Search

Fig. S5. No downregulation in protein expression of GFAP and NKCC1 was found in immunodeficient SB28-GFP tumors in peri-tumor layer 3.

Supplementary Materials and Methods

More clinical evidence is needed regarding the relative priority of treatments for brain metastases (BMs) from EGFR/ALK-negative/unselected non-small cell lung cancer (NSCLC). PubMed, EMBASE, Web of Science, Cochrane Library, and ClinicalTrials.gov databases were searched. Overall survival (OS), central nervous system progression-free survival (CNS-PFS), and objective response rate (ORR) were selected for Bayesian network meta-analyses. We included 25 eligible randomized control trials (RCTs) involving 3,054 patients, investigating nine kinds of treatments for newly diagnosed BMs and seven kinds of treatments for previously treated BMs. For newly diagnosed BMs, adding chemotherapy, EGFR-TKIs, and other innovative systemic agents (temozolomide, nitroglycerin, endostar, enzastaurin, and veliparib) to radiotherapy did not significantly prolong OS than radiotherapy alone; whereas radiotherapy + nitroglycerin showed significantly better CNS-PFS and ORR. Surgery could significantly prolong OS (hazard ratios [HR]: 0.52, 95% credible intervals: 0.41–0.67) and CNS-PFS (HR: 0.32, 95% confidence interval: 0.18–0.59) compared with radiotherapy alone. For previously treated BMs, pembrolizumab + chemotherapy, nivolumab + ipilimumab, and cemiplimab significantly prolonged OS than chemotherapy alone. Pembrolizumab + chemotherapy also showed better CNS-PFS and ORR than chemotherapy. In summary, immune checkpoint inhibitor (ICI)-based therapies, especially ICI-combined therapies, showed promising efficacies for previously treated BMs from EGFR/ALK-negative/unselected NSCLC. The value of surgery should also be emphasized. The result should be further confirmed by RCTs.

Continuous intra-operative neurophysiological monitoring (CINM) that provides precise mapping of neural anatomy through the entire microsurgery process is essential to preserve the structural and functional integrity of the nerve. However, bulky and rigid electrodes used currently in clinics are usually unable to reliably maintain continuous and stable contacts with the vulnerable and complex nerve networks, thus often resulting in detrimental post-operative complications, such as hemiplegia and sensory disturbances. Here, we describe a biomechanically compatible, suture-free, and individually reconfigurable CINM based on soft and stretchable organic electronic materials. Due to both low impedance and modulus of our conducting polymer electrodes, we achievedfor the first timecontinuous recording of near-field action potential with high signal-to-noise ratio and minimal invasiveness during microsurgeries. Utilizing this unprecedented CINM modality, in conjunction with localized neurostimulation, we further demonstrated our approach in enabling optimal post-operative prognosis in preclinical animal models by preserving normal neural functions after a variety of tumor resection surgeries.

The aim of this study was to investigate an imaging biomarker based on contrast enhanced T1-weighted and T2-weighted magnetic resonance imaging (MRI) to determine the hearing loss related to acoustic neuromas (AN). In this retrospective study, 441 acoustic neuromas treated with microsurgery were included. The diagnostic and follow-up MRI and audiometry of these patients were compared. We discovered a new MRI grading biomarker based on the percentage of tumor filling the inner auditory canal (TFIAC classification). The area under the receiver operating characteristics (AUROC) curve was highest for TFIAC (0.675), followed by period of observation (0.615) and tumor size (0.6) (P

Glioma is one of the most common primary malignant tumors of the central nervous system accounting for approximately 40% of all intracranial tumors. Temozolomide is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk gliomas, including grade II to grade IV. Our bioinformatic analysis of The Cancer Genome Atlas and Chinese Glioma Genome Atlas datasets and immunoblotting assay show that SLC12A2 gene and its encoded Na+-K+-2Cl− cotransporter isoform 1 (NKCC1) protein are abundantly expressed in grade II–IV gliomas. NKCC1 regulates cell volume and intracellular Cl− concentration, which promotes glioma cell migration, resistance to temozolomide, and tumor-related epilepsy in experimental glioma models. Using mouse syngeneic glioma models with intracranial transplantation of two different glioma cell lines (GL26 and SB28), we show that NKCC1 protein in glioma tumor cells as well as in tumor-associated reactive astrocytes was significantly upregulated in response to temozolomide monotherapy. Combination therapy of temozolomide with the potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated the cytotoxic effects of temozolomide, decreased tumor-associated reactive astrogliosis, and restored astrocytic GLT-1 and GLAST glutamate transporter expression. The combinatorial therapy also led to suppressed tumor growth and prolonged survival of mice bearing GL26 glioma cells. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in the pathogenesis of glioma tumors and presents as a therapeutic target for reducing temozolomide-mediated resistance and tumor-associated astrogliosis.

Purpose. The immune checkpoint inhibitor is approved for breast cancer treatment, but the low expression of PD-L1 limits the immunotherapy. CD155 is another immune checkpoint protein in cancers and interacts with ligands to regulate immune microenvironment. This study is aimed at investigating the expression of CD155 and the association with prognosis and pathological features of breast cancer. Methods. 126 patients were recruited this cohort study consecutively, and CD155 expression on tumor cells was detected by immunohistochemistry. The Kaplan-Meier survival curve and Cox hazard regression model were used to estimate the association. Results. 38.1% patients had an overexpression of CD155, and the proportion of tumor cells with CD155 overexpression was 17%, 39%, 37%, and 62% among Luminal A, Luminal B, HER2-positive, and triple negative breast cancer cases, respectively (p<0.05). Patients with CD155 overexpression had the Ki-67 index significantly higher than that of patients with low expression (42% vs. 26%). Though the number of tumor-infiltrating lymphocytes was higher among patients with CD155 overexpression (144/HPF vs. 95/HPF), the number of PD-1+ lymphocytes was significantly higher (52/HPF vs. 25/HPF, p<0.05). Patients of CD155 overexpression had the disease-free and overall survival decreased by 13 months and 9 months, respectively (p<0.05). CD155 overexpression was associated with an increased relapse (HR=13.93, 95% CI 2.82, 68.91) and death risk for breast cancer patients (HR=5.47,1.42,20.99). Conclusions. Overexpression of CD155 was correlated with more proliferative cancer cells and a dysfunctional immune microenvironment. CD155 overexpression introduced a worse relapse-free and overall survival and might be a potential immunotherapy target for breast cancer.

Objective. This study is aimed at investigating the association of exhausted CD8+ tumor-infiltrating lymphocytes with clinic-pathological factors. Methods. 133 patients diagnosed with primary invasive ductal breast cancer were recruited into the cross-sectional study consecutively. Immunohistochemistry was used to detect biomarker expression on formalin-fixed and paraffin-embedded sections. Double staining of CD8 and PD-1 was conducted on lymphocytes. Results. The proportion of CD8+/PD-1- TILs was 16% among patients with axillary lymph node metastasis, significantly lower than those without metastasis (24%). The expression of CK7, CK20, or Ki-67 was not related with the proportion of phenotypes of CD8/PD-1 TILs. Younger patients had more cell counts of CD8+/PD-1- TILs than elderly patients (18/HPF vs. 9/HPF, p<0.05). Patients with axillary lymph node metastasis had less CD8+/PD-1- TILs than those without metastasis (11/HPF vs. 27/HPF, p<0.05). Median counts of CD8+/PD-1- TILs among patients with CK20 and E-Cad expression were 33/HPF and 14/HPF, significantly higher than those among patients with negative CK20 (16/HPF) and E-Cad expression (6/HPF). Ki-67 index had a significant correlation with cell counts of CD8+/PD-1+ TILs and CD8+/PD-1- TILs, and the correlation coefficients were 0.19 and 0.21 (p<0.05), respectively. Conclusion. The proportion of CD8+/PD-1- TILs was related with metastatic status of the axillary lymph node but cell counts of CD8+/PD-1- TILs were related with metastatic status of the axillary lymph node and expression of CK7, CK20, E-Cad, and Ki-67. Absolute cell counts, not proportion of CD8/PD-1 TILs, were more likely to distinguish clinic and pathologic characteristics of breast cancer.

Background: CTLA4 has been widely reported to contribute to downregulation of immune responses to anti-tumor activity. Anti-CTLA4 provides a novel strategy to improve the outcome of cancer immunotherapy, but the role of CTLA4 needs to be addressed in glioblastoma (GBM). To investigate the role of CTLA4 in GBM, we analyzed the sequencing data of CTLA4 and evaluated its clinical prognostic value. Methods: 467 GBM cases were enrolled into this study from 4 cohorts. In their works, the Cancer Genome Atlas (TCGA) cohort was divided into training set and the Chinese Glioma Genome Atlas (CGGA) cohort, REMBRANDT, and GSE84465 cohorts were divided into validation set. Then, the role of CTLA4 in tumor microenvironment (TME) of GBM was comprehensively investigated. Findings: Significant differences exist in immunological characteristics between low and high CTLA4 expression groups. Mutation analysis found distinct genomic patterns associated with CTLA4 expression. Next, network analysis found the module named c1-1562 including CTLA4 correlated with over survival (OS) in GBM. We also developed a predictive model to calculate the risk score of every GBM case and the risk score was independently related to OS. Furthermore, expression of CTLA4 was positively related to the infiltration level and function of macrophage in GBM TME based on seven independent algorithms and single cell RNA-seq data. Interpretation: This study confirmed that CTLA4 plays an obviously key role in clinical, genomic, and TME status. This will contribute to stratifying clinical treatment and enhancing the efficiency of cancer immunotherapy. Funding Information: This project was supported by National Natural Science Foundation of China (No. 82071996, No. 81802483 and No. 82003075), the Beijing Municipal Health Commission of China (No. PXM2019_026280_000002), the Capital’s Funds for Health Improvement and Research (No. 2018-1-1071), and Beijing Hospitals Authority Youth Program (No. QML20190507) Declaration of Interests: The authors have declared that no competing interest exists. Ethics Approval Statement: The study was conducted in accordance with good clinical practice guidelines and the declaration of Helsinki, which has been approved by Capital Medical University Institutional Review Board.

Glycolysis refers to one of the critical phenotypes of tumor cells, regulating tumor cell phenotypes and generating sufficient energy for glioma cells. A range of noticeable genes [such as isocitrate dehydrogenase (IDH), phosphatase, and tensin homolog (PTEN), or Ras] overall impact cell proliferation, invasion, cell cycle, and metastasis through glycolysis. Moreover, long non-coding RNAs (LncRNAs) are increasingly critical to disease progression. Accordingly, this study aimed to identify whether glycolysis-related LncRNAs have potential prognostic value for glioma patients. First, co-expression network between glycolysis-related protein-coding RNAs and LncRNAs was established according to Pearson correlation (Filter: |r| > 0.5 & P < 0.001). Furthermore, based on univariate Cox regression, the Least Absolute Shrinkage and Selection Operator (LASSO) analysis and multivariate Cox regression, a predictive model were built; vital glycolysis-related LncRNAs were identified; the risk score of every single patient was calculated. Moreover, receiver operating characteristic (ROC) curve analysis, gene set enrichment analysis (GSEA), GO and KEGG enrichment analysis were performed to assess the effect of risk score among glioma patients. 685 cases (including RNA sequences and clinical information) from two different cohorts of the Chinese Glioma Genome Atlas (CGGA) database were acquired. Based on the mentioned methods, the risk score calculation formula was yielded as follows: Risk score = (0.19 × EXPFOXD2-AS1) + (−0.27 × EXPAC062021.1) + (−0.16 × EXPAF131216.5) + (−0.05 × EXPLINC00844) + (0.11 × EXPCRNDE) + (0.35 × EXPLINC00665). The risk score was independently related to prognosis, and every single mentioned LncRNAs was significantly related to the overall survival of patients. Moreover, functional enrichment analysis indicated that the biologic process of the high-risk score was mainly involved in the cell cycle and DNA replication signaling pathway. This study confirmed that glycolysis-related LncRNAs significantly impact poor prognosis and short overall survival and may act as therapeutic targets in the future.

The aim of this study was to investigate an imaging biomarker based on contrast enhanced T1-weighted and T2-weighted magnetic resonance imaging (MRI) to determine the hearing loss related to acoustic neuromas (AN).In this retrospective study, 441 acoustic neuromas treated with microsurgery were included. The diagnostic and follow-up MRI and audiometry of these patients were compared.We discovered a new MRI grading biomarker based on the percentage of tumor filling the inner auditory canal (TFIAC classification). The area under the receiver operating characteristics (AUROC) curve was highest for TFIAC (0.675), followed by period of observation (0.615) and tumor size (0.6) (P 0.001). The percentage of patients in TFIAC grade III (90.1%) experiencing hypoacusis prior to microsurgery was significantly higher than that in TFIAC grade I (72.7%, P = 0.037) and TFIAC grade IV patients had a higher rate of non-serviceable hearing compared to TFIAC grade III patients (P 0.001). During the follow-up, TFIAC grade IV patients experienced a significantly higher rate of non-serviceable hearing than TFIAC grade III patients in all ANs (P 0.001) and in serviceable hearing acoustic neuroma cases prior to surgery (TFIAC grade IV 55.4%, TFIAC grade III 69.0%, P = 0.045). The TFIAC grade IV patients experienced a significantly higher rate of facial nerve dysfunction than TFIAC grade III patients after surgery (grade IV 48.0%, grade III 26.1%, P 0.001).The TFIAC classification serves as a potential imaging biomarker for preoperative and postoperative hearing prediction in ANs, which may aid neurosurgeons in predicting hearing loss and selecting optimal surgical strategies.

The weak immunogenicity of gliomas presents a barrier for effective immunotherapy. Na/H exchanger isoform 1 (NHE1) maintains alkaline intracellular pH (pHi) of glioma cells and acidic microenvironment. In addition, NHE1 is expressed in tumor-associated microglia and tumor-associated macrophages (TAMs) and involved in protumoral communications between glioma and TAMs. Therefore, we hypothesize that NHE1 plays a role in developing tumor resistance and immunosuppressive tumor microenvironment. In this study, we investigated the efficacy of pharmacological inhibition of NHE1 on combinatorial therapies. Here we show that temozolomide (TMZ) treatment stimulates NHE1 protein expression in two intracranial syngeneic mouse glioma models (SB28, GL26). Pharmacological inhibition of NHE1 potentiated the cytotoxic effects of TMZ, leading to reduced tumor growth and increased median survival of mice. Blockade of NHE1 stimulated proinflammatory activation of TAM and increased cytotoxic T cell infiltration into tumors. Combining TMZ, anti-PD-1 antibody treatment with NHE1 blockade significantly prolonged the median survival in the mouse glioma model. These results demonstrate that pharmacological inhibition of NHE1 protein presents a new strategy for potentiating TMZ-induced cytotoxicity and increasing tumor immunogenicity for immunotherapy to improve glioma therapy.

Steroid sulfatase (STS), a desulfating enzyme that converts steroid sulfates to hormonally active steroids, plays an important role in the homeostasis of sex hormones. STS is expressed in the adipose tissue of both male and female mice, but the role of STS in the development and function of adipose tissue remains largely unknown. In this report, we show that the adipose expression of Sts was induced in the high-fat diet (HFD) and ob/ob models of obesity and type 2 diabetes. Transgenic overexpression of the human STS in the adipose tissue of male mice exacerbated the HFD-induced metabolic phenotypes, including increased body weight gain and fat mass, and worsened insulin sensitivity, glucose tolerance, and energy expenditure, which were accounted for by adipocyte hypertrophy, increased adipose inflammation, and dysregulation of adipogenesis. The metabolic harm of the STS transgene appeared to have resulted from increased androgen activity in the adipose tissue, and castration abolished most of the phenotypes. Interestingly, the transgenic effects were sex specific, because the HFD-fed female STS transgenic mice exhibited improved metabolic functions, which were associated with attenuated adipose inflammation. The metabolic benefit of the STS transgene in female mice was accounted for by increased estrogenic activity in the adipose tissue, whereas such benefit was abolished upon ovariectomy. Our results revealed an essential role of the adipose STS in energy homeostasis in sex- and sex hormone-dependent manner. The adipose STS may represent a therapeutic target for the management of obesity and type 2 diabetes.

The role of astrocytes in dysregulation of blood-brain barrier (BBB) function following ischemic stroke is not well understood. Here, we investigate the effects of restoring the repair properties of astrocytes on the BBB after ischemic stroke. Mice deficient for NHE1, a pH-sensitive Na(+)/H(+) exchanger 1, in astrocytes have reduced BBB permeability after ischemic stroke, increased angiogenesis and cerebral blood flow perfusion, in contrast to wild-type mice. Bulk RNA-sequencing transcriptome analysis of purified astrocytes revealed that ~177 genes were differentially upregulated in mutant astrocytes, with Wnt7a mRNA among the top genes. Using a Wnt reporter line, we confirmed that the pathway was upregulated in cerebral vessels of mutant mice after ischemic stroke. However, administration of the Wnt/β-catenin inhibitor, XAV-939, blocked the reparative effects of Nhel-deficient astrocytes. Thus, astrocytes lacking pH-sensitive NHE1 protein are transformed from injurious to “protective” by inducing Wnt production to promote BBB repair after ischemic stroke.

Aims To investigate the roles of Claudin-5 (CLDN5) in regulating the permeability of the blood-brain barrier (BBB) during lung cancer brain metastasis. Results By silencing and overexpressing the CLDN5 gene in human brain vascular endothelial (hCMEC/D3) cells, we demonstrated the attenuation of cell migration ability and CLDN5's significant positive role in cell proliferation in CLDN5-overexpressing hCMEC/D3 cells and observed the opposite result in the CLDN5 knockdown group. The reinforced CLDN5 expression reduced the paracellular permeability of hCMEC/D3 cells and decreased the invasion of lung adenocarcinoma A549 cells. Overall, 1685 genes were found to be differentially expressed between the CLDN5-overexpressing cells and the control cells using the Affymetrix Human Transcriptome Array 2.0 (HTA 2.0), and the function of these genes was determined by Gene Ontology and pathway analyses. The possible biological functions of the 1685 genes include cell proliferation, adhesion molecules, and the Jak-STAT, PI3K-Akt, Wnt, and Notch signaling pathways. The identified sets of mRNAs that were specific to CLDN5-overexpressing hCMEC/D3 cells were verified by a qRT-PCR experiment. Conclusion CLDN5 regulates the permeability of BBB by regulating the proliferation, migration, and permeability of hCMEC/D3 cells, especially through the cell adhesion molecule signaling pathway, to enhance the function of the tight junctions, which was involved in reducing the formation of lung cancer brain metastasis.

CTLA-4, identified as an immune checkpoint, has been widely reported to contribute to downregulation of immune responses to anti-tumor activity. Anti-CTLA-4 provides a novel strategy to improve the outcome of cancer immunotherapy, but the role of CTLA-4 needs to be addressed in glioma. To investigate the role of CTLA-4 in diffuse gliomas, we analyzed the transcriptome level data of CTLA-4 and evaluated its clinical prognostic value. 1022 glioma samples with transcriptome data were enrolled into this study, including 325 RNA-seq data from CGGA and 697 RNA-seq data from TCGA. Clinical and molecular data were also obtained. R project was the main tool for statistical analysis and graphical work. High-grade gliomas exhibited high expression of CTLA-4 while IDH wildtype gliomas showed CTLA-4 enrichment. According to TCGA transcriptional classification in gliomas, mesenchymal subtype displayed significantly higher CTLA-4 expression than other molecular subtypes. Gene ontology analysis demonstrated that CTLA-4 and correlated genes were involved in immune responses and inflammatory activities in diffuse gliomas. Through the analysis of transcriptomic data, CTLA-4 expression strongly correlated with immune cell populations and inflammatory activity. Survival analysis suggested that high expression of CTLA-4 was associated with poor prognosis in gliomas and may serve as an indicator of outcome in gliomas. High CTLA-4 expression is closely related to malignancy in diffuse gliomas and predicts poor outcome. Meanwhile, CTLA-4 plays an important role in regulating T cell activation and antitumor responses in gliomas. Therefore, blocking CTLA-4 is a potential target for developing the immunotherapy of gliomas. Funding: This work was supported by the capital health research and development of special (2014-2-1072); Beijing Municipal Natural Science Foundation (7142054) and National Natural Science Foundation of China (81471229). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by Capital Medical University Institutional Review Board (IRB).

Background: Immunotherapy is a promising therapeutic approach for cancers. However, the immune background and the interactions between pituitary adenomas (PAs) and the host immune system remain unclear. Methods: Poly-A enriched RNA sequencing was performed on 115 snap frozen PA samples. Estimating the Proportion of Immune and Cancer cells (EPIC) was used to detect all the main cell types (B cells, CD8+ T cells, CD4+ T cells, macrophages and NK cells) in a tumor sample from the expression of genes. Immunohistochemical (IHC) staining was used to validate the main findings. Findings: In RNA sequencing data, PA samples showed immune infiltration, including B cells, CD8+ T cells, CD4+ T cells, macrophages and NK cells. The results were further validated by IHC. Meanwhile, pathway analysis showed that TNFR superfamily (TNFRSF), IL-10 family and TGF beta (TGFβ) family were the potential mechanisms of immune infiltration in PAs. In order to search how tumor making progression with high infiltration of immune cells, gene-expression data revealed that tumor progression was related to increased expression of PD-1/PD-L1 and was associated with higher infiltration of CD8+ T cells. Besides, pathway enrichment analysis showed that regulation of death/apoptosis processes and the JAK-STAT pathway were found to be significantly enriched in samples with higher immune infiltration. At last, analysis of cancer-testis antigens (CTAs) expression in CD8+ T cells suggested that CTAG2 and TSPYL6 were potential immunotherapeutic targets in GH and non-functioning PAs, respectively. Interpretation: Our work provides new angles for understanding tumor-infiltrating immune cells. The results may inform effective checkpoint blockade and cancer vaccine therapies and make it possible to take immunotherapy into invasive functioning PAs. Funding Statement: This work was supported by the National Natural Science Foundation of China under Grant number 81471229; Capital Medical Development Research Foundation under Grant number 201811071. Declaration of Interests: No potential conflicts of interest are declared. Ethics Approval Statement: The PA tissue collection was approved by the Institutional Review Board of Beijing Tiantan Hospital, Capital Medical University. All procedures involving human participants conformed to the ethical standards of the national research committee and the 2013 Helsinki Declaration, which were approved by the Institutional Review Board of Beijing Tiantan Hospital, Capital Medical University. Informed consent was obtained from all patients prior to their enrollments in this study.

Background: Decision-making concerning the treatment of choroid plexus tumor (CPT) in pediatric patients remains a topic of considerable debate. The aim of this work was to describe clinical features and prognostic risk factors of CPT in the pediatric population and to provide theoretical opinions regarding clinical decisions for CPT. Methods: The data of 96 patients with CPT and younger than 14 years were retrospectively analyzed. Clinical characteristics such as pathological type of CPTs, rate and severity of hydrocephalus, treatment and outcome, and recurrence were investigated. For categorical variables, the Pearson's Chi-square test was performed. The Mann–Whitney U-test was used for comparisons between nonnormally distributed parameters. Log-rank test was used for progression-free survival (PFS). Results: The study included 70 choroid plexus papilloma (CPP) cases, 17 atypical choroid plexus papilloma (aCPP) cases, and 9 choroid plexus carcinoma (CPC) cases. Compared with patients with CPP or aCPP, patients with CPC had a shorter disease course (median: CPP, 4 months; aCPP, 2 months; CPC, 1 month; H: 23.5, P < 0.001), higher rate of acute hydrocephalus (CPP, 27.1%; aCPP, 52.9%; CPC, 77.8%; χ2 = 10.9, P < 0.05), and lower incidence of cure rate (CPP, 85.7%; aCPP, 70.5%; CPC, 33.3%; χ2 = 13.5, P < 0.05). The severity of hydrocephalus with tumor in the lateral or third ventricle was significantly higher than that with tumors in the fourth ventricle (severe hydrocephalus: lateral ventricle, 51.7%; third ventricle, 47.0%; fourth ventricle, 11.1%; χ2 = 26.0, P < 0.001). Patients with gross total surgical resection had no better PFS than those with partial resection because of the use of adjuvant therapy in the latter (χ2 = 4.0, P > 0.05). Patients with CPC experienced shorter time for recurrence than those with CPP or aCPP (χ2 = 40.1, P < 0.0001). Conclusions: Our results indicated that CPP in the fourth ventricle could trigger serious clinical symptoms at an early stage, requiring early intervention. Adjuvant treatment might be necessary for patients with partially resected CPP, aCPP, and CPC to achieve a favorable outcome.

Background Sodium/hydrogen exchanger 1 (NHE1), encoded by the SLC9A1 gene (SoLute Carrier family 9A1) in humans, is the main H+ efflux mechanism in maintaining alkaline intracellular pH (pHi) and Warburg effects in glioma. However, to date, there are no clinical studies exploring pharmacological inhibition of NHE1 protein in cancer treatment. In this study, we investigated NHE1 expression in gliomas and its relationship with glioma clinical outcome. Methods The Chinese Glioma Genome Atlas (CGGA) dataset containing transcriptome sequencing data of 325 glioma samples and the Cancer Genome Atlas (TCGA) with 698 glioma mRNAseq data were analyzed in this study. Mouse SB28 and GL26 intracranial syngeneic glioma models in C57BL/6 J mice were established to investigate NHE1 expression and impact of NHE1 protein inhibition with its inhibitor HOE642 on tumorigenesis and anti-PD1 therapy. Tumor angiogenesis, immunogenicity, and progression were assessed by immunofluorescence staining and flow cytometric profiling. Results Analysis of SLC9A1 mRNA expression in two data sets, CGGA and TCGA, reveals significantly higher SLC9A1 mRNA levels in higher grade gliomas. The SLC9A1 mRNA expression was especially enriched in isocitrate dehydrogenase (IDH)1/2 wild-type glioblastoma (GBM) and in mesenchymal glioma subtypes. Worsened survival probabilities were correlated with the elevated SLC9A1 mRNA levels in gliomas. The underlying mechanisms include promoting angiogenesis, and extracellular matrix remodeling. Increased SLC9A1 mRNA expression was also associated with tumor-associated macrophage accumulation. NHE1 inhibitor HOE642 reduced glioma volume, invasion, and prolonged overall survival in mouse glioma models. Blockade of NHE1 protein also stimulated immunogenic tumor microenvironment via activating CD8 T-cell accumulation, increasing expression of interferon-gamma (Ifng), and sensitized animals to anti-PD-1 therapy. Conclusion Our findings strongly suggest that NHE1 protein emerges as a marker for tumorigenesis and prognosis in glioma. Blocking NHE1 protein is a novel strategy for adjuvant anti-cancer therapies. Electronic supplementary material The online version of this article (10.1186/s13046-018-0923-z) contains supplementary material, which is available to authorized users.

Low-grade gliomas (LGGs) present a high incidence of epilepsy, infiltrative growth, resistance to therapy, and high risk of transforming into high-grade gliomas (HGGs). Temozolomide (TMZ) is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk LGGs. Na+-K+-2Cl- co-transporter 1 (NKCC1) regulates cell volume and intracellular Cl- concentration which promotes glioma cell migration, resistance to TMZ, and tumor-associated epilepsy. Our new bioinformatic analysis of TCGA and CGGA datasets shows that LGGs expressed higher SLC12A2 gene mRNA (encoding NKCC1 protein) than HGGs, which was confirmed at NKCC1 protein levels. In syngeneic mouse glioma models with intracranial transplantation of two different mouse glioma cell lines (GL26 and SB28), we detected upregulated NKCC1 protein expression in glioma tumor cells as well as in peri-tumor reactive astrocytes in response to TMZ monotherapy. This provided a rationale for us to test the efficacy of combining NKCC1 pharmacological blockade with TMZ on improving therapeutic outcomes. 5-day combination therapy of TMZ with potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated TMZ-mediated apoptosis, and decreased peri-tumor reactive astrocyte formation, which collectively led to suppressed tumor growth and prolonged survival of tumor-bearing mice. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in regulating the pathogenesis of LGGs and its blockade presents therapeutic potentials for reducing TMZ-mediated resistance.

Immunosuppressive tumor microenvironment (TME) in glioblastoma (GBM) attributes to poor efficacy of chemotherapy and/or immunotherapy. We recently reported that SLC9A1 gene expression (Na/H exchanger 1, NHE1) in GBM tumor correlates with increased macrophage tumor infiltration and worsened patient survival. Temozolomide (TMZ) monotherapy, the standard care therapy for GBM, stimulates NHE1 protein expression in mouse syngeneic glioma models. We speculate that in response to TMZ therapy, NHE1 promotes immunosuppressive TME development via deregulation of glucose metabolism and anti-tumor function of immune cells. CD57BL/6 mice bearing GL26 glioma tumor were treated with NHE1 inhibitor HOE-642 (H, 0.3 mg/kg/day), TMZ (T, 2.5 mg/kg/day), and/or anti-PD-1 (10 mg/kg/day) monotherapy or in combination. The T+H+anti-PD-1 combination therapy increased glucose uptake (2-NBDG) and mitochondrial mass (mitotracker fluorescent intensity) of tumor infiltrating CD8+ T-cells by ~2-fold compared to the monotherapies. This suggests that NHE1 protein is involved in metabolic transformation of T-cells and affects their responses to chemo and/or immunotherapy. Furthermore, we detected that specific deletion of Nhe1 in Cx3Cr1CreErT2r+/-;Nhe1f/f mice (Nhe1 KO) alters activation of glioma-associated microglia/macrophages (GAM) and T-cells in response to TMZ therapy. TMZ monotherapy stimulated macrophage (CD11b+CD45hi) infiltration by ~2.5-fold in Cx3Cr1CreErT2-/-;Nhe1f/f (Nhe1 Con) mice bearing SB28-GFP glioma tumor with abundant proinflammatory CD16/32+ GAMs. In contrast, TMZ treated Nhe1 KO mice displayed ~3-fold reduction in tumor infiltrating GAMs with nearly abolished Ym-1+ tolerogenic macrophages, ~3-fold reduction of tumor-promoting CD4+CD25+FoxP3+ regulatory T-cells (Treg) and increased CD8+/Treg ratio by ~2-fold than the Nhe1 Con mice. Importantly, TMZ followed by anti-PD-1 therapy prolonged survival of Nhe1 KO mice but not Nhe1 Con mice. Taken together, our study suggests that NHE1 is involved in transformation of GAMs and PD-1 check point activation of T-cells. Blocking NHE1 function in combination with TMZ and anti-PD-1 may restore glucose metabolism of immune cells necessary for their anti-tumor function.

CMTM6, a previously uncharacterized protein, was identified as a critical regulator of PD-L1, which is reported as an immune checkpoint inhibitor, to modulate the T cell activities both in vitro and in vivo of other tumors. However, the role of CMTM6 has so far remained unclear in glioma. To investigate the role of CMTM6 in gliomas, we analyzed the transcriptome level, genomic profiles and its relationship with clinical practice. 1862 glioma samples with transcriptome data were enrolled in this study, including CGGA RNA-seq, TCGA RNA-seq, CGGA-microarray, GSE16011 and IVY GBM databases. Clinical information and genomic profiles containing somatic mutations and DNA copy numbers were also obtained. We found that CMTM6 expression was highly correlated with major clinical and molecular characteristics. Cases with high CMTM6 expression were more likely to be predicted as malignant entities and frequent with genomic aberrations of driver oncogenes. Moreover, gene ontology analysis based on significantly correlated genes of CMTM6 expression exhibited that CMTM6 was associated with immune responses and inflammatory activities. CMTM6 was synthetic with other immune checkpoint inhibitors. Additionally, CMTM6 was involved in immune functions via modulating T-lymphocyte-mediated anti-tumor immunity. Finally, high CMTM6 expression was associated with reduced survival time and may serve as a strong indicator of poor prognosis in gliomas. In brief, High level of CMTM6 expression is closely related to high malignant gliomas. Meanwhile, CMTM6 plays an important role in regulating T cell activation and antitumor responses. Therefore, CMTM6 is a promising target for developing immunotherapy of gliomas.

Table S1. The information of human glioma tissue (DOCX 23 kb)

The cholesterol sulfotransferase SULT2B1b converts cholesterol to cholesterol sulfate (CS). We previously reported that SULT2B1b inhibits hepatic gluconeogenesis by antagonizing the gluconeogenic activity of hepatocyte nuclear factor 4α (HNF4α). In this study, we showed that the SULT2B1b gene is a transcriptional target of HNF4α, which led to our hypothesis that the induction of SULT2B1b by HNF4α represents a negative feedback to limit the gluconeogenic activity of HNF4α. Indeed, downregulation of Sult2B1b enhanced the gluconeogenic activity of HNF4α, which may have been accounted for by the increased acetylation of HNF4α as a result of decreased expression of the HNF4α deacetylase sirtuin 1 (Sirt1). The expression of Sult2B1b was also induced by HNF4α upon fasting, and the Sult2B1b null (Sult2B1b-/-) mice showed increased gluconeogenic gene expression and an elevated fasting glucose level, suggesting that SULT2B1b also plays a restrictive role in HNF4α-mediated fasting-responsive gluconeogenesis. We also developed thiocholesterol, a hydrolysis-resistant derivative of CS, which showed superior activity to that of the native CS in inhibiting gluconeogenesis and improving insulin sensitivity in high-fat-diet-induced diabetic mice. We conclude that the HNF4α-SULT2B1b-CS axis represents a key endogenous mechanism to prevent uncontrolled gluconeogenesis. Thiocholesterol may be used as a therapeutic agent to manage hyperglycemia.

Acute kidney injury (AKI) is associate with high mortality. Despite evidence of AKI-induced distant organ injury, a relationship between AKI and liver injury has not been clearly established. The goal of this study is to investigate whether renal ischemia-reperfusion (IR) can affect liver pathophysiology. We showed that renal IR in mice induced fatty liver and compromised liver function through the downregulation of constitutive androstane receptor (CAR; −90.4%) and inhibition of hepatic very-low-density lipoprotein triglyceride (VLDL-TG) secretion (−28.4%). Treatment of mice with the CAR agonist 1,4-bis[2-(3,5 dichloropyridyloxy)] benzene (TCPOBOP) prevented the development of AKI-induced fatty liver and liver injury, which was associated with the attenuation of AKI-induced inhibition of VLDL-TG secretion. The hepatoprotective effect of TCPOBOP was abolished in CAR(−/−) mice. Interestingly, alleviation of fatty liver by TCPOBOP also improved the kidney function, whereas CAR ablation sensitized mice to AKI-induced kidney injury and lethality. The serum concentrations of interleukin-6 (IL-6) were elevated by 27-fold after renal IR, but were normalized in TCPOBOP-treated AKI mice, suggesting that the increased release of IL-6 from the kidney may have mediated the AKI responsive liver injury. Taken together, our results revealed an interesting kidney-liver organ cross-talk in response to AKI. Given the importance of CAR in the pathogenesis of renal IR–induced fatty liver and impaired kidney function, fatty liver can be considered as an important risk factor for kidney injury, and a timely management of hepatic steatosis by CAR activation may help to restore kidney function in patients with AKI or kidney transplant.

Despite the multidisciplinary integration in the therapeutic management of glioblastoma multiforme (GBM), the prognosis of GBM patients is poor. There is growing recognition that the cells in the tumor microenvironment play a vital role in regulating the progression of glioma. Astrocytes are an important component of the blood-brain barrier (BBB) as well as the tripartite synapse neural network to promote bidirectional communication with neurons under physiological conditions. Emerging evidence shows that tumor-associated reactive astrocytes interact with glioma cells and facilitate the progression, aggression, and survival of tumors by releasing different cytokines. Communication between reactive astrocytes and glioma cells is further promoted through ion channels and ion transporters, which augment the migratory capacity and invasiveness of tumor cells by modifying H+ and Ca2+ concentrations and stimulating volume changes in the cell. This in part contributes to the loss of epithelial polarization, initiating epithelial-mesenchymal transition. Therefore, this review will summarize the recent findings on the role of reactive astrocytes in the progression of GBM and in the development of treatment-resistant glioma. In addition, the involvement of ion channels and transporters in bridging the interactions between tumor cells and astrocytes and their potential as new therapeutic anti-tumor targets will be discussed.

Glioblastoma is the most common and aggressive brain tumor with a median survival of ~15 months. Clinical trials of patients for immunotherapy showed an inferior response, which can in part attribute to an immunosuppressive, acidic tumor microenvironment (TME) composed of glioma-associated microglia/macrophages (TAMs), regulatory T-cells (Treg), and expression of immune checkpoint molecules. NHE1 maintains alkaline pHi in glioma and TAMs, promoting their migration and proliferation. Temozolomide (TMZ) therapy concurrently induces upregulation of NHE1 in glioma and alters TME immunogenicity. In this study, we investigated the impact of selective deletion of Nhe1 from microglia/macrophage using Cx3Cr1(CreEr+/-);Nhe1(f/f) mice (Nhe1 KO). Non-immunogenic SB28-GFP glioma cells were transplanted intracranially, and tamoxifen (75 mg/kg, i.p.) was administered at 1–5 days post-glioma injection (d.p.i.). Cx3Cr1(CreEr-/-);Nhe1(f/f) mice receiving identical treatment served as controls (Con). These mice were subsequently treated with DMSO or TMZ (2.5 mg/kg, i.p.) at 2–6 d.p.i. TAM infiltration and T-cell profile were analyzed by flow-cytometry at 10 d.p.i. TMZ monotherapy in Con mice elevated the tumor infiltrated microglia by 2-fold and macrophage numbers by 2.5-fold. In contrast, Nhe1 KO mice treated with TMZ did not exhibit a significant increase of microglia or macrophage infiltration. Ym-1(+) tolerogenic macrophages in tumors from TMZ-treated Nhe1 KO mice were nearly abolished. TMZ treatment in Nhe1 KO mice also reduced the tumor-promoting CD4(+)CD25(+)FoxP3(+) Treg infiltration but did not change CD8(+)GZMb(+) and CD8(+)IFNγ(+) populations. Moreover, combining TMZ with selective deletion of Nhe1 in Cx3Cr1(+) cells significantly elevated CD4(+)PD-1(+) and CD8(+)PD-1(+) cell population. Most importantly, Nhe1 KO mice treated with combinatorial TMZ and anti-PD-1 therapy prolonged the animal survival. Taken together, our study suggests that NHE1 is involved in modulating TME and PD-1 expression. Blockade of NHE1 function in combination with TMZ and anti-PD-1 could improve TME and provide an alternative treatment strategy for glioblastoma. This was supported by NIH-grant R01NS75995(DS).

The impact of sexual dysfunction (SD) is distressing to many male patients with pituitary adenomas which affect both physical and psychological health. The research explored to identify risk factors affecting sexual function and the prognosis of male patients with pituitary adenomas. Two hundred and fifty-four male patients, who aged between 18 and 60 (mean ± s.d.: 44.16 ± 10.14) years and diagnosed with pituitary adenomas, were retrospectively analyzed. One hundred and fifty-nine patients (62.6%) complained of SD prior to surgery. The mean International Index of Erectile Function (IIEF-5) in patients with giant adenomas was 16.13 ± 2.51, much smaller than those with microadenomas or macroadenomas (P < 0.05). All the patients showed significant improvement in terms of erectile dysfunction (ED) following surgery (P < 0.05). In addition, complete resection achieved a higher degree of SD relief than partial resection. The incidence of SD in functioning pituitary adenomas (FPAs) was much higher than that in nonfunctioning pituitary adenomas (NFPAs) (P < 0.05). In addition, compared with NFPAs, males with prolactinomas (82.8%) had the higher prevalence of SD and significantly improvement following surgical intervention (P < 0.05). An inverse relationship was identified between decreasing testosterone levels and increasing incidence of SD before surgery (P < 0.05). There was no significant difference between 6 months and 12 months after surgery in serum testosterone level (P > 0.05). Our results indicated that surgical therapy could be optimized for improvements in SD and that testosterone levels can be used as a sensitive indicator to predict the recovery rate of sexual function in patients with pituitary adenomas following surgery and the serum testosterone level will stay stable in 6 months after surgery.

Supplemental Methods. Immunostaining image analysis, Cell counting, Fluorescence intensity quantification. Figure S1. Representative images of secondary antibody only staining. Figure S2. Differential NHE1 protein expression in human gliomas. Figure S3. SLC9A1 mRNA expression is related with angiogenesis in gene ontology analysis in TCGA dataset. Figure S4. Inhibition of NHE1 decreases the tumor vessel density in SB28 gliomas. Figure S5. HOE642 treatment decreases the resident-microglia TAMs and bone-marrow-derived TAMs in SB28 gliomas. Figure S6. Inhibition of NHE1 decreases the cytokines of TAMs in SB28 gliomas. Figure S7. Blockade of NHE1 increases the CD8 T cell infiltration. Figure S8. HOE642 treatment reduced the accumulation of MDSC in SB28 gliomas. Figure S9. Inhibition of NHE1 increases T cell anti-tumor immunity in SB28 glioma model. Figure S10. HOE642 plus anti-PD-1 combination therapy increases the tumor-associated macrophages infiltration in GL26 gliomas. Figure S11. HOE plus anti-PD-1 combination therapy stimulates T cell immunity in GL26 tumor. Figure S12. The effect of HOE642 treatment on the ratio of M1/M2 tumor-associated macrophages. (DOCX 3855 kb)

Supplemental Methods. Immunostaining image analysis, Cell counting, Fluorescence intensity quantification. Figure S1. Representative images of secondary antibody only staining. Figure S2. Differential NHE1 protein expression in human gliomas. Figure S3. SLC9A1 mRNA expression is related with angiogenesis in gene ontology analysis in TCGA dataset. Figure S4. Inhibition of NHE1 decreases the tumor vessel density in SB28 gliomas. Figure S5. HOE642 treatment decreases the resident-microglia TAMs and bone-marrow-derived TAMs in SB28 gliomas. Figure S6. Inhibition of NHE1 decreases the cytokines of TAMs in SB28 gliomas. Figure S7. Blockade of NHE1 increases the CD8 T cell infiltration. Figure S8. HOE642 treatment reduced the accumulation of MDSC in SB28 gliomas. Figure S9. Inhibition of NHE1 increases T cell anti-tumor immunity in SB28 glioma model. Figure S10. HOE642 plus anti-PD-1 combination therapy increases the tumor-associated macrophages infiltration in GL26 gliomas. Figure S11. HOE plus anti-PD-1 combination therapy stimulates T cell immunity in GL26 tumor. Figure S12. The effect of HOE642 treatment on the ratio of M1/M2 tumor-associated macrophages. (DOCX 3855 kb)