B7-H4 reduces the infiltration of CD8+T cells and induces their anti-tumor dysfunction in gliomas.

B7-H4 is a promising immune checkpoint molecule in tumor immunotherapy. Our previous study showed that high B7-H4 expression was strongly correlated with deficiency in tumor infiltrated lymphocytes (TILs) in glioma patients. On this basis, we investigated the impact of B7-H4 on CD8+TILs in gliomas and the associated molecular mechanism here. B7-H4-positive tumor samples (n=129) from our glioma cohort were used to assess B7-H4 expression and CD8+TIL quantification by immunohistochemistry. CD8+TILs from five glioma patients cultured with B7-H4 protein were used to evaluate anti-tumor dysfunction by flow cytometry and ELISpot. An orthotopic murine glioma model was used to investigate the role of B7-H4 in glioma CD8+TILs by immunohisto- chemistry and flow cytometry. CD8+TILs from glioma patients cultured with B7-H4 protein were used to explore the potential molecular mechanism by RNA sequencing and western blot. Our results showed that glioma CD8+TIL density was negatively correlated with B7-H4 expression both in glioma patient cohort (P < 0.05) and orthotopic glioma murine model (P < 0.01). B7-H4 also lowered the expression of CD137 and CD103 (P < 0.05 for both) in glioma CD8+TILs and reduced their secretion of the anti-tumor cytokines IFN-γ and TNF-α (P < 0.01 for both) in a dose-dependent manner. Furthermore, B7-H4 was found to induce early dysfunction of glioma CD8+TILs by downregulating the phosphorylation of AKT and eNOS (P < 0.05 for both). In conclusion, B7-H4 reduced the infiltration of glioma CD8+TILs and induced an anti-tumor dysfunction phenotype. B7-H4 may also impair the anti-tumor function of glioma CD8+TILs via the AKT-eNOS pathway. These results indicated that B7-H4 may serve as a potential target in future glioma immunotherapy.

Integration of 3D bioprinting and multi-algorithm machine learning identified glioma susceptibilities and microenvironment characteristics.

Glioma, with its heterogeneous microenvironments and genetic subtypes, presents substantial challenges for treatment prediction and development. We integrated 3D bioprinting and multi-algorithm machine learning as a novel approach to enhance the assessment and understanding of glioma treatment responses and microenvironment characteristics. The bioprinted patient-derived glioma tissues successfully recapitulated molecular properties and drug responses of native tumors. We then developed GlioML, a machine learning workflow incorporating nine distinct algorithms and a weighted ensemble model that generated robust gene expression-based predictors, each reflecting the diverse action mechanisms of various compounds and drugs. The ensemble model superseded the performance of all individual algorithms across diverse in vitro systems, including sphere cultures, complex 3D bioprinted multicellular models, and 3D patient-derived tissues. By integrating bioprinting, the evaluative scope of the treatment expanded to T cell-related therapy and anti-angiogenesis targeted therapy. We identified promising compounds and drugs for glioma treatment and revealed distinct immunosuppressive or angiogenic myeloid-infiltrated tumor microenvironments. These insights pave the way for enhanced therapeutic development for glioma and potentially for other cancers, highlighting the broad application potential of this integrative and translational approach.

MRI-derived radiomics assessing tumor-infiltrating macrophages enable prediction of immune-phenotype, immunotherapy response and survival in glioma.

BACKGROUND: The tumor immune microenvironment can influence the prognosis and treatment response to immunotherapy. We aimed to develop a non-invasive radiomic signature in high-grade glioma (HGG) to predict the absolute density of tumor-associated macrophages (TAMs), the preponderant immune cells in the microenvironment of HGG. We also aimed to evaluate the association between the signature, and tumor immune phenotype as well as response to immunotherapy. METHODS: In this retrospective setting, total of 379 patients with HGG from three independent cohorts were included to construct a radiomic model named Radiomics Immunological Biomarker (RIB) for predicting the absolute density of M2-like TAM using the mRMR feature ranking method and LASSO classifier. Among them, 145 patients from the TCGA microarray cohort were randomly allocated into a training set (N=101) and an internal validation set (N=44), while the immune-phenotype cohort (N=203) and the immunotherapy-treated cohort (N=31, patients from a prospective clinical trial treated with DC vaccine) recruited from Huashan Hospital were used as two external validation sets. The immunotherapy-treated cohort was also used to evaluate the relationship between RIB and immunotherapy response. Radiogenomic analysis was performed to find functional annotations using RNA sequencing data from TAM cells. RESULTS: An 11-feature radiomic model for M2-like TAM was developed and validated in four datasets of HGG patients (area under the curve = 0.849, 0.719, 0.674, and 0.671) using MRI images of post contrast enhanced T1-weighted (T1CE). Patients with high RIB scores had a strong inflammatory response. Four hub-genes (SLC7A7, RNASE6, HLA-DRB1 and CD300A) expressed by TAM were identified to be closely related to the RIB, providing important evidence for biological interpretation. Only individuals with a high RIB score were shown to have survival benefits from DC vaccine [DC vaccine vs. Placebo: median progression-free survival (mPFS), 10.0 mos vs. 4.5 mos, HR=0.17, P=0.0056, 95%CI=0.041-0.68; median overall survival (mOS), 15.0 mos vs. 7.0 mos, HR=0.17, P =0.0076, 95%CI=0.04-0.68]. Multivariate analyses also confirmed that treatment by DC vaccine was an independent factor for improved survival in the high RIB score group. However, in the low RIB score group, DC vaccine was not associated with improved survival. Furthermore, a radiomic nomogram based on the RIB score and clinical factors could efficiently predict the 1-, 2-, and 3-year survival rates, as confirmed by ROC curve analysis (AUC for 1-, 2- and 3-year survival: 0.705, 0.729 and 0.684, respectively). CONCLUSIONS: The radiomic model could allow for non-invasive assessment of the absolute density of TAM from MRI images in HGG patients. Of note, our RIB model is the first immunological radiomic model confirmed to have the ability to predict survival benefits from DC vaccine in gliomas, thereby providing a novel tool to inform treatment decisions and monitor patient treatment course by radiomics.

Signal-transducing adaptor protein 1 (STAP1) in microglia promotes the malignant progression of glioma.

BACKGROUND: Glioma is the most malignant primary brain tumor with a poor survival time. The tumour microenvironment, especially glioma-associated microglia/macrophages (GAMs), plays an important role in the pathogenesis of glioma. Currently, microglia (CD11b+/CD45Low) and macrophages (CD11b+/CD45High) are distinguished as distinct cell types due to their different origins. Moreover, signal-transducing adaptor protein 1 (STAP1) plays a role in tumourigenesis and immune responses. However, to date, no studies have been reported on STAP1 in GAMs. METHODS: The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases were used to investigate the association between STAP1 mRNA levels and clinical parameters (grades, mutations in isocitrate dehydrogenase, and overall survival). RNA-sequencing, qRT-PCR, Western blotting, immunohistochemistry and immunofluorescence analyses were performed to detect the expression level of STAP1 and related proteins. BV-2 cells were used to construct a STAP1-overexpressing cell line. Phagocytosis of BV-2 cells was assessed by flow cytometry and fluorescence microscopy. C57BL/6 mice were used to establish orthotopic and subcutaneous glioma mouse models. Glioma growth was monitored by bioluminescence imaging. RESULTS: STAP1 expression in glioma-associated microglia is positively correlated with the degree of malignancy and poor prognosis of glioma. Moreover, STAP1 may promote M2-like polarisation by increasing ARG1 expression and inhibiting microglial phagocytosis of microglia. Increased ARG1 may be associated with the IL-6/STAT3 pathway. Impaired phagocytosis may be associated with decreased cofilin and filopodia. CONCLUSION: STAP1 is positively associated with the degree of glioma malignancy and may represent a potential novel therapeutic target for glioma.

Identification of an IL-4-Related Gene Risk Signature for Malignancy, Prognosis and Immune Phenotype Prediction in Glioma.

BACKGROUND: Emerging molecular and genetic biomarkers have been introduced to classify gliomas in the past decades. Here, we introduced a risk signature based on the cellular response to the IL-4 gene set through Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis. METHODS: In this study, we provide a bioinformatic profiling of our risk signature for the malignancy, prognosis and immune phenotype of glioma. A cohort of 325 patients with whole genome RNA-seq expression data from the Chinese Glioma Genome Atlas (CGGA) dataset was used as the training set, while another cohort of 667 patients from The Cancer Genome Atlas (TCGA) dataset was used as the validating set. The LASSO model identified a 10-gene signature which was considered as the optimal model. RESULTS: The signature was confirmed to be a good predictor of clinical and molecular features involved in the malignancy of gliomas. We also identified that our risk signature could serve as an independently prognostic biomarker in patients with gliomas (p < 0.0001). Correlation analysis showed that our risk signature was strongly correlated with the Tregs, M0 macrophages and NK cells infiltrated in the microenvironment of glioma, which might be a supplement to the existing incomplete innate immune mechanism of glioma phenotypes. CONCLUSIONS: Our IL-4-related gene signature was associated with more aggressive and immunosuppressive phenotypes of gliomas. The risk score could predict prognosis independently in glioma, which might provide a new insight for understanding the IL-4 involved mechanism of gliomas.

Enhanced B7-H4 expression in gliomas with low PD-L1 expression identifies super-cold tumors.

BACKGROUND: Characterizing expression profiles of different immune checkpoint molecules are promising for personalized checkpoint inhibitory immunotherapy. Gliomas have been shown as potential targets for immune checkpoint inhibitors recently. Our study was performed to determine coexpression levels of two major B7 immune regulatory molecules programmed death ligand 1 (PD-L1) and B7-H4, both of which have been demonstrated to inhibit antitumor host immunity in gliomas. METHODS: We assessed tumor tissues from stage II-IV primary gliomas (n=505) by immunohistochemistry (IHC) for protein levels of both PD-L1 and B7-H4. Gene coexpression analysis assessing clusters based on extent of PD-L1/B7-H4 classifier genes expression were investigated in two transcriptome datasets (The Cancer Genome Atlas and Chinese Glioma Genome Atlas). In addition, levels of immune cell infiltrates were estimated with IHC and RNA-seq data for assessing the tumor immune microenvironment of PD-L1/B7-H4 subgroups. RESULTS: High expression of PD-L1 and B7-H4 in gliomas was 23% and 20%, respectively, whereas coexpression of two proteins at high levels was limited to 2% of the cases. Comparable results were seen in RNA-seq datasets where PD-L1 mRNA expression levels negatively correlated with that of B7-H4. Gene coexpression modules clustered within each grade of gliomas demonstrated lack of double-high modules (cluster with high expression of both PD-L1 and B7-H4 classifier genes). B7-H4 mRNA expression levels showed negative correlation with extent of immune cell infiltration and High-B7-H4 module gliomas (high B7-H4 but low PD-L1 classifier genes expression) had less tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). IHC assessment also showed few TILs and TAMs in High-B7-H4 subgroup gliomas. CONCLUSIONS: The majority of gliomas express PD-L1 or B7-H4, however, coexpression of both at high levels is minimal. The high-B7-H4 patients could be considered as 'super-cold' gliomas with significantly deficient in TILs, suggesting that B7-H4 might inhibit T-cell trafficking into the central nervous system. This study demonstrated that PD-L1 and B7-H4 may serve as mutually compensatory immune checkpoint molecules in gliomas for immune targeted or active-specific immunotherapy. The distinct B7-H4 pathways modulating T-cell function and immune evasion in glioma patients deserved to be further explored in the future during immunotherapy.

Bioinformatic Profiling Identifies a Fatty Acid Metabolism-Related Gene Risk Signature for Malignancy, Prognosis, and Immune Phenotype of Glioma.

Cancer cells commonly have metabolic abnormalities. Aside from altered glucose and amino acid metabolism, cancers cells often share the attribute of fatty acid metabolic alterations. However, fatty acid metabolism related-gene set has not been systematically investigated in gliomas. Here, we provide a bioinformatic profiling of the fatty acid catabolic metabolism-related gene risk signature for the malignancy, prognosis and immune phenotype of glioma. In this study, a cohort of 325 patients with whole genome RNA-seq expression data from the Chinese Glioma Genome Atlas (CGGA) dataset was used as training set, while another cohort of 667 patients from The Cancer Genome Atlas (TCGA) dataset was used as validating set. After confirmed that fatty acid catabolic metabolism-related gene set could distinguish clinicopathological features of gliomas, we used LASSO regression analysis to develop a fatty-acid metabolism-related gene risk signature for glioma. This 8-gene risk signature was found to be a good predictor of clinical and molecular features involved in the malignancy of gliomas. We also identified that this 8-gene risk signature had high prognostic values in patients with gliomas. Correlation analysis showed that our risk signature was closely associated with the immune cells involved in the microenvironment of glioma. Furthermore, the fatty acid catabolic metabolism-related gene risk signature was also found to be significantly correlated with immune checkpoint members B7-H3 and Tim-3. In summary, we have identified a fatty acid metabolism-related gene risk signature for malignancy, prognosis, and immune phenotype of glioma; and our study might contribute to better understanding of metabolic pathways and further developing of novel therapeutic approaches for gliomas.

[Basis of Tumor Microenvironment Relevant to Immunotherapies for Brain Metastases of NSCLC].

Brain is the most frequent site for distant metastases of non-small cell lung cancer (NSCLC). Brain metastasis (BM) is also the leading cause of disabilities and death in advanced NSCLC. In recent years, the application and effectiveness of small-molecule tyrosine kinase inhibitors has formed the basis for the treatment of NSCLC brain metastases with driver gene mutations. With the development of programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) inhibitors and relevant combination therapies, immunotherapy has become an important choice for non-classic oncogene addicted NSCLC BM patients. Also, the roles of relevant biomarkers are increasingly standing out. By reason of the particular immunopathological features of NSCLC brain metastases and its microenvironment, the aim of this review is to summarize relevant research progresses and provide more references for combination strategies of different therapeutic methods as well as the development of novel immunotherapies.
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Targeting hypoxia downstream signaling protein, CAIX, for CAR T-cell therapy against glioblastoma.

BACKGROUND: Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. METHODS: First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. RESULTS: We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. CONCLUSIONS: By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

Clinically relevant circulating microRNA profiling studies in pancreatic cancer using meta-analysis.

BACKGROUND: Pancreatic cancer (PaCa) is the most lethal gastrointestinal (GI) tumor. Although many studies on differentially expressed miRNAs as candidate biomarkers of pancreatic cancer have been published, reliability of these findings generated from investigations performed in single laboratory settings remain unclear. RESULTS: There were 29 articles with a total of 2,225 patients and 1,618 controls included in this meta-analysis. The pooled sensitivity was 82% (95% CI, 79-85%); the specificity was 85% (95% CI, 79-89%); and area under the curve (AUC) was 0.89 (95% CI, 0.86-0.92). Subgroup analyses indicated that there were significant divergences between Caucasian and Asian subgroups for circulating miRNA analysis. MATERIALS AND METHODS: To comprehensively investigate the potential utility of miRNAs as biomarkers of the disease, we searched publications diagnosing PaCa using miRNAs from PubMed, Medline, Embase, Google Scholar and Chinese National Knowledge Infrastructure (CNKI) databases. The sensitivity (SEN), specificity (SPE), and summary receiver operating characteristic (SROC) curve were used to examine the overall test performance, and heterogeneity was analyzed with the I2 test. CONCLUSIONS: Our analysis demonstrated that multiple miRNAs (SEN: 85%; SPE: 89%; AUC: 0.93) were more accurate for diagnosing PaCa than a single miRNA (SEN: 78%; SPE: 79%; AUC: 0.84), and future studies are still needed to confirm the diagnostic value of these pooled miRNAs for PaCa.

Soluble cpg15 from Astrocytes Ameliorates Neurite Outgrowth Recovery of Hippocampal Neurons after Mouse Cerebral Ischemia.

The present study focuses on the function of cpg15, a neurotrophic factor, in ischemic neuronal recovery using transient global cerebral ischemic (TGI) mouse model and oxygen-glucose deprivation (OGD)-treated primary cultured cells. The results showed that expression of cpg15 proteins in astrocytes, predominantly the soluble form, was significantly increased in mouse hippocampus after TGI and in the cultured astrocytes after OGD. Addition of the medium from the cpg15-overexpressed astrocytic culture into the OGD-treated hippocampal neuronal cultures reduces the neuronal injury, whereas the recovery of neurite outgrowths of OGD-injured neurons was prevented when cpg15 in the OGD-treated astrocytes was knocked down, or the OGD-treated-astrocytic medium was immunoadsorbed by cpg15 antibody. Furthermore, lentivirus-delivered knockdown of cpg15 expression in mouse hippocampal astrocytes diminishes the dendritic branches and exacerbates injury of neurons in CA1 region after TGI. In addition, treatment with inhibitors of MEK1/2, PI3K, and TrkA decreases, whereas overexpression of p-CREB, but not dp-CREB, increases the expression of cpg15 in U118 or primary cultured astrocytes. Also, it is observed that the Flag-tagged soluble cpg15 from the astrocytes transfected with Flag-tagged cpg15-expressing plasmids adheres to the surface of neuronal bodies and the neurites. In conclusion, our results suggest that the soluble cpg15 from astrocytes induced by ischemia could ameliorate the recovery of the ischemic-injured hippocampal neurons via adhering to the surface of neurons. The upregulated expression of cpg15 in astrocytes may be activated via MAPK and PI3K signal pathways, and regulation of CREB phosphorylation.SIGNIFICANCE STATEMENT Neuronal plasticity plays a crucial role in the amelioration of neurological recovery of ischemic injured brain, which remains a challenge for clinic treatment of cerebral ischemia. cpg15 as a synaptic plasticity-related factor may participate in promoting the recovery process; however, the underlying mechanisms are still largely unknown. The objective of this study is to reveal the function and mechanism of neuronal-specific cpg15 expressed in astrocytes after ischemia induction, in promoting the recovery of injured neurons. Our findings provided new mechanistic insight into the neurological recovery, which might help develop novel therapeutic options for cerebral ischemia via astrocytic-targeting interference of gene expression.

Transcriptomic and functional pathways analysis of ascorbate-induced cytotoxicity and resistance of Burkitt lymphoma.

Ascorbate is a pro-oxidant that generates hydrogen peroxide-dependent cytotoxity in cancer cells without adversely affecting normal cells. To determine the mechanistic basis for this phenotype, we selected Burkitt lymphoma cells resistant to ascorbate (JLPR cells) and their ascorbate-sensitive parental cells (JLPS cells). Compared with JLPS cells, the increased glucose uptake in JLPR cells (with upregulated glucose transporters, increased antioxidant enzyme activity, and altered cell cycling) conferred ascorbate-induced cytotoxicity and resistance. Transcriptomic profiles and function pathway analysis identified differentially expressed gene signatures for JLPR cells and JLPS cells, which differential expression levels of five genes (ATF5, CD79B, MHC, Myosin, and SAP18) in ascorbate-resistant cells were related to phosphoinositide 3 kinase, cdc42, DNA methylation and transcriptional repression, polyamine regulation, and integrin-linked kinase signaling pathways. These results suggested that coordinated changes occurred in JLPR cells to enable their survival when exposed to the cytotoxic pro-oxidant stress elicited by pharmacologic ascorbate treatment.

Exosomes for Immunoregulation and Therapeutic Intervention in Cancer.

Exosomes, as a subset of extracellular vesicles, function as a mode of intercellular communication and molecular transfer, and facilitate the direct extracellular transfer of proteins, lipids, and miRNAs/mRNAs/DNAs between cells. Cancers have adapted exosomes and related microvesicles as a pathway that can suppress the immune system and establish a fertile local and distant environment to support neoplastic growth, invasion, and metastasis; these tumor-derived exosomes affect immunoregulation mechanisms, including immune activation and immune suppression. Immune cell-derived exosomes can modulate the immune response in cancer, which supports the belief that these membranous vesicles are immunotherapeutic reagents. In this review, we discuss the recent advances in the cancer immunotherapy, roles of exosomes in cancer, immunoregulation of tumor-derived exosomes, and immunomodulation by immune cell-derived exosomes. The topics covered here highlight novel insights into the development of efficient exosome-based cancer vaccines for cancer therapeutic intervention.

Splicing factor NSSR1 reduces neuronal injury after mouse transient global cerebral ischemia.

This study focuses on the function of NSSR1, a splicing factor, in neuronal injury in the ischemic mouse brain using the transient global cerebral ischemic mouse model and the cultured cells treated with oxygen-glucose deprivation (OGD). The results showed that the cerebral ischemia triggers the expression of NSSR1 in hippocampal astrocytes, predominantly the dephosphorylated NSSR1 proteins, and the Exon3 inclusive NCAM-L1 variant and the Exon4 inclusive CREB variant. While in the hippocampus of astrocyte-specific NSSR1 conditional knockdown (cKD) mice, where cerebral ischemia no longer triggers NSSR1 expression in astrocytes, the expression of Exon3 inclusive NCAM-L1 variant and Exon4 inclusive CREB variant were no longer triggered as well. In addition, the injury of hippocampal neurons was more severe in astrocyte-specific NSSR1 cKD mice compared with in wild-type mice after brain ischemia. Of note, the culture media harvested from the astrocytes with overexpression of NSSR1 or the Exon3 inclusive NCAM-L1 variant, or Exon4 inclusive CREB variant were all able to reduce the neuronal injury induced by OGD. The results provide the evidence demonstrating that: (1) Splicing factor NSSR1 is a new factor involved in reducing ischemic injury. (2) Ischemia induces NSSR1 expression in astrocytes, not in neurons. (3) NSSR1-mediated pathway in astrocytes is required for reducing ischemic neuronal injury. (4) NCAM-L1 and CREB are probably mediators in NSSR1-mediated pathway. In conclusion, our results suggest for the first time that NSSR1 may provide a novel mechanism for reducing neuronal injury after ischemia, probably through regulation on alternative splicing of NCAM-L1 and CREB in astrocytes.