Cell Biochem Biophys (2014) 70:1625–1631 DOI 10.1007/s12013-014-0105-3

ORIGINAL PAPER

Expression of Tumor-Associated Macrophage in Progression of Human Glioma Peng Ding • Weimin Wang • Jinkun Wang Zhiyong Yang • Liping Xue



Published online: 3 September 2014 Ó Springer Science+Business Media New York 2014

Abstract The aim of this study is to investigate the expression of tumor-associated macrophages (TAMs) M1, M2 phenotypic in human glioma tissues, and to explore the clinical significance and prognostic value of TAMs in glioma patients. A total of 50 glioma samples were obtained from patients diagnosed in our hospital from 2007 to 2010. Clinical follow-up was conducted via return visits and telephone interviews after discharge. Progression free survival (PFS) was calculated based on tumor progression by MRI and CT examination from the primary operation. Overall survival (OS) time was calculated from the initial surgical operation date to end date of follow-up or death. Kaplan–Meier methodology was used to evaluate the survival of patients and log-rank test for comparing differences between groups. The expression levels of CD16 and CD206 were investigated in the 4 lm serial paraffin sections by immunohistochemistry. M1-type macrophages filtrated in all the grades of glioma samples, and the lower expression level was associated with high grade glioma. A negative correlation was found between WHO pathological grades and the expression of M1-type macrophages by Spearman correlation analysis. M2-type macrophages filtrated in all the grades of glioma samples with the higher

Peng Ding and WeiMin Wang contributed equally to this study. P. Ding  W. Wang  J. Wang  Z. Yang Department of Neurosurgery, The First Affiliated Hospital of Kunming Medical University, 295 XiChang Rd, Kunming 650032, People’s Republic of China L. Xue (&) Department of Ophthalmology, Yunnan NO. 2 Provincial People’s Hospital, 176 Qingnian Rd, Kunming 650021, People’s Republic of China e-mail: [email protected]

expression level associated with high grade glioma. A positive correlation was found between WHO pathological grades and the expression of M2-type macrophages by Spearman correlation analysis. The PFS and OS among patients with high levels of M1-type macrophages (CD16???) were significantly higher than those with less expression. The PFS and OS among patients with high levels of M2-type macrophages (CD206???) were significantly lower than those with low expression. M1-type macrophages may inhibit the tumor growth and improve the therapeutic outcome of glioma patients. M2 ratios are associated with tumor proliferation and poor prognosis. TAMs phenotypes of glioma samples are the potential biomarkers in assessing the degree of malignancy, tumor invasion, and patient prognosis in clinic. Keywords Glioma  Immunohistochemistry  Tumorassociated macrophages  Prognosis

Introduction Glioma, which arises from ectodermal layer, is the most common intracranial tumor, accounting for 40–50 % of all brain tumors [1]. Due to the capability of escaping from host immune recognition, glioma is generally known as the most lethal malignancies in central nervous system. Based on the cellular origins and histological characters, gliomas are classified by the World Health Organization (WHO) into pilocytic astrocytoma, diffuse astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme [2]. Despite the advances in surgical resection, chemotherapy, and radiation therapy, the prognosis remains poor with the median survival of diagnosed glioma patients of about 15 months. Recently, a growing number of studies have focused on the

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prognosis analysis in glioma to explore the host-dependent and tumor-dependent factors that determine the outcomes of treatment and prognosis of patients [3–5]. However, up to now, the results remain controversial. Therefore, a better understanding of the cellular origins, tumor-host immune responses, and molecular mechanisms of glioma may be crucial to develop the effective individualized strategies. Macrophages/microglia that infiltrate cancer tissues are so called tumor-associated macrophages (TAMs) and have shown to be involved in the formation of the tumor microenvironment and functioned in the promoting tumorigenesis, cell growth, invasion, metastasis, immune suppression as well as anti-tumor process [6, 7]. The anti-inflammatory M2type macrophages and activated proinflammatory M1-type macrophages have been identified as the two types of categorization of glioma TAMs. Classically, activated M1-type macrophages are thought to be involved in the interaction with immunogenic antigens by secreting proinflammatory mediators. In contrast, M2-type macrophages known as alternatively activated are significantly involved in immunomodulation and tumor cell proliferation [8]. Although it is generally known that TAMs are associated with the poor clinical prognosis in many kinds of malignant tumors, to our knowledge, the role of TAMs in the pathological grade and prognosis of glioma in Chinese people has rarely been reported. In this study, CD16 and CD206 are used as the standard biomarker of M1- and M2type macrophages representatively to identify the different types of TAMs in glioma tissues with immunohistochemistry, and the clinical significance and prognostic value of TAMs in glioma patients will be explored.

Materials and Methods Patients and Sample Collection A total of 50 glioma samples were obtained from the patients hospitalized in the department of Neurosurgery of the First Affiliated Hospital of Kun Ming Medical University between Jan 2007 and Jun 2010. Fifty adult patients (mean age 54.2 ± 9.7 years; median age 54 years; age range 36–71 years; 26 males and 24 females) were recruited in the study. All the cases were histologically diagnosed and categorized by two neuropathologists according to the WHO grades of 2007. Twelve patients of each WHO grades ranged from I to IV were enrolled, and all cases were primary diagnosis without surgery, chemotherapy, or radiation therapy history. Patients with diabetes, rheumatoid, hyperthyroidism, and other diseases were excluded. Clinical follow-up was conducted via return visits and telephone interviews after discharge in all patients. Progression free survival (PFS) was calculated

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based on tumor progression by MRI and CT evaluation from the primary operation. Overall survival (OS) time was calculated from the initial surgical operation date to end date of follow-up or death. All the tissue samples were fixed in 10 % formaldehyde, embedded in paraffin, and cut into 4-lm-thick serial sections. Immunohistochemistry The expression levels of CD16 and CD206 were investigated in the 4-lm serial paraffin sections. Immunohistochemistry was carried out according to the manufacturer protocol of Streptavidin-Peroxidase (SP) Two-step Kit. The sections were boiled in EDTA (1 mM; pH 8.0) for 15 min in a microwave oven for antigen retrieval. Mouse monoclonal antibody against CD16 and CD206 (1:100 dilution, Beijing Biosynthesis Biotechnology Co., LTD; China) was used as primary antibodies with the PBS as the negative control. Quantitative Scoring of Immunohistochemistry The degree of infiltration of CD16 and CD206 positive cells was scored using the method previously described in Al-Shibli’s report. Positive CD16 and CD206 were defined as strong nuclear/cytoplasm yellow–brown staining with clear and compact cellular structures. Imagines were analyzed by the Image-ProPlus 6.0 software (Media Cybemetics, USA). Positive stained cells were counted manually from five randomly chosen fields per section under 4009 high-power magnification, and the mean integrated optical density (IOD) values and areas of each region were calculated. The immunoreactivity intensity was determined by the ratio of IOD divided by the region area as mean IOD. Results of immunohistochemical analysis were categorized into strongly positive reaction (???) and mild positive reaction subgroups. Statistical Analysis Statistical analysis was performed using SPSS 17.0 software. The homogeneity of variance and normal distribution of samples were assessed before hypothesis test. The homogeneity of variance was estimated by Levene test, and the distribution of samples was examined by K–S test. p \ 0.05 was considered as statistically significant. All the data form a normal distribution. One-way ANOVA was used to estimate the quantitative data from multiple groups, and LSD-t test was used to compare the difference between two groups. Pearson correlation coefficient or Spearman rank correlation was used to assess the correlations between CD16/CD206 expression and pathological grades. Kaplan–Meier methodology was used to examine the survival of patients and log-rank test for comparing

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differences between groups. p \ 0.05 was considered as significant difference.

Results Expression Levels of CD16 and CD206 in Human Glioma In order to verify the invasion of activated proinflammatory M1-type macrophages and anti-inflammatory M2-type macrophages in human glioma tissue, sectioned materials of grade I (13), grade II (12), grade III (13), and grade IV (12) glioma were analyzed by immunohistochemistry. Protein expression levels of CD16 (marker for M1-type macrophages) and CD206 (marker for M2-type macrophages) were found in all samples from human glioma (Figs. 1, 2). The positive staining of both CD16 and CD206 was mainly localized in cytoplasm and/or nucleus. Marked cellular heterogeneity was found. Correlation Between Expression of CD16/CD206 in Human Glioma Tumor Tissue and WHO Pathological Grades The mean IOD value of CD16 was 0.1050 ± 0.0055 in grade I tumors and decreased to 0.0437 ± 0.0030 in grade

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IV tumors (Table 1). The expression level of CD16 was significantly decreased in I–IV grade tumors (F = 503.463, p = 0.000), and LSD tests showed that the difference between two groups were statistically significant (p = 0.000). A negative correlation was found between WHO pathological grades and the CD16 positive staining intensity in human glioma tumor tissues (Spearman r –0.705; p = 0.000) (Table 1). The mean IOD value of CD206 was 0.0448 ± 0.0030 in grade I tumors and increased to 0.1065 ± 0.0069 in grade IV tumors (Table 1). The expression level of CD206 was significantly increased in grade I–IV tumors (F = 345.753, p = 0.000), and LSD tests showed that the difference between two groups were statistically significant (p = 0.000). A positive correlation was found between WHO pathological grades and the CD16 positive staining intensity in human glioma tumor tissues (Spearman r 0.857; p = 0.000) (Table 1). Correlation Between the Expression of CD16/CD206 in Tumor Tissues and Pathological Features of Primary Glioma Patients The expression levels of CD16 and CD206 were significantly correlated with the WHO grades of glioma (p = 0.000) (Table 2). The expression levels of CD16 and CD206 were not significantly correlated with gender, age,

Fig. 1 Immunohistochemical staining for CD16 in grade I (a), grade (II) (b), grade (III) (c), and grade (IV) (d) primary human glioma tumor tissue (SP 9400). Positive intranuclear and diffuse intracytoplasmic staining together with obvious cellular heterogeneity were found

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Fig. 2 Immunohistochemical staining for CD206 in grade I (a), grade (II) (b), grade (III) (c), and grade (IV) (d) primary human glioma tumor tissue (SP 9400). Positive intranuclear and diffuse intracytoplasmic staining together with obvious cellular heterogeneity were found

Table 1 Correlation between CD16, CD206 expression levels and WHO grades of patients with glioma WHO grades

n

Mean IOD (CD16) x ± s

Mean IOD (CD206) x ± s

I II

12 13

0.1050 ± 0.0055 0.0807 ± 0.0027

0.0448 ± 0.0030 0.0675 ± 0.0065

III

13

0.1189 ± 0.1839

0.0841 ± 0.0084

IV

12

0.0437 ± 0.0030

0.1065 ± 0.0069

F

503.463

345.753

Pa

0.000

0.000

a

a

Difference between two groups was compared with the LSD-t test

tumor size, tumor sites, or crystallization in patients with glioma (p [ 0.05) (Table 2). Correlation Between the Expression of CD16/CD206 in Tumor Tissues and Prognosis of Primary Glioma Patients The staining intensity of CD16/CD206 was classified into low and high expression levels according to the median value of Mean IOD. To investigate the variables with potential prognostic significance in patients with glioma, univariate analysis using log-rank test of Kaplan–Meier methodology was performed. Therefore, the median value of

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immunohistochemically detected variables of CD16 0.0767 was used as cut-off. Twenty-three cases were divided into high expression group, and another 27 were in low expression group. The PFS and OS of CD16 high expression group were 707.565 ± 46.152 and 996.043 ± 54.091 days, respectively. The PFS and OS of CD16 low expression group were 331.520 ± 35.023 and 498.480 ± 51.901 days, respectively. The PFS and OS among patients with high levels of CD16 were significantly higher than those with lowlevel expression (p = 0.000) (Fig. 3). The median value of staining intensity of CD206 in Mean IOD was 0.0747; 23 cases were defined as high expression group, and 27 were low expression group. The PFS and OS of CD206 high expression group were 322.308 ± 32.562 and 484.077 ± 48.548 days, respectively. The PFS and OS of CD206 low expression group were 735.545 ± 41.475 and 1035.682 ± 44.811 days, respectively. The PFS and OS among patients with high levels of CD206 were significantly lower than those with low-level expression (p = 0.000) (Fig. 4).

Discussion Gliomas which accounts for the most common malignant brain tumors are associated with the poor prognosis among

Cell Biochem Biophys (2014) 70:1625–1631 Table 2 Correlations between tumor tissue expression levels of CD16, CD206 and clinical characters of patients with glioma

Variables

1629

n

CD16-positive cells Low

High

Gender

v2

3.281

p

CD206-positive cells Low

High

0.067

male

26

10

16

14

12

Female

24

14

10

10

14

v2

p

2.350

0.125

1.823

0.196

Age C50

27

12

15

\50

23

14

9

\5 cm

31

14

17

C5 cm

19

13

6

Tumor size

2.345

Grades I–II III–IV

16.324 24 26

5 23

19 3

Supratentorial

36

19

Infratentorial

14

yes No

0.2134

14

13

9

14

17

14

7

12

1.234

0.000*

31.47 20 2

4 24

17

20

16

7

7

6

8

16

7

9

7

9

34

19

15

16

18

Site

0.031

Crystallization *Difference between two categories was compared using v2 tests, p \ 0.05

1.4133

0.015

0.796

0.923

0.000*

0.003

0.868

0.005

0.973

Fig. 3 Survival curve of 50 glioma patients with different CD16 expression levels is shown. Kaplan–Meier survival estimates of patients were performed according to IHC positivity of multiple CD16 (low vs. high) expression levels. Patients expressing high CD16

level (CD16???) in tumor tissue showed significant improved progression free survival (a) and overall survival (b) than low CD16 level (CD16?) (p = 0.000, log-rank test)

all human cancers [9, 10]. As one of the major features, overcoming host immune defense makes glioma one of the most lethal malignances. However, the underlying mechanisms about how glioma escapes from immune recognition are still quite unclear. Increasing studies were designed to explore the potential biomarkers and clarify the relationship between molecular environment and clinical characteristics, but most of those studies were based on the westerner and Caucasian peoples, and few of them were well defined in Chinese [11]. The mainstream management strategies combine surgical resection with chemotherapy

and radiotherapy, but the survival rate is still low in primary glioma patients [12]. Therefore, identifying prognostic factors that correlated with beneficial Chinese patient survival are important for better understanding of the molecular environment involved in tumor progression and developing novel therapeutic target. The occurrence and development of tumor is considered as a highly complicated process that involves the interaction between tumor cells and surrounding stroma of host, as well as many distinct angiogenesis growth factors and enzymes [13]. High-grade brain malignances that regarded

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Fig. 4 Survival curve of 50 glioma patients with different CD206 expression levels is shown. Kaplan–Meier survival estimates of patients were performed according to IHC positivity of multiple CD206 (low vs. high) expression levels. Patients expressing low

CD206 level (CD206?) in tumor tissue showed significant improved progression free survival (a) and overall survival (b) than high CD206 level (CD206???) (p = 0.000, log-rank test)

as heterogeneous are composed of bona fide tumors cells and a range of intermingling parenchymal cells. Glioma tumor tissue that obtained from surgical resection usually contains multiple cell types, including cancer cells as well as a large number of nontransformed cells, which is considered to influence tumor microenvironment and secrete a number of factors to promote their tumorigenicity and pluripotency [14]. The failure of standard therapies for malignant glioma is believed to attribute to phenotypic heterogeneity and specific immune-privileged environment of the brain [3]. Increasing evidences have shown that TAMs represent the predominant infiltrating immune cells in the tumor environment and crucial contributors to cancer-induced inflammation. The permission of intrinsic immune response of central nervous system as well as the successful isolation of circulating tumor specific antibodies and cytotoxic T lymphocytes [15] has opened a new window for the investigation into TAMs as a potential therapeutic target against glioma. The important role of TAMs in facilitating tumor growth, progression, and invasion has been well documented. Accumulating evidence has demonstrated the clinical importance of TAMs in various cancer types including lung cancers, liver cancers, Hodgkin’s lymphoma, malignant mesothelioma, and melanoma [4, 6, 16– 19]. When responding to specific stimulation signals such as cancer cells or cytokines, macrophages derived from circulating monocytes and myeloid progenitor cells infiltrated local tissues and polarized in a range of phenotypes according to the host microenvironment. Macrophages are generally regarded as tumor-associated macrophages and the predominant immune cell infiltration in the tumor microenvironment of malignant glioma, accounting for up to 30–50 % of the tumor stromal [7, 20]. In glioma, regarding the plasticity and capacity to inhibit or promote

tumor growth, the TAMs are generally developed into two distinct phenotypes: tumor-inhibitory M1-type with tumoricidal potential (also known as classically activated macrophage, caMphi) and tumor-promoting M2-type with immunosuppressive ability (alternatively activated macrophage, aaMphi). In general, M1-type macrophages act as the fighter in tumor biology, which protect the host from viral and microbial infections, generate large amounts of inflammatory cytokines (like IL-1, IL-6, IL-12, IL-23, and TFN), chemokine (CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, and CXCL10), and fight against tumors, and regulate the Th1-type immune response [21]. In contrast, M2-type macrophages produce large amount of IL10, IL-4, and IL-13, highly express DECTIN-1 (a kind of b-glucan receptor), suppress inflammation and type I immune response, inhibit T cells production and promote tumor growth, angiogenesis, and remodeling and repair of wounded/damaged tissues [22]. Recently, accumulating evidence support that TAMs, under the influence of glioma cells, play a critical role in promoting the tumor angiogenesis and subsequent progression and invasion locally. Clinical and experimental studies have showed the association of increasing infiltrating TAMs with poor prognosis and tumor resistance to therapies in cancer patients [7, 23]. The same results were also found in our present study. In this study, we highlight the emerging data related to the tumor grade and phenotype of TAMs in glioma microenvironment, as well as the prognosis and clinical features. We found that M1-type macrophages filtrated in all the grades of glioma samples, and the lower expression level in high grade glioma. A negative correlation was found between WHO pathological grades and the expression of M1-type macrophages (CD16 positive) in human glioma tumor tissues by Spearman correlation analysis,

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indicating the association of the M1-type with degree of glioma malignancy. We also found that the M2-type macrophages filtrated in all the grades of glioma samples, and the higher expression level in high grade glioma. A positive correlation was found between WHO pathological grades and the expression of M2-type macrophages (CD206 positive) in human glioma tumor tissues by Spearman correlation analysis, suggesting the association of the M2-type with degree of glioma malignancy. Additionally, we discuss the potential of TAMs to be associated with the patient prognosis. Results indicated that the PFS and OS among patients with high levels of M1type macrophages (CD16???) were significantly higher than those with less expression, suggesting that the M1type macrophages may inhibit the tumor growth and optimize the therapeutic outcome of glioma patients. On the other hand, the PFS and OS among patients with high levels of M2-type macrophages (CD206???) were significantly lower than those with low expression, indicating that the M2 ratios are associated with tumor proliferation and poor prognosis. In summary, TAMs phenotypes of glioma samples are the potential biomarkers in assessing the degree of malignancy, tumor invasion, and patient prognosis in clinic. Further studies on the relationship between oncogenic signals of TAMs and malignant transformation of epithelial cells will clarify more detailed information about the mechanisms of tumorigenesis. A better understanding of the bidirectional regulating of TAMs in glioma will prove a novel therapeutic target in glioma treatment [24]. However, this study has only small sample size and is a retrospective study; more randomized, prospective studies with a large sample size are needed. Acknowledgments This study was supported by a special grant for High-Level Training of Yun Nan (D201230) and a special joint grant of Yunnan Provincial Science and Technology Department and Kunming Medical University (2013FZ281 and 2010CD157).

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Expression of tumor-associated macrophage in progression of human glioma.

The aim of this study is to investigate the expression of tumor-associated macrophages (TAMs) M1, M2 phenotypic in human glioma tissues, and to explor...
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