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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 4  |  Page : 882-888

Tim-3 expression in glioma cells is associated with drug resistance


1 Department of Neurosurgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China
2 Department of Neurosurgery, Tumor Hospital Affiliated of Xinjiang Medical University, Xinshi District, China
3 Department of Rehabilitation, The Second Affiliated Hospital of Xinjiang Medical University, Urumqi, China
4 Department of Urology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China
5 Department of Neurosurgery, Shunde Hospital, Southern Medical University (The First People's Hospital of Shunde Foshan), Foshan, China
6 Department of Neurosurgery, The First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou, China

Date of Web Publication14-Aug-2019

Correspondence Address:
Xiao Bing Xu
Department of Neurosurgery, Shunde Hospital, Southern Medical University (The First People' s Hospital of Shunde Foshan), Foshan
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_630_18

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 > Abstract 


Objective: T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) has been widely recognized as a negative regulator of antitumor immunity. However, the mechanism by which Tim-3 suppresses antitumor treatment in gliomas remains unclear. This study aims to explore whether Tim-3 is expressed and to evaluate its effect in drug-fasted glioma cells.
Subjects and Methods: U87 and U251 glioma cell lines were tested. Cell proliferation activity, cell viability, and the protein and mRNA levels of Tim-3 were detected using CCK-8, flow cytometry, Western blotting, and reverse transcription-quantitative polymerase chain reaction, respectively. Enhancement of the sensitivity of glioma cells to chemotherapeutic agents was tested after inhibiting Tim-3 expression using Tim-3 small interfering RNAs (siRNA).
Results: As temozolomide (TMZ) concentration increased, the ratio of apoptotic cells also increased accordingly. However, the level of Tim-3 expression in living cells from the high-dose group was higher than in the low- and middle-dose groups. After interfering with the expression of Tim-3 using siRNA against Tim-3, the killing effect of TMZ rose through an increase in apoptosis.
Conclusions: The presence of Tim-3 mRNA and protein in glioma cells was detected. Significantly, knocking down Tim-3 expression improved the potential of TMZ treatment.

Keywords: Drug resistance, glioma cell, small interfering RNAs, Tim-3


How to cite this article:
Zhang J, Zhu ZQ, Li YX, Zhuang QF, Lai Y, Li SF, Xu XB, Liu JM. Tim-3 expression in glioma cells is associated with drug resistance. J Can Res Ther 2019;15:882-8

How to cite this URL:
Zhang J, Zhu ZQ, Li YX, Zhuang QF, Lai Y, Li SF, Xu XB, Liu JM. Tim-3 expression in glioma cells is associated with drug resistance. J Can Res Ther [serial online] 2019 [cited 2019 Nov 20];15:882-8. Available from: http://www.cancerjournal.net/text.asp?2019/15/4/882/264297




 > Introduction Top


Glioblastoma is the most malignant and devastating primary brain tumor in adults.[1] Current treatments remain insufficient, as these tumors tend to recur despite extensive removal and subsequent radiochemotherapy. Studies have revealed a relationship between the expression of Tim-3 and tumor-related immunosuppression.[2],[3],[4] Several independent studies described drug resistance that resulted in final treatment failure. Identifying the cause of drug tolerance and adopting intervention measures to combat it are extremely necessary to prolong the survival of these patients. The investigation of resistance mechanisms to different drug modalities is gaining traction in the glioma research.[5] We investigated the causes of glioma drug resistance against temozolomide (TMZ).

Apart from its role in immunoreaction, Tim-3 also has a biological role in tumors.[6] Melanoma, human colorectal carcinoma, and cervical cancer tissues were shown to contain Tim-3-positive mast cells, which advance tumor infiltration.[6],[7],[8] Although an increasing body of evidence about Tim-3 expression has been documented in many different human tumors, its effect in glioma cells has not yet been determined. Tim-3 can uniquely discriminate between Th1 cells and Th2 cells in both humans and mice.[9] Evidence supporting Tim-3 expression in some tumor cells has been documented in recent years.[7] However, whether Tim-3 is expressed in cancer cells from nonhematologic cancers remains unclear.

The protein and miRNA levels of Tim-3 in glioma cell lines were initially determined using western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses, respectively. Tim-3 expression in glioma cells was significantly related to cell death resistance. Knockdown of Tim-3 expression remarkably decreased the proliferative abilities of glioma cells and sensitized them to TMZ by enhancing apoptosis. Tim-3 is a crucial factor in the drug-resistance of glioma cells and can be a potential therapeutic target for the treatment of malignant glioma.


 > Subjects and Methods Top


Agents

TMZ was purchased from Sigma-Aldrich (Sigma-Aldrich Corporation, St. Louis, Missouri, United States), stored at −20°C, dissolved in dimethyl sulfoxide (DMSO), and diluted in serum-free medium at indicated concentrations for treatment. The stock concentration was 200 mmol/L.

Cell culture and antibodies

U87 and U251 cells were purchased from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere at 37°C with 5% CO2. When the cells covered 80%–90% of the bottom of the culture plate, we began adding various concentrations of TMZ, then cultured the cells for an additional 24, 48, and 72 h. For the transfection assays, we transfected the cells with small interfering RNAs (siRNAs) using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and anti-Tim-3 goat polyclonal antibody (Santa Cruz Biotechnology, Inc.) and biotinylated secondary antibody were purchased from Santa Cruz Biotechnology, Inc.

Cell viability assay

Following exposure to TMZ, the cell viability of U87 and U251 cells was evaluated using the Cell Counting Kit 8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). In brief, U87 and U251 cells were planted in flat-bottomed 96-well plates at a density of 3 × 104 in 180 μl of set medium per well. Ten microliters of CCK-8 solution were added to each well after incubation with increasing concentrations of TMZ (DMSO control or 150–2400 μmol/L) at 37°C in a humidified 5% CO2 atmosphere for 72 h. The absorbance was quantified using a spectrophotometer at 450 nm. Each assay was performed three times, and each experiment was repeated in triplicate.

RNA isolation and reverse transcription-quantitative polymerase chain reaction analysis

In brief, total RNA was obtained from the two glioma cell lines. A 5' sense primer (5'-CTGCTGCTACTACTTACAAGGTC-3') and a 3' antisense primer (5'-GCAGGGCAGATAGGCATTCT-3') were used to expand Tim-3 transcripts. A 5' sense primer (5'-CTCACGAAACTGGAATAAGC-3') and a 3' antisense primer (5'-AAGCCACACGTACTAAAGGT-3') were used to enhance the β-actin internal control. The primers utilized for PCR examination of Tim-3 were designed to span introns in order to avert false-positive amplifications from DNA. We also used total RNA product without reverse transcription as a template for PCR as a negative control to prevent genomic DNA pollution.

Flow cytometry

Fluorescence-activated cell sorting buffer (PBS with 3% FBS; Gibco; Thermo Fisher Scientific Inc.) was used to resuspend U87 and U251 cell lines, which were blocked for 20 min at 4°C with 10% human health serum (Beijing Red Cross Blood Center, Beijing, China) and then stained in the dark with fluorescence-conjugated antibodies for 30 min at 4°C. The BD FACSCalibur™ (BD Biosciences) was used to perform flow cytometry analysis. In total, ~10,000 cells were collected, and the data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA). The fluorescence-conjugated antibodies used were as follows: phycoerythrin (PE)-conjugated human anti-Tim-3 rat IgG2a monoclonal antibody (catalog no. FAB2365P; dilution, 50 μg/ml; RandD Systems China Co., Ltd., Shanghai, China); PerCp-Cy5.5-conjugated anti-human cluster of differentiation CD3 mouse monoclonal antibody (catalog no. 45-0037-42; dilution, 10 μg/ml −1; eBioscience, San Diego, CA, USA); allophycocyanin-conjugated antihuman CD8 mouse monoclonal antibody (catalog no. 17-0086-42; dilution, 10 μg/ml − 1; eBioscience); and PE-conjugated rat IgG2a isotype control antibody (catalog no. IC006P; dilution, 50 μg/ml −1; RandD Systems China Co., Ltd.).

Western blotting

Lysis buffer replenished with protease inhibitors was used to lyse the two glioma cell lines. The proteins from the glioma cell lines were quantified using a Bicinchoninic Acid kit (Thermo Fisher Scientific, Inc.). Then, a total of 50 μg protein was run on a 10% SDS-PAGE gel for partition and subsequently transferred onto a nitrocellulose membrane. After blocking in 5% milk at room temperature for 1 h, the membrane was incubated with primary antibodies against Tim-3 (1:1,000; cat. no. ab185703; Abcam) and GAPDH (1:1,000; cat. no. sc-365062; Santa Cruz Biotechnology, Inc.) at 4°C overnight. The next day, TBS with Tween-20 was used to wash the membrane three times, after which the membrane was incubated with secondary antibodies (1:1,000; cat. no. sc-3836; Santa Cruz Biotechnology, Inc.) at 37°C for another hour. Using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.), quantified protein levels and images were taken using the LAS-3000 imaging system (Fujifilm Corporation, Tokyo, Japan).

Statistical analysis

The GraphPad Prism 5.0 software (Chenkai Network Technology Co., Ltd, Shanghai, China) was used for all statistical analyses. The data are expressed as the mean ± standard error. Student's t-test was used to confirm any statistically significant differences. P < 0.05 was the cutoff for a statistically significant difference.


 > Results Top


Tim-3 expression has a positive correlation with glioma cell resistance to temozolomide

TMZ, when given in both concomitant and adjuvant phases, has been widely used as an effective therapy in patients with high-grade glioma (HGG). It prolongs survival and delays progression without increasing early adverse events. Tim-3 has been implicated previously in tumor biology. However, the expression of Tim-3 in glioma cell lines is completely unclear. The present study first explored the expression of Tim-3 in glioma cell lines through Western blotting and RT-qPCR. The results showed that Tim-3 was expressed at a high level in the glioma cell lines, compared with noncancerous cells [Figure 1].
Figure 1: Differential expression of Tim-3 was shown in glioma cell lines. The 293T cell line was included as a control. Western blot analysis revealed that the protein level of Tim-3 was higher in glioma cell lines than in the control. The RNA level of Tim-3 was detected by reverse transcription-quantitative polymerase chain reaction and was significantly higher in glioma cell lines than in the control

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Although TMZ has been considered one of the most effective chemotherapeutic agents for HGG, many glioma cells survive after TMZ treatment. The IC50 for TMZ in U87 and U251 glioma cell lines was measured to be 1200 μmol/L [Table 1]. Using this viability assay, we observed that U87 and U251 glioma cells could partially survive even after incubation with a high concentration of TMZ [Figure 2]. U87 and U251 glioma cell lines demonstrated a remarked insensitivity to TMZ.
Table 1: The viability of cells in different concentration of temozolomide

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Figure 2: The cell viability of glioma cells in different concentrations of temozolomide. *P < 0.05, ***P < 0.01, compared to the control group (dimethyl sulfoxide only)

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The total RNA obtained from the glioma cells was subjected to RT-qPCR analysis. It was noted that the mRNA level of Tim-3 in glioma cells was higher than in the control [Figure 3]. It was shown that the protein level of Tim-3 was also significantly higher than that in the paired noncancerous samples [Figure 4]. Our data indicated that Tim-3 was expressed at a high level in the drug-resistance glioma cells.
Figure 3: Reverse transcription-quantitative polymerase chain reaction analysis indicated that the mRNA level of Tim-3 in high concentrations of temozolomide was higher than in low concentrations, *P < 0.05, ***P < 0.01, compared to the control group (dimethyl sulfoxide only)

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Figure 4: Differential expression of Tim-3 was shown in glioma cell lines. Western blot analysis revealed that the protein level of Tim-3 in the high concentration group was significantly higher than in the others

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Repressing Tim-3 expression increased apoptosis in U87 and U251 cell lines

To knock down the expression of Tim-3 in cultured glioma cell lines, siRNA against Tim-3 was designed. To further clarify the link between Tim-3 expression and drug-resistance, we transfected glioma cells with Tim-3 siRNA. As a result, Tim-3 expression levels notably decreased [Figure 5]. The TMZ concentration (1200 μmol/L) was used in the following experiments. After knockdown of Tim-3 expression, cell viability declined noticeably, compared to the TMZ-only treatment group [Figure 6]. These results strongly suggest that downregulating Tim-3 significantly reduces glioma cell resistance to TMZ.
Figure 5: The effect of Tim-3 siRNA on U87 cells. Reverse transcription- quantitative polymerase chain reaction was performed to detect Tim-3 expression in U87 cells transfected with Tim-3 siRNA or treated with “scramble” siRNA, ***P < 0.05

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Figure 6: Knockdown of Tim-3 with siRNA in cultured glioma cells. Western blot analysis revealed that the protein level of Tim-3 in the Tim-3 siRNA group significantly decreased; the expression of Tim-3 increased in the 1200 μM temozolomide group, but after transfection with Tim-3 siRNA, the expression of Tim-3 did not increase. *P < 0.05, compared to the control group

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Next, CCK-8 assays were used to monitor cell viability. As expected, cells treated with both Tim-3 siRNA and TMZ had lower viability than cells treated with TMZ alone [Figure 6]. Although TMZ induces the apoptosis of glioma cells, it does not markedly affect the growth of resistant cells.

The interference efficiency of Tim-3 siRNA was assessed by Western blotting and RT-qPCR. Seventy-two hours after transfection, the RNA and protein were isolated from cells and subjected to RT-qPCR and Western blotting, respectively. As shown in [Figure 6], the mRNA and protein levels of Tim-3 in these cells were notably reduced, compared to the control groups. These results clearly show the efficiency of transfection and the high specificity of Tim-3 siRNA.

Knockdown of Tim-3 expression sensitized the glioma cell lines to TMZ via the enhancement of apoptosis. We noticed that TMZ alone led to a moderate increase in apoptosis, while the addition of Tim-3 knockdown greatly increased apoptosis. Quantitative analysis demonstrated that the proportion of dead cells increased from 26.6% ± 6.2% to 23.8% ± 5.1% in TMZ-treated cell lines to 60.9% ± 5.7% and 57.3% ± 9.2% in cells with combined treatment (TMZ + Tim-3 siRNA). This finding supported the idea that knocking down the expression of Tim-3 in glioma cells causes an evident induction of apoptosis. To further quantify the induction of apoptosis, flow cytometric analysis was performed. This assay demonstrated that TMZ incubation after Tim-3 knockdown resulted in an observably greater percentage of apoptotic cells than in glioma cells treated without Tim-3 knockdown. These results revealed that reducing Tim-3 expression remarkably enhanced the sensitivity of glioma cells to TMZ and demonstrated a positive correlation between the expression of Tim-3 and glioma cell drug resistance [Figure 7].
Figure 7: Temozolomide treatment and Tim-3 siRNA induce apoptotic cell death. Dot-plot analysis from flow cytometry in 5% fetal bovine serum medium or serum-free medium. Percentage of cells according to viability and type of cell death: PE/7AAD− (viable cells), PE+/7AAD+ (late apoptosis), PE+/7AAD− (early apoptosis), and PE−/7AAD+ (necrosis). Data are represented as mean ± standard error of the mean (n = 6). *P < 0.05 versus control, #P < 0.05 versus 1200 μM temozolomide group

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 > Discussion Top


HGG remains one of the most intractable and lethal solid cancers in adults, and a helpful therapeutic molecular target has not yet been identified.[9] The median survival time of patients is roughly 15 months, even after surgical resection, radiotherapy, and chemotherapy.[5],[10],[11] TMZ can increase overall survival and has turned into the most important chemotherapeutic agent for these patients.[12] Regrettably, the antitumor effect of TMZ does not last due to acquired or intrinsic drug resistance. Therefore, the identification of the cause of drug resistance to TMZ is highly desired.

Tim-3, which can affect cancer therapy in the tumor microenvironment, is a surface molecule that can distinguish Th1 cells from Th2 cells in both mice and humans.[10] Although studies have indicated that Tim-3 is involved in the immune regulation of tumors, its direct expression and function in gliomas were unknown. Interestingly, we observed that glioma cell lines had Tim-3 expression, and that it was preferentially expressed in TMZ-treated cells. Although TMZ evidently restrained cell growth and promoted apoptosis in glioma cell lines, a portion of glioma cells are still resistant to it.[13] The coincidental finding that Tim-3 expression is associated with resistance to chemotherapy-induced apoptosis resulted in our efforts to investigate whether Tim-3 expression serves a functional role in driving drug resistance in glioma.

To explore this potential effect, drug resistance in glioma cells was investigated. In the present study, the expression of Tim-3 was found in the glioma cell lines, and knocking down Tim-3 expression enhanced the sensitivity of glioma cell lines to TMZ in vitro. This result showed that Tim-3 expression increased the resistance of glioma cells to TMZ and suggested the necessity for the implementation of therapeutic regimens that treat Tim-3 as a potential target. One study has suggested anti-Tim-3 monoclonal antibodies as a therapeutic option to damage cancer cells in other tumors. This approach may cause the enhancement of the efficacy of chemotherapeutic agents, along with the anticipated unique transference of therapeutic effect by interfering with the expression of Tim-3.[14]

These data support the idea that knockdown of Tim-3-directed regulation promotes decreased drug resistance and increased apoptosis. Tim-3 expression was recently documented in melanoma cells, lowering the adhesion ability of tumor cells and boosting tumor survival.[7] The author reported a significant effect of Tim-3 expression, which plays an independent prognostic role in nonsmall cell lung cancer patients, on tumor cells.[15] In this study, we have shown for the first time that Tim-3 is more highly-expressed in TMZ-treated glioma cell lines than in untreated cell lines. Recent research has supported the role of Tim-3 in T-cell exhaustion in cancer.[11] Blockade of Tim-3 pathways can more effectively suppress tumor growth than targeting either pathway alone, showing that these two pathways act synergistically to establish T-cell exhaustion.[16] Tim-3 knockdown had not been reported in glioma cell lines until our study. A functional effect of Tim-3 on cytotoxic resistance has not been confirmed in glioma cell lines. The mechanisms for regulating the expression of Tim-3 in glioma cells have not been yet established. In agreement with our study, some published studies have confirmed that Tim-3 overexpression was related to shorter overall survival times in epithelial cancers involving cervical cancer and lung cancer.[15] Even more importantly, Cao et al. observed that knocking down Tim-3 expression in Hela cells decreased both invasion and migration.[8] These findings demonstrate that Tim-3 not only inhibits tumor treatment but also directly boosts cancer progression.

These experiments showed that it is possible to reduce drug-resistance by knocking down Tim-3 expression, which results in some protection against drug resistance in tumor cell lines, although the protection is not as complete as in the original cells. Despite great progress in our understanding of the involvement of Tim-3 in tumor immunity, the link between tumor cells themselves and Tim-3 expression has not yet been explored. One of our most outstanding findings is that when we applied Tim-3 siRNA to downregulate Tim-3 expression in glioma cell lines, cell death increased significantly. Further research is required to elucidate the maze of variables potentially involved in drug resistance. It is feasible that if immunization followed chemotherapy, even without complete immune protection, cells might be more sensitized to chemotherapy and the outgrowth of resistant tumor cells could be restrained. This type of experimentation could also be performed for other tumor cell lines with drug resistance. Overall, the data provide evidence that cancer cells with Tim-3 expression are resistant to chemotherapy.


 > Conclusion Top


The expression of Tim-3 in glioma cell lines was observed and was found to correlate with drug-resistance. We utilized siRNA to knock down Tim-3 expression in glioma cell lines in order to understand its mechanism of action and to make available additional therapeutic targets to treat malignant gliomas.

Acknowledgment

This study was funded by Project of Guangdong Medical Science and Technology Research Foundation (A2018017), and Project of Science and Technology Department of Xinjiang Uygur Autonomous Region (2018D01C251). Foundation of the Science and Technology Program of Guangzhou, People's Republic of China (Grant No. 201607010365) and High Level University Construction Project of Guangzhou University of Traditional Chinese Medicine (No. A1-AFD018171Z11072).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Liu Y, Shete S, Hosking F, Robertson L, Houlston R, Bondy M. Genetic advances in glioma: Susceptibility genes and networks. Curr Opin Genet Dev 2010;20:239-44.  Back to cited text no. 1
    
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Anderson AC. Tim-3, a negative regulator of anti-tumor immunity. Curr Opin Immunol 2012;24:213-6.  Back to cited text no. 2
    
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Sakuishi K, Jayaraman P, Behar SM, Anderson AC, Kuchroo VK. Emerging Tim-3 functions in antimicrobial and tumor immunity. Trends Immunol 2011;32:345-9.  Back to cited text no. 3
    
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Norde WJ, Hobo W, van der Voort R, Dolstra H. Coinhibitory molecules in hematologic malignancies: Targets for therapeutic intervention. Blood 2012;120:728-36.  Back to cited text no. 4
    
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Endicott JA, Ling V. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem 1989;58:137-71.  Back to cited text no. 5
    
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Yu M, Lu B, Liu Y, Me Y, Wang L, Zhang P. Tim-3 is upregulated in human colorectal carcinoma and associated with tumor progression. Mol Med Rep 2017;15:689-95.  Back to cited text no. 6
    
7.
Wiener Z, Kohalmi B, Pocza P, Jeager J, Tolgyesi G, Toth S. TIM-3 is expressed in melanoma cells and is upregulated in TGF-beta stimulated mast cells. J Invest Dermatol 2007;127:906-14.  Back to cited text no. 7
    
8.
Cao Y, Zhou X, Huang X, Li Q, Gao L, Jiang L, et al. Tim-3 expression in cervical cancer promotes tumor metastasis. PLoS One 2013;8:e53834.  Back to cited text no. 8
    
9.
Yang DY, Bu XY, Zhou ZL, Yan ZY, Ma CX, Qu MQ, et al. Enhanced antitumor effects of radiotherapy combined local nimustine delivery rendezvousing with oral temozolomide chemotherapy in glioblastoma patients. J Cancer Res Ther 2018;14:78-83.  Back to cited text no. 9
    
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Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, et al. Th1-specific cell surface protein tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 2002;415:536-41.  Back to cited text no. 10
    
11.
Buckner JC. Factors influencing survival in high-grade gliomas. Semin Oncol 2003;30:10-4.  Back to cited text no. 11
    
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Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009;10:459-66.  Back to cited text no. 12
    
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Wang Q, Qi F, Song X, Di J, Zhang L, Zhou Y, et al. A prospective longitudinal evaluation of cognition and depression in postoperative patients with high-grade glioma following radiotherapy and chemotherapy. J Cancer Res Ther 2018;14:S1048-S1051.  Back to cited text no. 13
    
14.
Smith LM, Nesterova A, Ryan MC, Duniho S, Jonas M, Anderson M, et al. CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers. Br J Cancer 2008;99:100-9.  Back to cited text no. 14
    
15.
Zhuang X, Zhang X, Xia X, Zhang C, Liang X, Gao L, et al. Ectopic expression of TIM-3 in lung cancers: A potential independent prognostic factor for patients with NSCLC. Am J Clin Pathol 2012;137:978-85.  Back to cited text no. 15
    
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Hafler DA, Kuchroo V. TIMs: Central regulators of immune responses. J Exp Med 2008;205:2699-701.  Back to cited text no. 16
    


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