circMMD reduction following tumor treating fields inhibits glioblastoma progression through FUBP1/FIR/DVL1 and miR-15b-5p/FZD6 signaling.

BACKGROUND: Tumor treating fields (TTF) is the latest treatment for GBM. Circular RNA (circRNA) has been demonstrated to play critical roles in tumorigenesis. However, the molecular mechanism of TTF remained largely unknown and the role of circRNA in TTF was not reported. The aim of this study was to elucidate the role and mechanism of circMMD in TTF treatment of GBM. METHODS: Divergent primer was designed to verify the existence of circMMD in GBM cells. The prognostic role of circMMD was explored in glioma specimens. The knockdown and overexpressed plasmids were used to evaluate the effect of circMMD on GBM cell proliferation and TTF efficacy. RNA pull-down and RNA immunoprecipitation were performed to identify binding proteins of circMMD. Subcutaneous and intracranial tumor models were established to validate findings in vivo. RESULTS: The expression of circMMD was elevated in GBM and its high expression indicated poor prognoses. TTF intervention could reduce circMMD synthesis, which suppressed GBM proliferation and increased TTF-mediated apoptosis. The reduction of circMMD promoted the interaction between FUBP1 and FIR, which decreased DVL1 transcription. Meanwhile, decreased circMMD would promote the activity of miR-15b-5p to degrade FZD6. Finally, the diminished expression of DVL1 and FZD6 expression suppressed the activation of Wnt/ß-catenin pathway. CONCLUSIONS: Our study revealed a novel mechanism of TTF that TTF-mediated reduction of circMMD could inhibit Wnt/ß-catenin pathway to suppress GBM proliferation.

The schemes, mechanisms and molecular pathway changes of Tumor Treating Fields (TTFields) alone or in combination with radiotherapy and chemotherapy.

Tumor Treating Fields (TTFields) is a physical therapy that uses moderate frequency (100-300 kHz) and low-intensity (1-3 V/cm) alternating electric fields to inhibit tumors. Currently, the Food and Drug Administration approves TTFields for treating recurrent or newly diagnosed glioblastoma (GBM) and malignant pleural mesothelioma (MPM). The classical mechanism of TTFields is mitotic inhibition by hindering the formation of tubulin and spindle. In addition, TTFields inhibits cell proliferation, invasion, migration and induces cell death, such as apoptosis, autophagy, pyroptosis, and cell cycle arrest. Meanwhile, it regulates immune function and changes the permeability of the nuclear membrane, cell membrane, and blood-brain barrier. Based on the current researches on TTFields in various tumors, this review comprehensively summarizes the in-vitro effects, changes in pathways and molecules corresponding to relevant parameters of TTFields (frequency, intensity, and duration). In addition, radiotherapy and chemotherapy are common tumor treatments. Thus, we also pay attention to the sequence and dose when TTFields combined with radiotherapy or chemotherapy. TTFields has inhibitory effects in a variety of tumors. The study of TTFields mechanism is conducive to subsequent research. How to combine common tumor therapy such as radiotherapy and chemotherapy to obtain the maximum benefit is also a problem that's worthy of our attention.

Whole transcriptome and proteome analyses identify potential targets and mechanisms underlying tumor treating fields against glioblastoma.

Glioblastoma (GBM) is one of the most malignant types of brain cancer. Tumor treating fields (TTFields) is the up-to-date treatment for GBM. However, its molecular mechanism requires additional investigation. Herein, a novel TTFields system was developed (CL-301A) and its efficiency in suppressing GBM cell proliferation and inducing cell apoptosis was demonstrated. Through the whole proteomic and transcriptomic analyses, a multitude of differentially expressed proteins (1243), mRNAs (4191), miRtNAs (47), lncRNAs (4286), and circRNAs (13,903) were identified. Bioinformatic analysis indicated that TTFields mainly affected nuclear proteins and interrupt cell mitosis-related events. Moreover, the inhibition of autophagy could significantly enhance the anti-GBM activity of TTFields. And CDK2-AS1 might be a target of TTFields to mediate cell cycle arrest via regulating CDK2 mRNA stability. This study provided valuable resources for understanding the mechanism of TTFields, which might further assist the investigation of TTFields in GBM treatment.

Exploring the efficacy of tumor electric field therapy against glioblastoma: An in vivo and in vitro study.

AIMS: Tumor electric fields therapy (TTFields) is emerging as a novel anti-cancer physiotherapy. Despite recent breakthroughs of TTFields in glioma treatment, the average survival time for glioblastoma patients with TTFields is <2 years, even when used in conjugation with traditional anti-cancer therapies. To optimize TTFields-afforded efficacy against glioblastoma, we investigated the cancer cell-killing effects of various TTFields paradigms using in vitro and in vivo models of glioblastoma. METHODS: For in vitro studies, the U251 glioma cell line or primary cell cultures prepared from 20 glioblastoma patients were treated with the tumor electric field treatment (TEFT) system. Cell number, volume, and proliferation were measured after TEFT at different frequencies (100, 150, 180, 200, or 220 kHz), durations (24, 48, or 72 h), field strengths (1.0, 1.5, or 2.2V/cm), and output modes (fixed or random sequence output). A transwell system was used to evaluate the influence of TEFT on the invasiveness of primary glioblastoma cells. For in vivo studies, the therapeutic effect and safety profiles of random sequence electric field therapy in glioblastoma-transplanted rats were assessed by calculating tumor size and survival time and evaluating peripheral immunobiological and blood parameters, respectively. RESULTS: In the in vitro settings, TEFT was robustly effective in suppressing cell proliferation of both the U251 glioma cell line and primary glioblastoma cell cultures. The anti-proliferation effects of TEFT were frequency- and "dose" (field strength and duration)-dependent, and contingent on the field sequence output mode, with the random sequence mode (TEFT-R) being more effective than the fixed sequence mode (TEFT-F). Genetic tests were performed in 11 of 20 primary glioblastoma cultures, and 6 different genetic traits were identified them. However, TEFT exhibited comparable anti-proliferation effects in all primary cultures regardless of their genetic traits. TEFT also inhibited the invasiveness of primary glioblastoma cells in transwell experiments. In the in vivo rat model of glioblastoma brain transplantation, treatment with TEFT-F or TEFT-R at frequency of 200 kHz and field strength of 2.2V/cm for 14 days significantly reduced tumor volume by 42.63% (TEFT-F vs. control, p = 0.0002) and 63.60% (TEFT-R vs. control, p < 0.0001), and prolonged animal survival time by 30.15% (TEFT-F vs. control, p = 0.0415) and 69.85% (TEFT-R vs. control, p = 0.0064), respectively. The tumor-bearing rats appeared to be well tolerable to TEFT therapies, showing only moderate increases in blood levels of creatine and red blood cells. Adverse skin reactions were common for TEFT-treated rats; however, skin reactions were curable by local treatment. CONCLUSION: Tumor electric field treatment at optimal frequency, strength, and output mode markedly inhibits the cell viability, proliferation, and invasiveness of primary glioblastoma cells in vitro independent of different genetic traits of the cells. Moreover, a random sequence electric field output confers considerable anti-cancer effects against glioblastoma in vivo. Thus, TTFields are a promising physiotherapy for glioblastoma and warrants further investigation.

Evaluation of a tumor electric field treatment system in a rat model of glioma.

OBJECTIVE: Glioma is a devastating disease lacking effective treatment. Tumor electric field therapy is emerging as a novel non-invasive therapy. The current study evaluates the efficacy and safety of a self-designed tumor electric field therapy system (TEFTS ASCLU-300) in a rat orthotopic transplantation model of glioma. METHODS: A model of intracranial orthotopic transplantation was established in rats using glioma C6 cells. For electric field therapy, glioma-bearing rats were exposed to alternating electric fields generated by a self-developed TEFTS starting on either 1st (Group 2) or 3rd (Group 3) day after transplantation, while other conditions were maintained the same as non-treated rats (Group 1). Glioma size, body weight, and overall survival (OS) were compared between groups. Immunohistochemical staining was applied to access tumor cell death and microvessel density within the tumor. In addition, the systemic effects of TEFTS on blood cells, vital organs, and hepatorenal functions were evaluated. RESULTS: TEFTS treatment significantly elongated the OS of tumor-bearing rats compared with non-treated rats (non-treated vs treated: 24.77 ± 7.08 days vs 40.31 ± 19.11 days, P = .0031). Continuous TEFTS treatment starting on 1st or 3rd day significantly reduced glioma size at 2 and 3 weeks after tumor cell inoculation (Week 2: Group 1:289.95 ± 101.69 mm3 ; Group 2:70.45 ± 17.79 mm3 ; Group 3:73.88 ± 33.21 mm3 , P < .0001. Week 3: Group 1:544.096 ± 78.53 mm3 ; Group 2:187.58 ± 78.44 mm3 ; Group 3:167.14 ± 109.96 mm3 , P = .0005). Continuous treatment for more than 4 weeks inhibited tumor growth. The TEFTS treatment promoted tumor cell death, as demonstrated by increased number of Caspase 3+ cells within the tumor (non-treated vs treated: 38.06 ± 10.04 vs 68.57 ± 8.09 cells/field, P = .0007), but had minimal effect on microvessel density, as shown by CD31 expression (non-treated vs treated: 1.63 ± 0.09 vs 1.57 ± 0.13% of positively stained areas, P > .05). No remarkable differences were observed in hepatorenal function, blood cell counts, or other vital organs between non-treated and treated groups. CONCLUSION: The TEFTS developed by our research team was proved to be effective and safe to inhibit tumor growth and improve general outcomes in a rat model of brain glioma.

The prognostic value of maximal surgical resection is attenuated in oligodendroglioma subgroups of adult diffuse glioma: a multicenter retrospective study.

PURPOSE: Maximal surgical resection is associated with survival benefit in the majority of studies in adult diffuse glioma. This study aims to characterize the prognostic value of surgical resection in molecular subgroups of diffuse glioma. METHODS: 1178 patients with diffuse glioma from our centers and 422 from TCGA dataset were collected. The Kaplan-Meier analysis and multivariable Cox regression models were conducted to identify the prognostic value of surgical resection through different histological and molecular stratifications. RESULTS: Firstly, we confirmed progression-free survival (PFS) benefit associated with gross total resection (GTR) over sub-total resection (STR) in lower-grade glioma (HR 1.49; 95% CI 1.17-1.90; P = 0.001). Intriguingly however, we were unable to detect a significant PFS or overall survival (OS) benefit in oligodendroglioma (N = 397; HR 1.36; 95% CI 0.86-2.14; P = 0.19 and HR 1.05; 95% CI 0.55-1.99; P = 0.89, respectively). Secondly, when analyzed in molecular subgroups, we were similarly unable to detect a significant PFS or OS benefit in IDH MT/codel subgroup (N = 269; HR 1.47; 95% CI 0.92-2.34; P = 0.11 and HR 1.54; 95% CI 0.78-3.05; P = 0.21, respectively), oligodendroglioma with IDH MT/codel subgroup (N = 233; HR 1.33; 95% CI 0.79-2.21; P = 0.28 and HR 1.16; 95% CI 0.53-2.54; P = 0.70, respectively) or other relevant subgroups. TCGA validation also showed a significant survival benefit in astrocytoma rather than oligodendroglioma. Exploratory RNAseq analysis displayed that fewer cell proliferation-related gene expression features were specific to oligodendroglioma. CONCLUSION: These results suggest that the benefit of maximal surgery may be attenuated in patients within oligodendroglioma relevant subgroups because of the chemosensitive and indolent nature. The aggressive surgery accompanying with risk of neurologic morbidity may be unnecessary for these patients given the lack of survival benefit with gross total resection.

Molecular subgroups and B7-H4 expression levels predict responses to dendritic cell vaccines in glioblastoma: an exploratory randomized phase II clinical trial.

Dendritic cell (DC)-based vaccination is a promising approach for active-specific immunotherapy, but is currently of limited efficacy. The safety and effectiveness of a DC vaccine (DCV) loaded with glioblastoma stem cell-like (GSC) antigens was assessed in glioblastoma multiforme (GBM) patients. In this double-blind, placebo-controlled phase II clinical trial, 43 GBM patients were randomized after surgery at a 1:1 ratio to receive either DCV (n = 22) or normal saline placebo (n = 21). Overall survival (OS) and progression-free survival (PFS) were analysed. Participants were stratified into different molecular subgroups based on the mutation (MT) status of isocitrate dehydrogenase (IDH1/2) and telomerase reverse transcriptase (TERT). Plasma cytokine levels, tumor-infiltrating lymphocyte numbers and immune co-inhibitory molecules PD-L1 and B7-H4 were also assessed. Multivariate Cox regression analysis revealed that DCV treatment significantly prolonged OS (p = 0.02) after adjusting for IDH1 and TERT promoter MT and B7-H4 expression, primary vs recurrent GBM. Among IDH1wild type (WT) TERTMT patients, DCV treatment significantly prolonged OS (p < 0.01) and PFS (p = 0.03) and increased plasma levels of cytokines CCL22 and IFN-γ compared with placebo. Patients with low B7-H4 expression showed significantly prolonged OS (p = 0.02) after DCV treatment. Therefore, IDH1WTTERTMT and low B7-H4 expression identified subgroups of GBM patients more responsive to GSC DCV-based specific active-immunotherapy.

A signature based on survival-related genes identifies high-risk glioblastomas harboring immunosuppressive and aggressive ECM characteristics.

OBJECTIVE: To seek survival-related genes in glioblastoma and establish a survival-gene signature for predicting prognoses of glioblastoma using public databases.
 Methods: Three independent glioma databases (GEO GSE53733, CGGA, TCGA) with whole genome expression data were included for analysis. Survival-related genes were obtained by comparing the long-term (>36 months) and short-term (<12 months) survivors in the database GSE53733. CGGA was used as the training set to develop the signature and TCGA was used as the validation set. Cox regression analysis and linear risk score assessment were conducted to look for prognostic signatures with survival-related genes. Principal components analysis, gene set enrichment analysis (GSEA), gene ontology (GO) and protein-protein interaction (PPI) analysis were performed to explore distinct expression profiles between risk grouped glioblastoma.
 Results: We totally found 211 survival-related genes and developed a signature with 17 survival-related genes for prognosis of glioblastoma. Based on this signature, the low-risk group had longer survival time while the high-risk group had shorter survival time. Additionally, the expression profiles between the high-risk and low-risk glioblastoma were different. Functional annotations revealed that the genes enriched in the high-risk glioblastoma were involved in immune systems and processes of extracellular matrix (ECM).
 Conclusion: The novel survival-gene signature can predict high-risk glioblastoma with shorter survival time, enhance immunosuppressive features, and increased invasion preferences.

[Up-regulation of TIM-3 on CD4+ tumor infiltrating lymphocytes predicts poor prognosis in human non-small-cell lung cancer].

OBJECTIVE: To characterize the expression of negative costimulatory molecule, T cell immunoglobulin and mucin-domain-containing molecules 3 (TIM-3), on tumor infiltrating lymphocytes (TILs) in human non-small-cell lung cancer (NSCLC) and explore the clinical significance of the expression. METHODS: A total of 56 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed NSCLC patients. Infiltrating lymphocytes from both tumor tissues and adjacent normal lung tissues were analyzed for TIM-3 expression by immunofluorescence staining and flow cytometry. Correlation analysis was performed between TIM-3 expression on TILs and the prognosis of the patients. RESULTS: The expression of TIM-3 on CD4+ TILs in tumor tissues [(28.64±10.46)%] was significantly higher than that on CD4+ T cells in adjacent normal tissues [(13.32±6.95)%]. Similarly, the expression of TIM-3 on CD8+ TILs in tumor tissues [(30.77±15.58)%] was up-regulated as compared with that on CD8+ T cells in adjacent normal tissues [(12.98±8.19)%]. Moreover, majority of the TIM-3 positive TILs from both adjacent normal tissues and tumor lung tissues were positive for another negative costimulatory molecule, programmed death 1 (PD-1). Importantly, TIM-3 expression on CD4+ TILs was correlated with poor prognosis of the patients. CONCLUSION: TIM-3, as a key negative regulator in the anti-tumor immunity, contributes to the tumor immune evasion. It has an adverse influence on the prognosis of NSCLC patients.