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IL-18, a therapeutic target for immunotherapy boosting, promotes temozolomide chemoresistance via the PI3K/AKT pathway in glioma

Journal of Translational Medicine volume 22, Article number: 951 (2024) Cite this article

Abstract

Interleukin-18, a member of the interleukin − 1 family of cytokines, is upregulated in glioma. However, its effects on glioma remain unclear. This study aimed to explore the role and underlying mechanisms of interleukin-18 expression in glioma. Here, we demonstrated that interleukin-18 enhanced resistance to temozolomide by increasing proliferation and inhibiting apoptosis in cultured glioma cells. Further in vivo studies revealed that interleukin-18 promoted temozolomide resistance in BALB/c nude mice bearing tumor. Mechanical exploration indicated that interleukin-18 stimulation could activate the PI3K/AKT signaling pathway in glioma cells, and PI3K inhibition could reduce the temozolomide resistance promoted by interleukin-18. We found that interleukin-18 upregulated CD274 expression in glioma, revealing its potential effects on the microenvironment. Furthermore, we established a tumor xenograft model and explored the therapeutic efficacy of anti-interleukin-18 monoclonal antibody. Targeting interleukin-18 prolonged survival and attenuated CD274 expression in the mice bearing tumor. Combined treatment with anti-interleukin-18 and anti-PD-1 monoclonal antibody showed better efficacy in suppressing tumor growth than either treatment alone in mice bearing tumor. Collectively, these data present that interleukin-18 promotes temozolomide chemoresistance in glioma cells via PI3K/Akt activation and establishes an immunosuppressive milieu by modulating CD274. This study highlights the therapeutic value of interleukin-18 in glioma.

Introduction

Glioma is the most common primary malignant brain tumor. Malignant glioma is characterized by a short lifetime and high mortality rate with limited options for standard treatment [1,2,3]. The alkylating drug temozolomide (TMZ) is the standard chemotherapy for glioma. However, most patients with malignant glioma eventually die of disease progression caused by tumor recurrence due to resistance to TMZ [4,5,6,7]. Strategies to overcome TMZ resistance are therefore crucial and significantly improve the overall survival of patients with glioma.

Interleukin-18 (IL-18) is a member of the interleukin-1 family. It was originally described as an inducer of interferon-γ. As a primary inflammatory cytokine, IL-18 is a key driver of tumor-promoting inflammation [8]. IL-18 is highly expressed in many tumor tissues, and it promotes tumor progression, including glioma, pancreatic cancer, breast cancer, gastric cancer, and multiple myeloma [9,10,11,12,13,14,15]. Recent studies indicate that IL-18 contributed to tumor chemoresistance [16, 17]. However, the in-depth role of IL-18 in chemoresistance of glioma remains to be elucidated. Here, we explored whether IL-18 is involved in TMZ resistance in glioma.

Due to the poor prognosis of glioma, an increasing number of researchers have focused on immune checkpoint inhibitors for glioma treatment. Furthermore, IL-18 induces programmed cell death ligand 1 (PD-L1, CD274) expression in diffuse large B-cell lymphoma and renal cell carcinoma, which contributes to immune escape in patients with cancer [18, 19]. However, no studies have reported the role of IL-18 in the regulation of CD274 expression in glioma.

In this study, we indicated that IL-18 promoted chemoresistance by activating the PI3K-AKT signaling pathway. We further investigated the association between IL-18 and CD274 expression, as well as the effect of IL-18 blockade on anti-PD1 therapy. These findings confirm that targeting IL-18 is a potential therapeutic strategy for the treatment of glioma.

Materials and methods

Clinical samples and ethical approval

Paraffin-embedded tissue sections were collected from glioma patients with written informed consent recruited from Zhujiang Hospital. Six typical glioma specimens were selected for further analyses (Supplement Table 1). This study was approved by the Ethics Committee of Zhujiang Hospital (2023-KY-022-02).

Immunohistochemistry (IHC) staining

Paraffin-embedded glioma sections were deparaffinized and dehydrated via a graded ethanol series, followed by antigen retrieval in boiling citric acid buffer. Non-specific binding was blocked with 5% bovine serum albumin. The sections were permeabilized through incubation in 0.01% Triton X-100, followed by incubation with the primary antibodies at 4 °C overnight. The following primary antibodies were used: IL-18 (Proteintech, IL, USA), CD274 (Proteintech, IL, USA), and CD4 and CD8 (Service Bio, China). Then the sections were stained with biotinylated secondary antibodies (Santa Cruz Biotechnology, CA, USA) for 2 h at room temperature. The samples were then counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) and observed through an upright microscope (DM2500, Leica). And we used ImageJ for the quantification of IHC.

Cell lines

To investigate the effects of exogenous IL-18 (Sino Biological, Beijing, China) on glioma, we have examined the endogenous expression of IL-18 in several glioma cells in our lab. The expression of IL-18 in U87 and U118 is lower than other cells (Figure S2A and B), which is suitable for exploring obvious effects of exogenous IL-18. And We chose these two cells for further research. U87, U118 and GL261 glioma cells were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and were tested for mycoplasma contamination. Our team established and maintained U87 TMZ-resistant (TR) glioma cells (U87TR) as described previously [20]. The TR cells were generated by repetitive pulse exposure of U87 cells to TMZ (48 h every 2 weeks) and with increasing TMZ concentrations for 6 months. For TR phenotype maintenance, U87TR cells were alternately treated with TMZ (500 µM) for 48 h [20]. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, CA, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT, USA) and antibiotics (100 µg/mL streptomycin and 100 U/mL penicillin) at 37 °C in a humidified air incubator (Thermo Scientific, Waltham, MA, USA) containing 5% CO2.

Enzyme-linked immunosorbent assay

U87 cells were incubated with PBS or 50 ug/mL TMZ for 48 h. And the supernatants of U87 cells were collected for ELISAs. ELISAs were performed to determine secreted IL-18 levels in glioma cell supernatants according to the manufacturer’s instructions of IL-18 enzyme-linked immunosorbent assay kit (MultiSciences Biotech, China). Absorbance was measured at 450 nm using an Ultra Multifunctional Microplate Reader (Tecan, Switzerland), and the IL-18 concentration was calculated using GraphPad Prism 8 software.

RNA isolation, reverse transcription, and quantitative real-time PCR

U87 and U118 cells were treated with DMSO, human IL-18 protein (75 ng/mL), or IL-18 + LY294002 (50 µmol/L) for 48 h. After that, total RNA from cells was isolated using Trizol Reagent (Takara Bio, Shiga, Japan) according to the manufacturer’s protocol. The yield and quality of RNA was determined by measuring the absorbance at 260 and 280 nm. Then, cDNA was synthesized according to protocol of the Prime Script RT reagent kit (Takara Bio, Shiga, Japan). Quantitative real-time PCR was performed by using the SYBR GREEN PCR Master Mix (Takara Bio, Shiga, Japan) on a 7900 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The quantitative PCR primers are human CD274 forward 5’-CTGCACTTTTAGGAGATTAGATC-3’ and reverse 5’-CTACACCAAGGCATAATAAGATG-3’. GAPDH was used as the endogenous control. The relative expression of gene was calculated via the relative quantification (ΔΔCt) method with at least three independent experiments.

Protein extraction and western blot analysis

Total proteins were obtained from cells according to the protocol of Whole Cell Lysis Assay (KeyGEN BioTECH, China) on ice and quantified via a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). The western blotting was performed according to standard procedures. The following antibodies were employed: IL-18 (Sino Biological, TX, USA), PI3K, (Abcam, MA, USA), P-Akt (Ser473), AKT, P-c-Raf, β-actin (Cell Signaling Technology, MA, USA), CDK2 (ImmunoWay, TX, USA), and CD274 (Proteintech, IL, USA). After incubation with the primary antibody, the membranes were incubated in appropriate horseradish peroxidase-labeled secondary antibodies (Cell Signaling Technology, MA, USA). Enhanced chemiluminescence (Biosciences, Foster City, CA, USA) was added to each membrane and the bands were visualized with ECL detection system. Finally, densitometry analysis of protein was performed via the Image J software and β-actin levels were used as loading control.

TMZ chemosensitivity and cell viability assay

Cell viability analysis was performed according the manufacturer’s instructions of Cell Counting Kit-8 (CCK-8) (KeyGEN Biotech, China). Firstly, Glioma cells were seeded in 96-well plates with human IL-18 protein at doses from 0 to 105 ng/mL for 48 h. Next, these cells were seeded in 96-well plates and treated with TMZ at different concentrations (0, 100, 200, 300, 400, and 500 µg/mL) for 48 h. These cells were then cultured in fresh medium with 10% CCK-8 solution for 2 h, and the absorbance at 450 nm was detected with Ultra Multifunctional Microplate Reader (Tecan, Switzerland). The IC50 (the half maximal inhibitory concentration) values of TMZ were obtained by using GraphPad Prism 8 software. For cell viability assay, glioma cells were incubated with 75ng/mL IL-18 for 24, 48, 72, and 96 h, followed by incubating with fresh medium containing 10% CCK-8 solution for 2 h.

5′-ethynyl-2′-deoxyuridine (EdU) staining assay

Glioma cells proliferation was assessed via EdU detection Kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol. LY294002 is a broad-spectrum inhibitor of PI3K, its formula is C19H17NO3 [21, 22]. Glioma cells were cultured in a cover glass of 6-well plates with DMSO, IL-18, or IL-18 + 50 µmol/L LY294002 for 48 h, followed by incubating with 10 µM EdU reagent for 2 h at 37 °C. The cells were fixed through immersing in 4% formaldehyde for 30 min. After washing, the cells were incubated with Apollo for 30 min, and the nuclei were stained with Hoechst 33,342. The EdU-positive cells were detected via confocal microscope (Leica, Germany). The ratio of Edu-positive cells was calculated via Image-Pro Plus software (version 6.0; Media Cybernetics Inc., Rockville, MD, USA).

Flow cytometry of cell apoptosis

The apoptosis of glioma cells was measured via an Annexin-V-FITC Apoptosis Detection Kit (KeyGEN BioTECH, China) according to the manufacturer’s protocol. Briefly, U87 and U118 cells were incubated with DMSO, IL-18, or IL-18 + 50 µmol/L LY294002 for 48 h. And then these cells treated with 50 µg/mL TMZ, the of apoptotic rate was measured via flow cytometric analysis of Annexin V-FITC and Propidium Iodide (PI) double staining. The cells were divided into four groups. The cells in Q3 were non-viable apoptotic cells and Q4 were viable apoptotic cells. The apoptosis rate is the sum of Q3 and Q4. We applied ANOVA for multivariate analysis.

Bioinformatics analysis

RNA-seq data and clinical information of patients in this study were obtained from the Chinese Glioma Genome Atlas (CGGA) database (http://www.cgga.org.cn/) and the Cancer Genome Atlas (TCGA) database (https://xenabrowser.net/). The differential expression analysis of IL-18 in different groups and grades were performed using one-way ANOVA, followed by Bonferroni correction for multiple comparisons. All patients were grouped into IL-18-upregulated and IL-18-downregulated groups according to the median IL-18 expression. The Kaplan–Meier survival curves and log-rank test were performed to evaluated prognosis of different patients. The correlation analyses of IL-18 expression were detected using Pearson’s correlation coefficient. The Differential expressed genes (DEGs) was obtained via the “limma” package in R software (version 4.30). A | fold-change | > 2 and adjusted P value < 0.05 were considered to the threshold for DEGs. The “clusterProfiler” package was used to analysis Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Gene set enrichment analysis (GSEA) was performed using the GSEA software (version 4.2.1). The cutoff criterion for significant enrichment gene sets was P < 0.05. The abundance of immune cells in glioma of TCGA was evaluated by CIBERSORT algorithm [23]. The significant differences between two groups were calculated by Student’s t-test. And all the results were presented using the “ggplot2” R package.

Tumor xenograft models and treatment experiments

After the approval from the Animal Ethics Committee of Zhujiang Hospital (LAEC-2021-053, LAEC-2021-088), 4–5 weeks old Female nude mice were purchased from the Laboratory Animal Center of Guangdong (Guangzhou, China). GL261 and U87 cells with luciferase reporter expression (GL261/luciferase or U87/luciferase cells) were used for the in vivo experiments. Before implantation, U87/luciferase cells were incubated with 75 ng/mL IL-18 (Sino Biological, TX, USA) for 7 days. And approximately 2.5 × 105 U87/luciferase cells were stereotactically implanted into the right brain of nude mice. These mice were randomly grouped into four groups (n = 5 per group) and raised in a specific pathogen-free facility. Then the mice were treated with dimethyl sulfoxide (DMSO), TMZ or LY294002 (PI3K inhibitor; MedChem Express, NJ, USA) according to their groups. One week after implantation, the mice were administered DMSO, TMZ (5 mg/kg), LY294002 (10 mg/kg), or TMZ (5 mg/kg) + LY294002 (10 mg/kg) via intraperitoneal injection twice weekly. With the application of an intraperitoneal injection of D-luciferin (150 mg/kg), the growth of xenograft tumors was detected weekly using bioluminescence imaging.

For the other tumor xenograft model, 1 × 105 GL261/luciferase cells were intracranially implanted into 4-weeks old male C57BL/6 mice. The mice were divided into four groups (n = 5 per group) according to different treatment of monoclonal antibody (mAB): 100 mg of anti-PD-1 mAB (RMP1-14, Bio X Cell, NH, USA), anti-mouse IL-18 mAB (Bio X Cell, NH, USA), mouse IgG (Bio X Cell, NH, USA), or a combination of anti-PD-1 mAB and anti-IL-18 mAB injected intratumorally on days 3, 7, 11, and 15 after GL261/luciferase cells implantation. All mice were monitored for survival and tumor volume. Survival results were analyzed using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). For evaluation of tumor growth, intracranial tumors were detected weekly using the method described above.

Statistical analysis

All results were presented as means ± standard deviation (SD) for three independent in vitro experiments. Student’s t-test was performed for pairwise comparison and ANOVA for multivariate analysis. Kaplan-Meier survival analysis was used to detect the correlation between different variates and survival rates. All statistical analyses were performed using GraphPad Prism 5.0 and SPSS 18.0 software. P < 0.05 was considered significant statistically.

Results

IL-18 upregulation is associated with poor prognosis in glioma

To identify if IL-18 is correlated with glioma, we analyzed the IL-18 mRNA expression among the glioma and normal brain tissues in TCGA database. We found that IL-18 was significantly high expressed in glioma tissues (Fig. 1A). Also, IL-18 was significantly increased in high-grade glioma from TCGA and CGGA database (Fig. 1B and C). In addition, Kaplan-Meier survival curve showed that the patients with glioma expressed high IL-18 expression levels presented shorter overall survival (Fig. 1D and E). Next, we measured the expression of IL-18 in patients with glioma at our hospital using IHC. IL-18 expression was upregulated in higher-grade glioma (Fig. 1F and G). These results indicate that the patients with glioma expressed high IL-18 is associated with poorer prognosis.

Fig. 1
figure 1

IL-18 upregulation is associated with poor prognosis in glioma. (A) The mRNA expression data of glioma compared with normal brain tissues in TCGA database; IL-18 expression was significantly increased in tumor tissues (****P < 0.0001). TCGA and CGGA database analysis showed that IL-18 expression level was positively correlated with glioma grade (B and C), but negatively correlated with prognosis (D and E). (F) Representative immunohistochemical staining images of IL-18 in different grade glioma (scale bars = 20 μm). (G) Quantitative analysis of IL-18 expression per field in immunohistochemical staining images as shown in F. Average Optical Density is value of the integrated optical density of target protein divided by distribution area. IL, interleukin; TCGA, The Cancer Genome Atlas; CGGA, Chinese Glioma Genome Atlas; WHO, World Health Organization

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IL-18 activates the PI3K-AKT signaling pathway in glioma

To investigate the potential mechanism of IL-18 in patients with glioma, we performed GSEA and KEGG pathway enrichment analysis of the DEGs between the IL-18-upregulated and IL-18-downregulated groups in TCGA cohorts. The pathway enrichment results showed that PI3K-Akt signaling pathway was the most significant (Fig. 2A). Moreover, the PI3K-Akt signaling-related genes were highly enriched in the IL-18-upregulated group (Fig. 2B). Based on these results, we hypothesized that IL-18 worsens glioma prognosis by regulating the PI3K/Akt signaling pathway. Then, U87 and U118 cells were stimulated with human IL-18 protein at doses ranging from 0 to 105 ng/mL for 48 h. The CCK-8 assay indicated that IL-18 significantly increased the viability of both cells in a dose-dependent manner, with a maximum effect at 75 ng/mL and 105 ng/mL (Fig. 2C). Therefore, 75 ng/mL was selected as the optimal dose for IL-18 stimulation. Western blotting showed that IL-18 markedly activated the PI3K/Akt signaling pathway and significantly increased the expression levels of downstream target genes, such as PI3K, PAKT, CDK2, and phospho-c-Raf, which are related to cell proliferation and apoptosis (Fig. 2D, E and Figure S2C, D). Taken together, these results suggest that IL-18 activates the PI3K-AKT signaling pathway in glioma.

Fig. 2
figure 2

IL-18 activates the PI3K-AKT signaling pathway in glioma. Patients with glioma were divided into IL-18-upregulated and IL-18-downregulated groups according to the mean value of IL-18 expression in The Cancer Genome Atlas cohorts. (A) Kyoto Encyclopedia of Genes and Genomes pathway analyses of the differentially expressed genes between the IL-18-upregulated and IL-18-downregulated groups. (B) Gene set enrichment analysis to compare PI3K-AKT signaling gene terms between the IL-18-upregulated and IL-18-downregulated groups. (C) U87 and U118 cells were stimulated with human IL-18 protein at different doses (0, 15, 45, 75, 105 ng/mL) for 48 h. Cell viability was observed using a CCK-8 assay. **P < 0.01 vs. 0 ng/mL. (D) U87 and U118 cells were incubated with 75 ng/mL of IL-18 at different time points, and the levels of PI3K, P-c-Raf (S259), PAKT (Ser473), AKT, CDK2 and β-actin were examined via western blotting. Data are presented as means ± SD (n = 5 in each dose). IL, interleukin

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IL-18 promotes TMZ resistance in glioma by promoting proliferation and suppressing apoptosis

We found that U87TR cells showed higher IL-18 expression than U87 cells (Figure S1A). Besides, the PI3K-AKT pathway plays a vital role in temozolomide resistance of glioma [24, 25]. To evaluate the role of IL-18 in TMZ resistance in glioma, U87 and U118 cells were stimulated with human IL-18 at concentrations ranging from 0 to 105 ng/mL, followed by treating with different doses of TMZ. The results of CCK8 assay indicated that IL-18 markedly increased IC50 of TMZ in a dose-dependent manner, with a maximum effect at 75 ng/mL (Fig. 3A). Besides, 75ng/mL IL-18 significantly increased proliferation of glioma cells in different duration compared with controls (Fig. 3B). Furthermore, U87 and U118 cells were treated with DMSO, human IL-18 protein, or IL-18 + LY294002 for 48 h. The EdU-positive cells significantly increased in the IL-18 group, which was reversed by LY294002 (Fig. 3C and D). Flow cytometry showed that IL-18 significantly inhibited cell apoptosis under TMZ (50 µg/mL) treatment (Fig. 3E and F). These data indicate that IL-18 enhances TMZ resistance in glioma by promoting proliferation and suppressing apoptosis.

Fig. 3
figure 3

IL-18 promotes TMZ resistance in glioma by promoting proliferation and inhibiting apoptosis. (A) U87 and U118 cells were incubated with human IL-18 protein at doses from 0 to 105 ng/mL for 48 h, and then treated with TMZ at different concentrations (0, 100, 200, 300, 400, and 500 µg/mL) for 48 h. The TMZ sensitivity of U87 and U118 treated with different IL-18 doses were indicated through a Cell Counting Kit-8 assay. **P < 0.01 vs. 0 ng/mL. (B) Cell viability of U87 and U118 incubated with 75 ng/mL IL-18 for 24, 48, 72, and 96 h. *P < 0.05 compared with control groups. (C) U87 and U118 cells were incubated with DMSO, IL-18, or IL-18 + LY294002 (PI3K inhibitor) for 48 h. The rate of EdU-positive cells in these groups was determined via an EdU staining assay. Scale bar = 100 μm. (D) EdU-positive cell rate. (E) Apoptosis rate. (F) U87 and U118 cells were incubated with DMSO, IL-18, or IL-18 + LY294002 (PI3K inhibitor) for 48 h. Apoptosis rate after TMZ (50 µg/mL) treatment for 48 h measured by the Annexin V-FITC/PI double staining. **P < 0.01. Data are presented as means ± S.D. of three independent experiments. IC50, the half maximal inhibitory concentration; IL, interleukin; TMZ, temozolomide; PI, Propidium Iodide

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IL-18 promotes TMZ resistance in vivo

To identify whether IL-18 promotes TMZ resistance in vivo, mice were orthotopically implanted with 2.5 × 105 U87/luciferase cells following pre-incubation with PBS or IL-18 for 7 days. After treatment of TMZ, bioluminescence imaging results indicated that IL-18 treatment markedly promoted tumor growth (Fig. 4A and B) and decreased the overall survival of tumor-bearing mice (Fig. 4C), compared to controls. These results suggest that IL-18 promotes TMZ resistance in vivo. To further assess the antitumor effect of combined TMZ and LY294002 treatment in vivo, we treated tumor-bearing mice with DMSO, LY294002, TMZ, or TMZ + LY294002. The results presented that combined TMZ and LY294002 treatment significantly suppressed tumor growth and prolonged the overall survival of tumor-bearing mice compared with the control group (Fig. 4D and E). Collectively, these results strongly suggest that IL-18 promotes TMZ resistance in glioma in vivo and could be a potential therapeutic target.

Fig. 4
figure 4

IL-18 promotes TMZ resistance in vivo. BALB/c mice were orthotopically implanted with 2.5 × 105 U87/luciferase cells following pre-incubation with PBS or IL-18 for 7 days, and then treated with TMZ (n = 5 per group). (A) Representative pseudocolor bioluminescence images of orthotopic tumors. (B) Representative hematoxylin and eosin staining of tumor-frozen sections (scale bars = 1 mm). (C) Kaplan-Meier survival curve of mice bearing tumor. BALB/c mice were orthotopically implanted with 2.5 × 105 U87/luciferase cells following pre-incubation with IL-18 for 7 days, and then treated with DMSO, LY294002, TMZ, or TMZ + LY294002 (n = 5 per group). (D) Representative pseudocolor bioluminescence images of orthotopic tumors. (E) Kaplan-Meier survival curve of mice bearing tumor. *P < 0.05, **P < 0.01. Data are presented as means ± S.D. IL, interleukin; TMZ, temozolomide

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IL-18 upregulates CD274 in glioma

Recent studies have presented that IL-18 induces CD274 expression in several cancers [18, 19]. Next, we investigated whether IL-18 presents the same effects in glioma. TCGA cohort analysis showed a positive correlation between IL-18 and CD274 (Fig. 5A). Patients with high IL-18 expression in TCGA cohort showed decreased CD4+ and CD8+ T cell infiltration in the microenvironment of glioma (Fig. 5B). Furthermore, we found that CD274 was upregulated at both the mRNA and protein levels in glioma cells after incubation with IL-18 (Fig. 5C-E). Therefore, we treated C57 mice harboring GL261 cells with mouse IgG, anti-IL-18 mAB, anti-PD-1 mAB or combination. Anti-IL-18 mAB significantly decreased CD274 expression (Fig. 5F). Taken together, these results indicate that IL-18 upregulates CD274 expression in glioma and subsequently promotes tumor cell immune evasion.

Fig. 5
figure 5

IL-18 upregulates CD274 in glioma. (A) IL-18 and CD274 expression in TCGA cohorts. (B) CD4 + and CD8 + T cell infiltration in patients with glioma from TCGA were estimated using CIBERSOFT. (C-D) Western blotting was used to verify the expression level of CD274 in glioma cells after co-incubation with PBS or IL-18. (E) CD274 expression after incubation with 75 ng/mL IL-18 in glioma cells indicated using RT-qPCR. (F) Quantitative analysis of CD274 expression per field in immunohistochemical staining images as shown in Fig. 6C. *P < 0.05, ****P < 0.0001. Data are presented as means ± S.D. IL, interleukin; TCGA, The Cancer Genome Atlas

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Anti-IL-18 combined with anti-PD-1 mAB treatment has better efficacy in vivo

To investigate whether anti-IL-18 and anti-PD-1 mAB combination could be a novel therapeutic strategy for glioma, C57 mice were orthotopically implanted with 105 luciferase-expressing GL261 mouse glioma cells. Bioluminescence imaging results indicated that anti-IL-18 mAB treatment significantly suppressed tumor growth and increased the overall survival of tumor-bearing mice (Fig. 6A and B), compared to the negative control group. These effects were further improved by combined treatment with anti-IL-18 and anti-PD-1 mAB (Fig. 6A and B). IHC staining results indicated that the combined treatment significantly promoted CD4+ and CD8+ T cell infiltration (Fig. 6C and D) Taken together, these results suggest that anti-IL-18 combined with anti-PD-1 mAB could be a new therapeutic strategy for immune evasion in glioma.

Fig. 6
figure 6

Anti-IL-18 combined with anti-PD-1 monoclonal antibody treatment has better efficacy in vivo. C57 mice were orthotopically implanted with 105 GL261/luciferase cells following pre-incubation with IL-18 for 7 days, and then treated with mouse immunoglobulin G, anti-PD-1 monoclonal antibody, anti-IL-18 monoclonal antibody, or combination treatment respectively (n = 5 per group). (A) Representative pseudocolor bioluminescence images of orthotopic tumors. (B) Kaplan-Meier survival curve of mice with intracranial xenografts. (C) Representative immunohistochemical staining images of CD274, tumor-infiltrating CD4 T cells, and CD8 T cells in glioma tissues harvested from tumor-bearing mice. Scale bar = 20 μm. (D) Quantitative analysis of the numbers of positive cells per field in immunohistochemical staining images as shown in C. *P < 0.05, ****P < 0.0001. Data are presented as means ± S.D. IL, interleukin; mIgG, mouse immunoglobulin G; aPD-1, anti-PD-1 monoclonal antibody; aIL-18, anti-IL-18 monoclonal antibody

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Discussion

This study has several major findings. We first detected IL-18 overexpression in higher-grade glioma and TR glioma cells and investigated its clinical relevance. Next, we explored the role of IL-18 in glioma cells and its underlying mechanisms of action. Finally, we verified that IL-18 upregulated CD274 expression in glioma cells to promote immune evasion. Figure 7 shows the mechanism underlying the role of IL-18 in glioma progression. Collectively, these results suggest that IL-18 promotes TMZ resistance by promoting cell proliferation and suppressing apoptosis via the PI3K-Akt signaling pathway. Furthermore, anti-IL-18 mAB combined with anti-PD-1 mAB resulted in a more profound blockage of tumor growth. All of results indicate that IL-18 may be a target for overcoming TMZ resistance and immune evasion.

Fig. 7
figure 7

Schematic diagram representing the role of interleukin (IL)-18 in glioma progression. TMZ, temozolomide; GBM, glioblastoma; aPD-1, anti-PD-1 antibody

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Chronic inflammation plays an important role in the malignant phenotype of many human cancers [26]. The IL-1 family is a prototypic inflammatory cytokine upstream of the cytokine cascade [27], upregulated in many cancers, including breast, colon, brain, lung, pancreas, and melanomas [28]. Thus, we explored the role of the IL-1 family in glioma. After multivariable adjustment for other clinical factors of glioma, including grade and sex, IL-18 was the most significant and independent prognostic indicator among the IL-1 family members (Figure S1B). Furthermore, the TR glioma cells established by our group showed IL-18 overexpression.

Here, we focused on IL-18, which was initially named IFN-γ-inducing factor and was later defined as a proinflammatory cytokine with the ability to induce IFN-γ [29]. IL-18 is correlated with worse prognosis in several tumors, including glioma, pancreatic cancer, breast cancer, gastric cancer, and multiple myeloma [9,10,11,12,13,14,15]. In addition, IL-18 is highly expressed in the serum of patients with glioma [30]. Our study showed that IL-18 was highly expressed in TR cells and their microenvironment, and that it promotes TMZ resistance in glioma cells by suppressing cell apoptosis and promoting proliferation both in vivo and in vitro. In contrast, TMZ treatment could induce IL-18 secretion in glioma cells (Figure S1C). In future studies, we will focus on this mechanism, how TMZ increases IL-18 secretion in glioma and the blockage of the possible loop between TMZ and IL-18.

IL-18-mediated signaling induces various inflammatory factors involved in several cancers [31]. Rex et al. [32] reported many mechanisms induced by IL-18, including NF-ƙB, mitogen-activated protein kinases (MAPKs) and PI3K/AKT signaling. So, we explored the underlying mechanism of IL-18 in glioma via bioanalysis, which presented upregulated PI3K/AKT signaling. Besides in recent studies, IL-18 also activated PI3K-AKT signaling in different diseases [32,33,34]. Furthermore, our previous researches and recent literatures indicated PI3K/AKT pathway plays a vital role in temozolomide resistance of glioma [24, 25, 35, 36]. Next, we focused on the PI3K-AKT signaling pathway. We demonstrated that IL-18 activated the PI3K-AKT signaling pathway both in vivo and in vitro. The PI3K inhibitor LY290042 reversed TMZ sensitivity in mice treated with IL-18. Our results show that LY290042 is a promising treatment for TR glioma in animal models, which suppresses the effects of IL-18.

Glioma are the majority of malignant brain tumors, which presents limitations to current treatment strategies [37]. A recent study showed that anti-PD-1 immunotherapy could improve overall survival in patients with glioma [38, 39]. Tang et al. [40] reported that IFN-γ upregulates CD274 in the tumor microenvironment to inhibit T cell activation. Moreover, the hyperactive PI3K pathway could upregulate CD274 [41]. IL-18 is the proinflammatory cytokine and induces IFN-γ production. Taken together, we hypothesized that IL-18 upregulates CD274 expression in glioma. Lu et al. [19] showed that IL-18 increases CD274 expression in the lymphoma via the IL-18/IFN-γ/CD274 pathway, coinciding with our assumption. Our study revealed that IL-18 upregulates CD274 expression in vivo and in vitro, and that combined treatment with anti-IL-18 and anti-PD-1 mAB presents more prominent glioma inhibition in vivo. Our results indicate IL-18 is a promising target for the treatment of glioma in animal models. However, the underlying molecular mechanisms of IL18 upregulated CD274 will need to be further explored in the future.

In conclusion, our results demonstrate that IL-18 promotes TMZ resistance in glioma by stimulating the PI3K/AKT signaling pathway. IL-18 upregulates CD274 expression in glioma. IL-18 inhibitors are a promising treatment for TMZ resistance and anti-PD-1 resistance in glioma. Taken together, our findings highlight a novel molecular mechanism underlying TMZ and anti-PD-1 resistance in glioma, providing a new target for glioma treatment. In future research, we will focus on the mechanism of action of TMZ/IL-18/CD274, which may provide alternative treatment strategies for glioma.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

Abbreviations

TMZ:

Temozolomide

IL-18:

Interleukin-18

PD-L1:

Programmed cell death ligand 1

IHC:

Immunohistochemistry

TR:

TMZ-resistant

CCK-8:

Cell Counting Kit-8

TCGA:

The Cancer Genome Atlas

DEG:

Differentially expressed gene

KEGG:

Kyoto Encyclopedia of Genes and Genomes

GSEA:

Gene set enrichment analysis

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Funding

This work was supported by the Key-Area Research and Development Program of Guangdong Province (No. 2023B1111020008), National Natural Science Foundation of China (82373347, 82073193, 82102946), and Guangdong Provincial Clinical Medical Centre for Neurosurgery (No. 2013B020400005).

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Authors and Affiliations

  1. Department of Neurosurgery, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China

    Huangyi Ji, Yufei Lan, Zhao Wang, Xiangyang Zhong, Wenhui Tang & Hongbo Guo

  2. Department of Neurosurgery, The First Affiliated Hospital of Shantou University Medical College, Shantou, 515041, China

    Huangyi Ji & Quantang Wei

  3. Department of Neurosurgery, Department of Neuro-Oncological Surgery, The National Key Clinical Specialty, The Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, The Neurosurgery Institute of Guangdong Province, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China

    Boyang Liu

  4. The Second Clinical School, Southern Medical University, Guangzhou, 510515, China

    Hongbin Chen

  5. ZhiXin High School, No. 152, ZhiXin South Road, Yuexiu District, Guangzhou, 510080, China

    Pengpeng Xing

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  1. Huangyi Ji
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  4. Zhao Wang
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Contributions

H.G. and B.L. designed the research study, H.J. and Y.L. performed most of the experiments with assistance from Z.W. and W.T., X.Z. and W.T. analyzed the data. H.J. and H.C. collected the clinical data. H.J. wrote the paper. All authors read and approved the final version of the manuscript. Huangyi Ji: investigation; data curation; Writing – Original Draft Preparation; validation. Yufei Lan: Data curation; investigation; Validation. Zhao Wang: Investigation. Xiangyang Zhong: Formal analysis. Wenhui Tang: Validation; investigation. Quantang Wei: Methodology. Pengpeng Xing and Hongbin Chen: Data curation. Boyang Liu: Conceptualization; Data curation; supervision; writing – review and editing. Hongbo Guo: Conceptualization; supervision; writing – review and editing.

Corresponding authors

Correspondence to Boyang Liu or Hongbo Guo.

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Ethical approval

Approval of the research protocol by an Institutional Reviewer Board: This study has been approved by the Ethics Committee of Zhujiang Hospital. Informed Consent: Human glioma specimens obtained from Zhujiang Hospital, Written informed consent was obtained from all patients and all studies using human tissue samples were approved by Zhujiang Hospital (2023-KY-022-02). All procedures involving mice were approved by the Ethics Committee of Zhujiang Hospital (LAEC-2021-053, LAEC-2021-088).

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Ji, H., Lan, Y., Xing, P. et al. IL-18, a therapeutic target for immunotherapy boosting, promotes temozolomide chemoresistance via the PI3K/AKT pathway in glioma. J Transl Med 22, 951 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05755-x

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Journal of Translational Medicine

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