Correlation and clinical significance of LC3, CD68+ microglia, CD4+ T lymphocytes, and CD8+ T lymphocytes in gliomas

Accepted Manuscript Title: Correlation and clinical significance of LC3, CD68+ microglia, CD4+ T lymphocytes, and CD8+ T lymphocytes in gliomas Authors: Weiguo Zhang, Shuhua Wu, Ke Guo, Zhongbo Hu, Jiangtao Peng, Jianmin Li PII: DOI: Reference:

S0303-8467(18)30095-7 https://doi.org/10.1016/j.clineuro.2018.02.044 CLINEU 4955

To appear in:

Clinical Neurology and Neurosurgery

Received date: Accepted date:

2-2-2018 28-2-2018

Please cite this article as: Zhang W, Wu S, Guo K, Hu Z, Peng J, Li J, Correlation and clinical significance of LC3, CD68+ microglia, CD4+ T lymphocytes, and CD8+ T lymphocytes in gliomas, Clinical Neurology and Neurosurgery (2010), https://doi.org/10.1016/j.clineuro.2018.02.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Correlation and clinical significance of LC3, CD68+ microglia, CD4+ T lymphocytes, and CD8+ T lymphocytes in gliomas

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Weiguo Zhanga, Shuhua Wub, Ke Guoa, Zhongbo Hua, Jiangtao Penga, Jianmin Lia

Department of Neurosurgery, Binzhou Medical University Hospital, Binzhou, Shandong Province

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256603, China.

Department of Pathology, Binzhou Medical University Hospital, Binzhou, Shandong Province 256603,

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China.

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Correspondence authors

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Jianmin Li Department of Neurosurgery, Binzhou Medical University Hospital, No. 661 Huanghe 2nd

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Road, Binzhou, Shandong Province 256603, China; Tel: 86-543-325-8658; Fax: 86-543-325-7792;

Highlights 

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E-mail: [email protected].

The expression of LC3 was positively correlated with CD68+ microglia, CD4+ T lymphocytes

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and CD8+ T lymphocytes, suggesting that changes in autophagy levels may be one of the factors that affect the cellular immune response in glioma.

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The expression of LC3, CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes were positively correlated with tumor grade in glioma. LC3, CD68+ microglia, CD8+ T lymphocytes were independent prognostic factors of glioma.

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Abstract Objective: To investigate the relationship between the expression of microtubule-associated protein LC3 and the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes, as well as

the clinical significance of those factors in gliomas. Patients and Methods: The study group consisted of 127 patients with gliomas who were operated to our hospital, we examined the expression of LC3 by Immunohistochemistry and Western blot, and we assessed the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes by Immunohistochemistry, we analyze the relationship between

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all the factors and explore the significance. Results: Immunohistochemistry and Western blotting showed that the expression of LC3 in normal brain tissue, low-grade gliomas, and high-grade gliomas

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are elevated to varying degrees (P<0.01); Immunohistochemical detection showed that the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes in gliomas was higher than that in

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normal brain tissues (P<0.01), and the high-grade gliomas were higher than those in low-grade gliomas

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(P<0.01); The results of Spearman correlation showed that the expression of LC3 was positively

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correlated with the numbers of CD68+ microglia, CD4+ T lymphocytes, and CD8+ T lymphocytes

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(P<0.05); Furthermore, survival analysis showed that LC3, CD68+ microglia, CD8+ T lymphocytes

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and tumor grade were independent prognostic factors of glioma. Conclusions: LC3 may be one of the factors that affect the tumor cellular immunity response in gliomas. The simultaneous detection of LC3,

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CD68+ microglia and CD8+ T lymphocytes can be used to assess the prognosis of glioma.

Keywords Glioma; LC3; CD68+ microglia; CD4+ T lymphocytes; CD8+ T lymphocytes

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1 Introduction

Glioma is the most common intracranial tumor and has the highest morbidity and mortality [1]; treatment is mainly based on surgery and chemotherapy, but the recurrence rate is still high. At present, tumor immunotherapy has become one of the most promising methods for tumor treatment because it

achieves a low recurrence rate and does not have severe side effects[2]; many patients with malignant tumors have benefited from immunotherapy[3-5]. Research has found that cellular immunity plays an important role in tumor immunity. Microglial cells in the central nervous system account for approximately 20% of glial cells; after activation, they can express major histocompatibility complex

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(MHC) Ⅱmolecules, which present antigens in the process of cell immunity[6]. CD8+ T lymphocytes are the main subtype that mediates the antitumor immune response. The target cells can be killed by the

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following three routes:  cell lysis induced by released perforin, serine protease-mediated apoptosis, and  the Fas/Fasl pathway. CD4+ T lymphocytes are mainly helper T lymphocytes that can mediate

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cytotoxicity by secreting IL-2, IL-3, IFN-γ, TNF-α and granulocyte macrophage colony stimulating

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factor.

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It is considered that many factors can affect cellular immunity of tumor, such as the abnormal

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expression of tumor surface antigen, surface major histocompatibility antigen system and the changes of autophagy in tumor cells. Now tumor cell autophagy is a hot topic. Autophagy is a unique biological

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phenomenon that is prevalent in eukaryotic cells. It is a process in which cells degrade their impaired

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organelles and macromolecules with lysosomes. Autophagy plays an important role in cell metabolism and the maintenance of cell homeostasis[7, 8]. LC3 is a homologue of yeast Atg8, which binds to phosphatidylethanolamine (PE) during autophagy to convert cytoplasmic LC3-I into membrane-bound

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LC3-II. LC3 is considered the most valuable of the many autophagy related-marker molecules[9, 10]. It has been found that autophagy plays a different role in different tumors. Autophagy related genes are highly expressed in cancers of the esophagus[11] and stomach[12], but the expression in pancreatic cancer[13] is suppressed. In addition, recent studies have found that tumor cell autophagy can affect the

cellular immunity by participating in the presentation and modification of tumor related antigens in tumors[14, 15]. However, the relationship between autophagy and cellular immunity in gliomas is not clear. Therefore, in-depth study of the relationship between autophagy and cellular immunity in gliomas, and exploring potential factors that influence glioma cellular immunity is a great value for

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In this study, we examined the expression of the autophagy-related marker LC3 by

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immunotherapy of glioma.

immunohistochemistry and Western blot, and we assessed the numbers of CD68+ microglia, CD4+ T

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lymphocytes and CD8+ T lymphocytes by immunohistochemistry in different grades of glioma; we

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studied the correlation between LC3 and CD68+ microglia, CD4+ T lymphocytes and CD8+ T

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lymphocytes and the relationship of LC3, CD68+ microglia, CD4+ T lymphocytes and CD8+ T

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lymphocytes with clinical pathological factors.We also evaluated the correlations between LC3

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expression, CD68+ microglia, CD4+ T lymphocyte, CD8+ T lymphocyte and prognosis in patients with gliomas. To our knowledge, this study is the first to investigate the correlation between the tumor

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cellautophagy and cellular immunity, as well as the relationship of those four variables with the

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prognosis of glioma patients.

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2 Materials and methods 2.1 Patients and tissue specimens 106 formalin-fixed, paraffin-embedded specimens from patients with gliomas between January 2010 and January 2013 were obtained from the archives of the Department of Pathology, Binzhou Medical University Hospital, China. 21 fresh-frozen tumor tissues and the normal brain tissues which must be

removed in the surgical approach that were obtained during surgery between January 2016 and January 2017 were used for protein preparation, the normal brain tissues were considered the control tissues and were verified as normal by the pathologist. All patients underwent surgical treatment in the Department of Neurosurgery, Binzhou Medical University Hospital, China. Before surgery, no patient

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had been treated with radiotherapy, chemotherapy or combined radiotherapy and chemotherapy, and all patients were free of systemic immune disease and other serious chronic diseases. All patients were

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suffering from objective causes and only received one cycle routine chemotherapy. Two highly

experienced pathologists used the 2016 World Health Organization (WHO) glioma classification

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criteria[16] to classify the gliomas by molecular typing in a double-blind manner. The tumors included

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59 high-grade (grade III, grade IV) gliomas and 68 low-grade (grade I, grade II) gliomas, from subjects

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aged 25-80 years (mean 48.8±9.5 years). The definition of follow-up was the time interval from

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surgery to death or last follow-up. The 106 patients were observed in January 2017, and the follow-up

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time was 7-96 months (median 26.7 months). The study was approved by the ethics committee of

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Binzhou Medical University Hospital, and all patients signed a consent form.

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2.2 Immunohistochemistry

The expression of LC3 and the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes in gliomas were detected by IHC and compared with the corresponding values from

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normal brain tissues. Formalin-fixed, paraffin embedded tissue samples were cut into 4-µm sections, dewaxed, rehydrated, blocked with hydrogen peroxide, and heated in an aqueous solution of EDTA for antigen retrieval. The sections were then incubated with rabbit polyclonal antibody against humanLC3 (Abcam, ab48394, Cambridge, UK, 1:200), CD68 (Abcam, ab125212, Cambridge, UK, 1:80), CD4

(Abcam, ab203034, Cambridge, UK, 1:200), and CD8 (Abcam, ab4055, Cambridge, UK, 1:100) overnight at 4°C. The sections were rewarmed for 20 min and then incubated with horseradish peroxidase-labeled secondary antibody at 37°C for 30 min. The sections were developed with diaminobenzidine tetrahydrochloride and counterstained with hematoxylin, then dehydrated in a graded

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ethanol series with 1% hydrochloric acid, cleared, and sealed with neutral gum. LC3 from Abcam was used as a positive control for expression, and negative controls were prepared by using PBS instead of

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an antibody.

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2.3 Immunohistochemistry evaluation

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In each slice, 10 high-magnification (400×) fields of view were randomly selected for evaluation, and

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the cell staining intensity and positive cell ratio were used to measure the expression of LC3.

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Immunostaining intensity was rated as follows: cytoplasm with no staining, 0 points; light yellow, 1

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point; tan, 2 points; brown, 3 points. The percentage of positively stained cells was graded as follows: 0%-5%, 0 points; 6%-25%, 1 point; 26%-50%, 2 points; 51%-75%, 3 points; 76%-100%, 4 points. An

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immunoreactivity score was calculated by multiplying the score for staining intensity by the score for

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percentage of positively stained cells. Tumors with an immunoreactivity score of 0-6 were designated negative, and those scoring 7-12 were classified as positive. CD68 was expressed in the cytoplasm of microglia, while CD4 and CD8 were expressed in the cytoplasm of T cells. Positive staining was

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visible as brownish-yellow or yellowish particles. The field of view was the same as for LC3. The number of target cells in each field was counted, and the average number of positive cells was used as the measure of expression. The median expression levels of CD68, CD4 and CD8 positive cells were used as cutoffs to define the positive and negative groups, greater than or equal to the median was

positive, less than the median was negative.

2.4 Western blot analysis The tissue was homogenized on ice in SDS buffer containing the protease inhibitor PMSF, and the

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protein concentration was measured with a bicinchoninic acid (BCA) assay. The homogenates were incubated on ice for 20 min and then centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was

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collected and added to an equal volume of 5×SDS buffer. The mixture was boiled for 10 minutes and stored at -20°C. The protein extracts (50 µg) were subjected to SDS-PAGE and then transferred onto

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polyvinylidene fluoride membranes (Millipore, USA).The membranes were blocked with 5% nonfat

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milk in Tris-buffered saline containing 0.1% TWEEN 20 at room temperature for 90 min and then

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immunoblotted with antibodies against LC3 (2 μg/mL, Abcam, ab48394, Cambridge, UK) and β-actin

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(0.5 μg/mL, Abcam, ab3280, Cambridge, UK) overnight at 4°C. The proteins bands were detected with

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secondary antibodies conjugated to horseradish peroxidase (1:5000, Abcam, UK) and visualized with enhanced chemiluminescence reagents. The optical density of each band was determined, and the result

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was the relative expression of each protein in the different samples.

2.5 Statistical analysis Statistical analysis was performed using the software SPSS 23.0 (SPSS Inc., Chicago, IL, USA).

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Pearson's chi-squared test was used to assess the correlation of LC3, CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes with clinicopathological parameters of patients with glioma. The correlations between the expression of LC3 and the numbers of CD68+ microglia, CD4+ T lymphocytes, CD8+ T and lymphocytes were analyzed by Spearman correlation. Survival analysis was

conducted using the Kaplan-Meier method, and groups were compared by the log-rank test. The Cox proportional hazards regression model was used to determine the effects of clinicopathological variables, LC3 expression, and the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes on survival. A difference was considered significant if its P value was below 0.05.

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3 Results 3.1 Immunohistochemistry was used to detect the expression of LC3 in gliomas and normal brain

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tissues

Immunohistochemical staining showed that LC3 was mainly expressed in the cytoplasm of glioma cells,

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which was brownish yellow or yellowish, whereas immunostaining signal was absent or very weak in

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normal brain tissues. The positive rate of LC3 was 71.12% in high-grade gliomas, 55.88% in low-grade

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gliomas, and 28.57% in normal brain tissues (P<0.01) (Figure 1, Table 1).

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3.2 Western blotting was used to detect the expression of LC3 in gliomas and normal brain tissues

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The expression levels of LC3 in gliomas and normal brain tissues were determined using Western blot

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analysis, and quantitative analysis was performed by Quantity One software. The relative expression level of LC3 was 0.685±0.062 in high-grade gliomas, 0.436±0.057 in low-grade gliomas and 0.268±0.031 in normal brain tissues (P<0.01) (Figure 2), in accordance with the immunohistochemistry

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results.

3.3 The numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes in gliomas and normal brain tissues

Immunohistochemical staining showed that CD68+ microglia were scattered in normal brain tissues and glioma tissues (Figure 1). A few CD4+ T lymphocytes and CD8+ T lymphocytes were distributed around the vessels in the normal tissues, while the CD4+ T lymphocytes and CD8+ T lymphocytes in the gliomas were distributed around both the tumor tissue and the blood vessels (Figure 1).Cell counts

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showed that the median of CD68 + microglia, CD4 + and CD8 + T lymphocytes in normal brain tissues were 10.8, 4.3 and 2.5, respectively. The median of CD68 + microglia, CD4 + and CD8 + T

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lymphocytes in low-grade gliomas , Respectively 22.1,6.6,4.0. The median numbers of CD68 + microglia, CD4 + and CD8 + T lymphocytes in high-grade gliomas were 30, 11, and 6,

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respectively.The positive rates of CD68 + microglia in normal brain tissues, low-grade gliomas and

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high-grade gliomas were 33.33%, 48.53%, 76.27%, the positive rates of CD4 + T lymphocytes were

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28.57%, 57.35% and 67.80%, respectively, the positive rates of CD8 + T lymphocytes were 19.05%,

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61.76%, 89.83%, respectively (P<0.01) (Table 1).

3.4 Correlation between the expression of LC3 and the numbers of CD68+ microglia, CD4+ T

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lymphocytes and CD8+ T lymphocytes in gliomas

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After immunohistochemical evaluation, Spearman correlation analysis showed that the expression of LC3 in glioma tissues was positively correlated with CD68+ microglia (P=0.017，r=0.227), CD4+ T

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lymphocytes (P=0.021，r=0.180) and CD8+ T lymphocytes (P=0.009，r=0.394) (P<0.05) (Table 2).

3.5 Association of LC3 expression and the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes with clinicopathological factors in gliomas The expression of LC3 and the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T

lymphocytes were positively correlated with tumor grade (P<0.05), but not with the age, sex, tumor size and location of the patients (P <0.05) (Table 3).

3.6 Kaplan-Meier analysis

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The median survival time of the 106 patients with glioma was 26.7 months, and the 5-year survival rate was 26.42%. Kaplan-Meier analysis showed that LC3, CD68+ microglia, CD4+ T lymphocytes and

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CD8+ T lymphocytes were associated with prognosis in glioma patients (P<0.05)(Figure 3). The 5-year survival rates were 31.34% and 17.95% (P<0.01) in LC3-positive and negative patients,

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respectively. The 5-year survival rates of CD68+ microglia-positive and negative patients were20.00%

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and 38.89% (P<0.01), respectively. The 5-year survival rates of CD4+ T lymphocyte-positive and

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negative patients were 31.82% and 17.5% (P<0.01), respectively, and the 5-year survival rates for

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CD8+ T lymphocyte-positive and negative patients were 31.25% and 11.54% (P<0.01), respectively.

3.7 Cox proportional hazards regression model analysis

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Cox proportional hazards model analysis showed that LC3, CD68+ microglia, CD8+ T lymphocytes

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and tumor grade were independent prognostic factors for patients with glioma (Table 4). The partial regression coefficient of LC3 was -1.818, the relative risk was 0.616, and the 95.0% CI for relative risk was 1.637-5.176; the partial regression coefficient of CD68+ microglia was 0.144, the relative risk was

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0.745, and the 95.0% CI for relative risk was 1.012-1.080; the partial regression coefficient of CD8+ T lymphocytes was -0.340, the relative risk was 0.415, and the 95.0% CI for relative risk is 1.068-1.239; the partial regression coefficient of tumor grade was 0.512, the relative risk was 1.669, and the 95.0% CI for relative risk was 1.100-2.534.

4 Discussion In this study, we investigated the correlation and significance of LC3 and CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes in the tumorigenesis and development of gliomas. The

CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes were detected by

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expression of LC3 was detected by immunohistochemistry and Western blotting, and the numbers of

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immunohistochemistry. The results showed that LC3, CD68+ microglia, CD4+ T lymphocytes and

CD8+ T lymphocytes expression in high-grade and low-grade glioma tissues were significantly higher

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than the normal tissues, and high-grade gliomas higher than low-grade gliomas, and the correlation

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analysis showed that the expression of LC3 was positively correlated with the numbers of CD68+

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microglia, CD4+ T lymphocytes and CD8+ T lymphocytes. The relationships between LC3 expression,

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CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes and clinical features were also

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analyzed, and the results showed that LC3, CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes were positively correlated with tumor grade but not with age, sex, tumor size and location.

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Survival analysis showed that LC3, CD68+ microglia, CD8+ T lymphocytes and tumor grade were

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independent prognostic factors of glioma prognosis.

Autophagy is a highly conserved biological phenomenon that is widespread in eukaryotic cells. It is the

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process of degrading long-lived proteins and damaged organelles through use of double-membraned autophagic vacuoles in the cell. Studies have shown that autophagy is associated with multiple malignancies. Wu et al.[17] found that LC3 is highly expressed in human colorectal cancer tissues and is associated with a favorable prognosis, whereas Jiang et al.[18] showed a significant reduction in LC3

expression in human lung cancer tissues. Thus, autophagy plays different roles in different tumors; however, there are few studies on autophagy in glioma. The results of immunohistochemistry and Western blotting in this study show that the expression of LC3 in glioma tissues is significantly higher than that in normal brain tissue, and the high-grade gliomas was significantly higher than the low-grade,

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suggesting that autophagy is activated in glioma tissues. Li et al.[19] found that in the process of tumor development, autophagy can continue to adapt the cell to changes in the tumor microenvironment,

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allowing it to effectively survive hypoxia, nutrient deprivation, and other harsh environmental

conditions that normally pose a threat of apoptosis. We hypothesized that activation of autophagy in

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glioma may serve as a compensatory mechanism to provide energy and a stable tumor

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microenvironment for glioma by participating in the metabolism of intracellular proteins.

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Given the presence of the blood-brain barrier, the lack of a lymphatic drainage system,

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antigen-presenting cells and other characteristics, the central nervous system was previously called an "immune-free zone",since Louveau et al.[20]has found lymphatic vessels in the brain tissue, the tumor

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immunity of the central nervous system has become a hot spot of research. Recent studies have shown

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that the blood-brain barrier is not an absolute barrier to lymphocytes[21], and activation of T lymphocytes outside the central nervous system makes it easy to detect antigens located in the parenchyma of the central nervous system[22], while Aloisi[23] found that microglia can express MHC

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class II molecules after activation and participate in cell immunity by antigen presentation. Therefore, the central "immune-free zone" concept has not been borne out by the evidence. In a recent study of glioma, CD4+ T lymphocyte counts in peripheral blood were significantly lower in glioma patients than in controls, while CD8+ T lymphocyte counts were significantly increased[24], but no research on

CD4+ T lymphocytes and CD8+ T lymphocytes in glioma tissue has been reported. The results of our experiments show that there is a significant increase in the number of CD68+ microglia and the infiltration of CD4+ T lymphocytes and CD8+ T lymphocytes in glioma tissues than in normal brain tissues, and the numbers of CD68+ microglia, CD4+ T lymphocytes, CD8+ T lymphocytes was

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is a change of cellular immune function in glioma as the tumor originates and develops.

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significantly higher in high-grade gliomas than in low-grade gliomas. These findings suggest that there

New research has found that autophagy plays an important role in tumor immune response[25, 26].

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Van den Boorn et al.[15] found that autophagy can induce the expression and release of tumor surface

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antigen and promote the identification and killing of tumor cells by immune cells. A study by Wang et

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al.[14] showed that activation of autophagy can significantly increase the antigen cross presentation of

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tumor cells and induce specific CD8+ T lymphocyte responses. In our study, by analyzing the

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correlation between autophagic activity and the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes in glioma tissues, we found that the expression of LC3 in glioma tissues was

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positively correlated with the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T

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lymphocytes, suggesting that changes in autophagy levels may be one of the factors that affect the cellular immune response in glioma. On the one hand, the process of autophagy in the degradation of damaged organelles and denatured proteins can induce tumor antigen production and expression and

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activate CD8+ T lymphocytes through direct presentation by MHC-I, or activate CD4+ T lymphocytes through presentation of the tumor antigen by MHC-II; on the other hand, autophagy can enhance cross-presentation of antigens in tumor cells and induce cellular immune response, but the mechanism remains to be further studied.

At present, there are few studies on the correlations between LC3, CD68+ microglia, CD4+ T lymphocytes, CD8+ T lymphocytes and clinicopathological factors in patients with glioma. We analyzed the data to show that LC3 expression, CD68+ microglia, CD4+ T lymphocytes and CD8+ T

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lymphocytes were positively correlated with tumor grade; the higher the tumor grade, the higher the expression or positive cell count. Our findings suggest that LC3 expression, CD68+ microglia, CD4+ T

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lymphocytes and CD8+ T lymphocytes are valuable for judging the degree of malignancy. We suggest that the grade of glioma are among the factors that affect their internal blood supply. The higher the

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grade of glioma, the more vigorous the metabolism of its cells, causing the blood supply inside the

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tumor to be relatively poor. Therefore, to a certain extent, the higher the grade, the higher the

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the activation of the cellular immune response.

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autophagic activity; autophagy, in turn, promotes the expression and presentation of tumor antigen and

This study also investigated the prognostic significance of LC3, CD68+ microglia, CD4+ T

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lymphocytes and CD8+ T lymphocytes in gliomas. Huang et al.[27] have shown that abnormal

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expression of LC3 is associated with poor prognosis in gliomas. Komohara et al.[28] have demonstrated that the infiltration of microglia in high-grade gliomas is associated with poor clinical outcomes. Han et al.[24] found that the relationship between CD4+ T lymphocytes or CD8+ T

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lymphocytes in peripheral blood of patients with glioma alone was not clinically significant. However, in our current study, the expression of LC3 and the number of CD8+ T lymphocytes were positively correlated with the prognosis of patients, while the number of CD68+ microglia was negatively correlated with the prognosis of patients. The reasons for the inconsistent results may be related to the

criteria for case selection, the size of the observed sample and the locations from which the specimens were taken. Zhai et al.[29] found that microglia were involved in creating a microenvironment conducive to glioma growth and promoting its growth and migration. However, in our correlation analysis, glioma autophagy was positively correlated with the number of CD68+ microglia; this

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suggests that CD68+ microglia may contribute to tumor antigen presentation and participate in anti-tumor processes; but survival analysis revealed that the number of CD68+ microglia was

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associated with poor prognosis. We suggest that CD68+ microglia have dual properties in the

development and progression of gliomas. On the one hand, activated CD68+ microglia can act as

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antigen-presenting cells of the central nervous system and participate in the cellular immune response

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to glioma; on the other hand, CD68+ microglia can promote the development of the tumor by secreting

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cytokines such as epidermal growth factor, vascular endothelial growth factor and matrix

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metalloproteinase-9. However, the latter effect on glioma is greater than the former in our study. At the

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same time, we also found that patients with high-grade gliomas have a poor prognosis. We presume that these factors are associated with the recurrence of glioma; high-grade gliomas are invasive, and

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likely.

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tumor tissue in functional areas cannot be completely removed, meaning that tumor recurrence is

5 Conclusions

In general, the expression of LC3 and the numbers of CD68+ microglia, CD4+ T lymphocytes and

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CD8+ T lymphocytes increased in glioma tissues, and the expression of LC3 was positively correlated with CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes. Furthermore, high expression of LC3 and CD8+ T lymphocytes was positively correlated with the prognosis of the patients, while CD68+ microglia were negatively correlated with prognosis. Our results suggest that glioma autophagy

may be one of the factors that affect cellular immunity and that LC3 expression of LC3 and the numbers of CD68+ microglia and CD8+ T lymphocytes can be used as prognostic indicators in patients with glioma.

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Authors' contributions JL, WZ and SW were involved in the study conception and design. KG, ZH and JP participated in data acquisition, analysis, and interpretation. WZ wrote the manuscript. All

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authors have read and approved the manuscript for publication.

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Ethical approval All procedures performed in studies involving human participants were in

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accordance with the ethical standards of the institutional and/or national research committee and with the

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1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Informed consent The pathologic examinations and further investigations were conducted after

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receiving the informed consent from the patients.

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Conflict of interest The authors declare that they have no competing interests.

Funding This research did not receive any specific grant from funding agencies in the public,

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commercial, or not-for-profit sectors.

Acknowledgements The authors would like to thank Xiangqian Gao for providing technical.

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Table 1. Positive expression of LC3, CD68+ microglia, CD8+ T lymphocytes and CD4+ T Lymphocytesin glioma tissues and normal brain tissues n (%) CD4+ T

CD8+ T

microglia

lymphocytes

lymphocytes

6 (28.57)

7 (33.33)

6 (28.57)

4 (19.05)

68

38 (55.88)*

33 (48.53)*

39 (57.35)*

42 (61.76)*

59

42 (71.12)*#

45 (76.27)*#

40 (67.80)*#

53 (89.83)*#

LC3

Normal brain tissues

21

Low-grade High-grade

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CD68+

n

*After a Chi-square test, P<0.01 for glioma tissues compared with normal brain tissues; #

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N

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After a Chi-square test, P<0.01 for high-grade glioma tissues compared with low-grade glioma tissues.

Table 2. Correlation between the expression of LC3 and the numbers of CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes in gliomas CD4+T

n

+

80

microglia +

-

61

19

LC3

r

P

0.227 -

47

17

30

CD8+T

lymphocytes +

-

64

16

0.017

r

0.180 15

lymphocytes

P

+

-

75

5

20

27

0.021

32

P

0.394

0.009

A

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PT

ED

M

A

N

U

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Abbreviations:+, positive expression; -, negative expression.

r

IP T

CD68+

Table 3. Correlation between LC3 expression, CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes and clinicopathologic parameters in 127 patients with glioma Characteristics

n

LC3+

59

37

P

CD68+ microglia

P

CD4+T lymphocytes

P

CD8+T lymphocytes

P

Age (years)

>50

36

37

0.328 68

43

70

44

43

0.311 42

0.332 42

Gender

Female

44

44

0.237 57

36

68

38

34

0.205 35

Tumor grade Low-grade High-grade

45

40

0.008 59

42

82

42

0.018 33

39

38

31

Frontal lobe

33

20

21

Temporal lobe

50

31 22

Other parts

9

7

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19 7

ED

35

31

0.180

PT

Parietal lobe

M

Tumor location

N

45

53

56 0.099

0.136

39

39

18

24

33 0.442

23 5

0.001

42

40

0.107

A

>3

U

47 0.076

0.432

41

0.011

Tumor size (cm) ≤3

54

0.243

SC R

Male

0.225 52

IP T

≤50

37 0.240

26 8

0.148

Table 4. Cox regression model analysis of overall survival in 106 glioma patients B

SE

Wald

df

Exp (B)

95.0% CI

P

LC3 (Positive vs. Negative)

-1.818

0.676

7.232

1

0.616

1.637-5.176

0.007

CD68+ microglia (Positive vs. Negative)

0.144

0.017

7.099

1

0.745

0.097-0.780

0.008

CD4+ T lymphocytes (Positive vs. Negative)

-0.698

0.311

5.028

1

2.011

0.460-3.316

0.318

CD8+ T lymphocytes (Positive vs. Negative)

-0.340

0.038

13.673

1

0.415

0.168-1.239

0.000

Age (>50 vs. ≤50)

-0.772

0.315

6.381

1

0.486

0.245-1.544

0.079

Gender (Male vs. Female)

0.811

0.350

9.495

1

0.514

0.494-1.663

0.150

Tumor grade (High-grade vs. Low-grade)

0.512

0.312

5.786

1

1.669

0.331-2.534

0.016

Tumor size (>3cm vs. ≤3cm)

-1.091

0.547

8.640

1

0.970

0.714-1.628

0.227

Frontal lobe vs. Temporal lobe

1.113

0.235

22.502

1

1.499

0.808-1.521

0.768

Frontal lobe vs. Parietal lobe

0.819

0.395

7.927

1

1.316

0.301-1.626

0.895

Frontal lobe vs. Other parts

0.754

0.317

8.441

1

0.910

0.377-1.905

0.461

A

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M

A

N

U

SC R

Tumor location

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Parameters

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Figure 1. Immunohistochemistry analysis was used to measure the abundance of LC3 expression, CD68+ microglia, CD4+ T lymphocytes and CD8+ T lymphocytes in glioma tissues and normal

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brain tissues. Magnification:×400.

Figure 2. Western blotting analysis the expression of LC3 in glioma tissues and normal brain

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tissues. 21gliomas were assessed by Western blot. (A) Proteins extracted from different grade glioma tissues (Grade I, II, III, and IV) and normal brain tissues (N) were subjected to Western blot analysis.

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The data were shown as the means±SE. (B) Data analysis showed that the expression of LC3 in glioma

A

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PT

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M

A

N

U

tissues and in normal brain tissues (P<0.05).

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Figure 3. Kaplan-Meier survival analysis of LC3, CD68+ microglia, CD4+ T lymphocytes, CD8+

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T lymphocytes, tumor grade and location in glioma patients (log-rank test). (A) The expression of LC3 and overall survival of glioma patients are shown. Positive expression [LC3 (+)], n = 67; negative expression [LC3 (-)], n = 39. (B) The expression of CD68+ microglia and the overall survival of

A

patients with gliomas are shown. Positive expression [CD68+ microglia (+)], n = 70; negative expression [CD68+ microglia (-)], n = 36. (C) The expression of CD4+ T lymphocytes and the overall survival of patients with gliomas are shown. Positive expression [CD4+ T lymphocytes (+)]; n = 66, negative expression [CD4+ T lymphocytes (-)], n = 40. (D) The expression of CD8+ T lymphocytes and overall survival of gliomas patients are shown. Positive expression [CD8+ T lymphocytes (+)], n = 80; negative expression [CD8+ T lymphocytes (-)], n = 26.