and 16.7% respectively. Moreover, the expression of CD133+ could not be detected in 44.4% (4 of 9) of diffuse

3.2. Distribution of TAM/Ms and correlation with GICs in malignant glioma

3.3. Primary cultured GICs and differentiated tumor cells Primary cultured GICs were obtained from three human glioblastoma specimens. The spheres were dissociated into single cell suspension, and cultured in stem cell medium. Most of the primary cells became adherent, a minority of floating cells formed neurosphere-like clones (Fig. 3A). Secondary spheres derived from single cells of these neurosphere-like clones were positive for stem cell markers, CD133, Nestin, and Oct4 (Fig. 3B–D). In order to obtain the primary AGCs, tumorspheres were cultured in DMEM/F12 containing 10% FBS. Primary AGCs (Fig. 3E) migrating out from tumorspheres after 72 h in culture were positive for differentiation markers, GFAP, MAP2 and MBP (Fig. 3F–H). 3.4. Production of tumor-derived chemoattractants by primary cultured GICs and their capacity to recruit primary microglia in vitro In order to confirm whether GICs have a greater capacity to direct the infiltration of TAM/Ms than AGCs, we test the mRNA expression levels of tumor-derived chemoattractants by Real-time PCR. It has been proven that the CC and CX3C chemokines, CCL2, CCL3, CCL5, CCL7, CCL8 and CX3CL1, CSF-1, VEGF-A and NTS are important mediators of TAM/M infiltration. Compared with AGCs, primary cultured GICs expressed 2- to 3-fold higher level of CCL2, CCL5 and CCL7 mRNA, 7-fold higher level of VEGF-A mRNA and nearly 50-fold higher level of NTS mRNA (Fig. 4A). Transwell chamber tests and antibody blocking assays showed, in comparison with Astrocyte-CM (Fig. 4C), AGCs-CM and GICs-CM

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Fig. 1. Synopsis of key immunocytochemical markers in gliomas and quantitative analyses derived from IHC. Iba1 immunoreactivity (arrows) was observed in diffuse astrocytoma (A), anaplastic astrocytoma (B), glioblastoma (C) and gliosarcoma (D). Immunolabeling was detected around blood vessels (arrowheads in B, C and D). (E) Mean percentages of Iba1+ cells in gliomas (including gliosarcoma) of grades II, III and IV. CD68 immunoreactivity (arrows) was observed in diffuse astrocytoma (F), anaplastic astrocytoma (G), glioblastoma (H) and gliosarcoma (I). (J) Mean percentages of CD68+ cells in gliomas (including gliosarcoma) of grades II, III and IV. Little CD133 immunoreactivity was observed in diffuse astrocytoma (K). CD133 immunoreactivity (arrows) was observed in anaplastic astrocytoma (L), glioblastoma (M) and gliosarcoma (N). Immunolabeling was detected around blood vessels (arrowheads in L, M and N). (O) Mean percentages of CD133+ cells in gliomas (including gliosarcoma) of grades II, III and IV. Scale bar = 50 μm (A–D, E–H, J–N). Hematoxylin counterstain. * p b 0.05, ** p b 0.01, exact Scheffe test.

promoted microglia migration up to 330 ± 81% (Fig. 4D) and 891±170% (Fig. 4E), which meant more than three times as many microglia migrated toward GICs-CM as compared to AGCs-CM. However, this effect could be impaired by application of an anti-CCL5-specific antibody (665± 108%; P = 0.083), an anti-VEGF-A-specific antibody (Fig. 4F, 537± 86%; P = 0.02) or an anti-NTS-specific antibody (Fig. 4F, 496±113%; P = 0.19). Placing GICs-CM in both the upper and lower chamber to eliminate the potential gradient decreased the migration of microglia, which is consistent with a chemotactic model of migration (Fig. 4B). These results indicated GICs from human glioma tissues had a

stronger potential to recruit microglia than AGCs, which involved increased production of chemoattractants derived from GICs. 3.5. TAM/M infiltration and the density of GICs in syngeneic orthotopic implantations In order to estimate the capacity of GICs to induce TAM/M infiltration in vivo, GL261-AC and GL261-NS cells were injected orthotopically into the brains of four-week-old female C57BL/6 mice. CD133 and CD68 expression were 0.2% ± 0.09% and 4.9% ± 1.1%,

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Fig. 2. Cellular distribution of CD133 and CD68 expression in GBM (A) Frozen sections were labeled with antibodies to CD133 (red). (B) Frozen sections were labeled with antibodies to CD68 (green). (C) DAPI-counterstained nuclei are in blue. (D) The merged image indicates that a large number of GICs and TAM/Ms were distributed in tumor core (TC) and along tumor margin (TM), but fewer in peritumoral region (PR). The majority of TAM/Ms were located around the hotspots (arrows) of GICs. (A–D) Laser confocal scanning microscopy. Scale bar = 100 μm. (E) Correlation analysis indicated that the location of TAM/Ms had a strong positive correlation with that of GICs, Cp = 0.819, p b 0.01. (F) Double label immunohistochemistry showed that GICs (brownish black, arrows) and TAM/Ms (scarlet, arrowheads) were both mainly focused around microvessels (star). Scale bar= 50 μm. (G) The percentage of CD133+ cells and CD68+ cells that were located in incremental distances of 20 μm from the nearest endothelial cell.

respectively, at the corresponding location of normal C57BL/6 mice (Fig. 5A, B, C), where tumor cells were implanted. However, elevated densities of CD133 and CD68 expression were determined by immunohistochemical staining with antibodies to CD133 and CD68

in tumors derived from GL261-AC cells (Fig. 5D, E, F). Meanwhile, tumors formed by GL261-NS cells contained a higher density of GICs, as labeled by CD133 (Fig. 5G), and much higher levels of TAM/M infiltration, as labeled by CD68 (Fig. 5H), compared to tumors formed

Fig. 3. GICs from primary glioblastoma and differentiated tumor cells from GICs (A) Primary cultured tumorspheres formed by GICs in stem cell medium. (B–D) Immunofluorescence of stem cell makers CD133 (B, red), Nestin (C, green) and OCT-4 (D, green). (E) AGCs creeping out from the edges of primary cultured tumorspheres at 72 h after plating in DMEM/F12 containing 10% FBS. (F–H) Immunofluorescence of differentiation makers GFAP (F, green), MAP2 (G, red) and MBP (H, red). (A, E) Phase contrast microscopy, scale bar = 50 μm. (B–D, F–H) Merged images, counterstained with DAPI showing nuclei in blue. Laser confocal scanning microscopy, scale bar= 100 μm.

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Fig. 4. The mRNA expression of tumor-derived chemoattractants and promoted migration of microglia toward GICs-CM in transwell chambers. (A) Quantitative RT-PCR was carried out in primary cultured GICs and AGCs in vitro to assess the mRNA expression levels for CCL2, CCL3, CCL5, CCL7, CCL8, CX3CL1, CSF-1, VEGF-A and NTS which were thought to ensure the TAM/M infiltration. * p b 0.05, ** p b 0.01, *** p b 0.001 by two-tailed t-test. (B) Transwell migration of microglia in chambers. Percentages of cells migrating toward AGCs-CM and GICs-CM relative to the migration toward Astrocytes-CM. GICs-CM U/L represents the case in which conditioned media were placed in both the upper (U) and lower (L) chamber, *** p b 0.001 vs. GICs-CM by two-tailed t-test. Relative to GICs-CM with appropriate IgG1/2 isotype control (GICs-CM+ IC), this effect could be impaired by preincubation of GICs-CM with neutralizing anti-VEGF-A or NTS antibodies (GICs-CM+ NA-VEGF-A and GICs-CM + NA-NTS), # p b 0.05 vs. GICs-CM + IC by two-tailed t-test.

by GL261-AC cells. These results indicated a more rigorous TAM/M infiltration in tumors derived from GL261-NS cells. 4. Discussion Our results confirmed that GICs play an important role in TAM/M infiltration into glioma. TAM/Ms are prominent in the stromal compartment of malignancies. Previous studies have shown that TAM/Ms account

for about 5% to 30% of cells within glioma(Graeber et al., 2002; Watters et al., 2005) and produce cytokines and signals that promote glioma growth, invasion and angiogenesis(Zeisberger et al., 2006; Wesolowska et al., 2008; Hong et al., 2009). We found that TAM/M infiltration varied largely in astrocytomas with different histological grades. The higher the histological grade of astrocytoma, the greater the number of infiltrating TAM/Ms in tumor tissue. Consistent with the past researches (Pallini et al., 2008; Zeppernick et al., 2008), the density of GICs positively correlated

Fig. 5. Coimmunofluorescence analysis of CD133 and CD68 expression in sections of orthotopic syngeneic implantations. (A) CD133 expression in the corresponding area of normal C57BL/6 mice where tumor cells were implanted in the experimental group. (B) CD68 expression in the corresponding area of normal C57BL/6 mice where tumor cells were implanted in the experimental group. (C) Merged image of A and B. (D) CD133 expression in the tumor derived from GL261-AC cells. Arrows represent CD133 staining (red). (E) CD68 expression in the tumor derived from GL261-AC cells. Arrowheads represent CD68 staining (green). (F) Merged image of D and E. (G) CD133 expression in the tumor derived from GL261-NS cells. Arrows represent CD133 staining (red). (H) CD68 expression in the tumor derived from GL261-NS cells. Arrowheads represent CD68 staining (green). (I) Merged image of G and H. DAPI-counterstained nuclei are in blue. (A–I) Laser confocal scanning microscopy. Scale bar = 50 μm. (J) Mean percentages of CD133+ cells and CD68+ cells in control tissues, tumors derived from GL261-AC and GL261-NS cells. * p b 0.01, compared with CD133 expression in control tissues; ** p b 0.01, compared with CD133 expression in tumors derived from GL261-AC cells; # p b 0.01, compared with CD68 expression in control tissues; ## p b 0.01, compared with CD68 expression in tumors derived from GL261-AC, by two-tailed t-test.

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with the histological grade of malignancies. Further investigation revealed the intimate relationship between GICs and TAM/Ms in malignant gliomas. GICs were arranged in nests or cords and, intriguingly, the majority of TAM/Ms were located around the hotspots of GICs (Fig. 2D). Tumor-derived chemoattractants are regarded as pivotal to the recruitment of TAM/Ms. Accumulating reports have indicated a highly positive correlation between TAM/M infiltration and the expression levels of these factors (Forstreuter et al., 2002; Martin et al., 2003; Platten et al., 2003; Kerber et al., 2008; Held-Feindt et al., 2010). Here, we found that TAM/Ms were mainly distributed around GICs, which produced more CCL2, CCL5, CCL7, VEGF-A and NTS than do the AGCs. The improved capacity of GICs to recruit microglia could be ruined by preincubation GICs-CM with neutralizing antibodies in vitro. Therefore, we hypothesized that accompanying with malignancy progression, the increased GICs which secrete more chemotactic factors are responsible for the boosted TAM/M infiltration. To verify this hypothesis, we isolated murine GICs (GL261-NS) from the GL261 mouse glioma cell line (GL261-AC) and striatally implanted either GL261-NS or GL261-AC cells into C57BL/6 mice to generate an orthotopic syngeneic glioma model. The results showed that TAM/M infiltration in GL261-NS tumors were much more obvious than in GL261-AC tumors, which supported our hypothesis. Neutralizing antibodies against VEGF-A and NTS evidently impaired microglia tropism to GICs in Transwell assay. Primary GICs expressed around 7-fold higher level of VEGF-A mRNA compared with AGCs. Bian et al. and Bao et al. have confirmed that GICs consistently secrete markedly elevated levels of VEGF-A and play a key role in triggering the “angiogenic switch” of malignance (Bao et al., 2006b; Yao et al., 2008). However, the chemotactic and immunosuppressive function of VEGF-A expressed by GICs have been paid relatively less attention. VEGF-A can induce recruitment and proliferation of microglia (Forstreuter et al., 2002) and can inhibit myeloid progenitor maturation to develop tumor associated macrophages which promote malignant progression (Johnson et al., 2009). The adverse effects of VEGF-A high expression by GICs may not only initiate angiogenesis in malignant glioma but may increase the TAM/Ms recruitment and improve their immunosuppressive capacity. NTS was identified as the other involved molecule in microglia migration toward GICs. It has been reported that neurotensin receptor-3 (NTR3) is functional in microglia and induces recruitment of microglia by a mechanism dependent on both phosphatidylinositol-3 kinase (PI3 kinase) and mitogen-activated protein (MAP) kinase pathways (Martin et al., 2005). GICs expressed much higher level of NTS mRNA than AGCs. But whether microglia migration toward GICs is also mediated by the same signal pathways needs to be further investigated. Meanwhile, NTS contributes to the growth and migration of tumor cells and promotes the malignant progression directly (Carraway and Plona, 2006). We infer that NTS may play multifunctional roles to regulate the biological behaviors of GICs and NTS signaling pathway may be a worthwhile source for the development of antitumor drugs targeting GICs. As a versatile component in tumor, TAM/Ms promote the proliferation of glioma cells in stromal areas, enhance the invasion of glioma cells at tumor margins and stimulate angiogenesis in perivascular areas (Lewis and Pollard, 2006). Thus, TAM/Ms regulate various aspects of malignant biological behaviors which are supposed to be determined by GICs. It has been indicated that most of the glioma cells beyond tumor margins are GICs and the invasive GICs are regarded as the root of glioma invasiveness (Yu and Bian, 2009; Sottoriva et al., 2010). Calabrese et al. discovered GICs were mainly located around microvessels (Calabrese et al., 2007). Interestingly, our research indicated that the majority of TAM/Ms were distributed along tumor margins (Fig. 2D) and around blood vessels, which corresponded to hotspots of GICs. Further study is needed to confirm whether the effects of TAM/Ms on the malignant characteristics of glioma are achieved through their interaction with GICs. On the other hand, the biological properties of GICs are regulated by the GIC niche.

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It has been reported that endothelial cells can organize the vascular niches to maintain the survival and stemness of GICs, depletion of endothelial cells from tumors obviously impairs self-renewal capacity of GICs (Calabrese et al., 2007). Meanwhile, inflammatory mediators and inflammatory cells in the stromal compartment of malignancy also improve the development of the CIC niche (Veeravagu et al., 2008; Ginestier et al., 2010). Endothelial cells produce inflammatory mediators to perform a “bridge” function between the immune system and the brain. Increasing evidence indicates that the bidirectional interaction between endothelium and microglia contributes to both the onset and the progression of neuroinflammation (Han and Suk, 2005). Thus, it is not surprising that the majority of TAM/Ms are present in the vascular niche of GICs. Additionally, microglia can modulate the neurogenic niche of neural stem cells (Battista et al., 2006; Nakanishi et al., 2007), and macrophages have been found to be an adaptive element of the epithelial progenitor niche (Pull et al., 2005). It is reasonable to believe that TAM/Ms accompanying with endothelial cells may be an important component of the GIC niche, further study is needed to confirm the role of TAM/ Ms in the architecture of GIC niche and the related molecular mechanism. Supplementary materials related to this article can be found online at doi:10.1016/j.jneuroim.2010.10.011. Conflict of interest The authors declare no conflict of interest. Acknowledgements We thank technicians Wei Sun and Li-ting Wang (Central Laboratory, Third Military Medical University, Chongqing, China) for their technical assistance in laser confocal scanning microscopy. This project was supported by the National Basic Research Program of China (973 Program, No. 2010CB529403) and grants from the National Natural Science Foundation of China (NSFC, No. 30901538 and NSFC, No. 30973494). References Badie, B., Bartley, B., Schartner, J., 2002. Differential expression of MHC class II and B7 costimulatory molecules by microglia in rodent gliomas. J. Neuroimmunol. 133, 39–45. Bao, S., Wu, Q., McLendon, R.E., Hao, Y., Shi, Q., Hjelmeland, A.B., Dewhirst, M.W., Bigner, D.D., Rich, J.N., 2006a. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756–760. Bao, S., Wu, Q., Sathornsumetee, S., Hao, Y., Li, Z., Hjelmeland, A.B., Shi, Q., McLendon, R. E., Bigner, D.D., Rich, J.N., 2006b. Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor. Cancer Res. 66, 7843–7848. Battista, D., Ferrari, C.C., Gage, F.H., Pitossi, F.J., 2006. Neurogenic niche modulation by activated microglia: transforming growth factor beta increases neurogenesis in the adult dentate gyrus. Eur. J. Neurosci. 23, 83–93. Calabrese, C., Poppleton, H., Kocak, M., Hogg, T.L., Fuller, C., Hamner, B., Oh, E.Y., Gaber, M.W., Finklestein, D., Allen, M., Frank, A., Bayazitov, I.T., Zakharenko, S.S., Gajjar, A., Davidoff, A., Gilbertson, R.J., 2007. A perivascular niche for brain tumor stem cells. Cancer Cell 11, 69–82. Carraway, R.E., Plona, A.M., 2006. Involvement of neurotensin in cancer growth: evidence, mechanisms and development of diagnostic tools. Peptides 27, 2445–2460. Dey, M., Ulasov, I.V., Lesniak, M.S., 2010. Virotherapy against malignant glioma stem cells. Cancer Lett. 289, 1–10. Forstreuter, F., Lucius, R., Mentlein, R., 2002. Vascular endothelial growth factor induces chemotaxis and proliferation of microglial cells. J. Neuroimmunol. 132, 93–98. Ginestier, C., Liu, S., Diebel, M.E., Korkaya, H., Luo, M., Brown, M., Wicinski, J., Cabaud, O., Charafe-Jauffret, E., Birnbaum, D., Guan, J.L., Dontu, G., Wicha, M.S., 2010. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. J. Clin. Invest. 120, 485–497. Graeber, M.B., Scheithauer, B.W., Kreutzberg, G.W., 2002. Microglia in brain tumors. Glia 40, 252–259. Han, H.S., Suk, K., 2005. The function and integrity of the neurovascular unit rests upon the integration of the vascular and inflammatory cell systems. Curr. Neurovasc. Res. 2, 409–423.

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