A Lactate-Depleting metal organic framework-based nanocatalyst reinforces intratumoral T cell response to boost anti-PD1 immunotherapy.

Infiltration and activation of intratumoral T lymphocytes are critical for immune checkpoint blockade (ICB) therapy. Unfortunately, the low tumor immunogenicity and immunosuppressive tumor microenvironment (TME) induced by tumor metabolic reprogramming cooperatively hinder the ICB efficacy. Herein, we engineered a lactate-depleting MOF-based catalytic nanoplatform (LOX@ZIF-8@MPN), encapsulating lactate oxidase (LOX) within zeolitic imidazolate framework-8 (ZIF-8) coupled with a coating of metal polyphenol network (MPN) to reinforce T cell response based on a "two birds with one stone" strategy. LOX could catalyze the degradation of the immunosuppressive lactate to promote vascular normalization, facilitating T cell infiltration. On the other hand, hydrogen peroxide (H2O2) produced during lactate depletion can be transformed into anti-tumor hydroxyl radical (•OH) by the autocatalytic MPN-based Fenton nanosystem to trigger immunogenic cell death (ICD), which largely improved the tumor immunogenicity. The combination of ICD and vascular normalization presents a better synergistic immunopotentiation with anti-PD1, inducing robust anti-tumor immunity in primary tumors and recurrent malignancies. Collectively, our results demonstrate that the concurrent depletion of lactate to reverse the immunosuppressive TME and utilization of the by-product from lactate degradation via cascade catalysis promotes T cell response and thus improves the effectiveness of ICB therapy.

Autopsy analysis reveals increased macrophage infiltration and cell apoptosis in COVID-19 patients with severe pulmonary fibrosis.

Clinical data indicates that SARS-CoV-2 infection-induced respiratory failure is a fatal condition for severe COVID-19 patients. However, the pathological alterations of different types of respiratory failure remained unknown for severe COVID-19 patients. This study aims to evaluate whether there are differences in the performance of various types of respiratory failure in severe COVID-19 patients and investigate the pathological basis for these differences. The lung tissue sections of severe COVID-19 patients were assessed for the degree of injury and immune responses. Transcriptome data were used to analyze the molecular basis in severe COVID-19 patients. Severe COVID-19 patients with combined oxygenation and ventilatory failure presented more severe pulmonary fibrosis, airway obstruction, and prolonged disease course. The number of M2 macrophages increased with the degree of fibrosis in patients, suggesting that it may be closely related to the development of pulmonary fibrosis. The co-existence of pro-inflammatory and anti-inflammatory cytokines in the pulmonary environment could also participate in the progression of pulmonary fibrosis. Furthermore, the increased apoptosis in the lungs of COVID-19 patients with severe pulmonary fibrosis may represent a critical factor linking sustained inflammatory responses to fibrosis. Our findings indicate that during the extended phase of COVID-19, antifibrotic and antiapoptotic treatments should be considered in conjunction with the progression of the disease.

Primary pulmonary hyalinizing clear cell carcinoma with fusions of both EWSR1::CREM and IRF2::NTRK3: report of a case with an aggressive behavior.

Primary pulmonary hyalinizing clear cell carcinoma (HCCC) is a rare salivary gland-type tumor newly recognized in recent years, with approximately 21 cases reported to date in the English literature, which constitutes a challenge in pathology diagnosis, particularly in small biopsy specimens. Here, we present a case of pulmonary HCCC diagnosed by computed tomography-guided percutaneous lung biopsy in a 70-year-old man's right lower lung. Although the morphology and immunophenotype of the tumor suggested the diagnosis of mucoepidermoid carcinoma, fluorescence in situ hybridization failed to reveal the rearrangement of MAML2 gene, which is characteristic of mucoepidermoid carcinoma. Instead, further molecular genetic testing showed that the tumor harbored a rare EWSR1::CREM fusion combined with a previously unreported IRF2::NTRK3 fusion. Pulmonary HCCC is commonly regarded as a low-grade malignant tumor with an indolent course, but this case has a different biological behavior, presenting extensive dissemination and metastases at the time of diagnosis, which expands our understanding of the prognosis of this tumor. The patient has had five cycles of combination chemotherapy and has been alive with the tumor for eight months.

Neutrophil infiltration and myocarditis in patients with severe COVID-19: A post-mortem study.

Aims: To investigate cardiac pathology in critically ill patients with coronavirus disease 2019 (COVID-19) and identify associations between pathological changes and clinical characteristics. Methods: The present autopsy cohort study included hearts from 26 deceased patients hospitalized in intensive care units due to COVID-19, and was conducted at four sites in Wuhan, China. Cases were divided into a neutrophil infiltration group and a no-neutrophil group based on the presence or absence of histopathologically identified neutrophilic infiltrates. Results: Among the 26 patients, histopathological examination identified active myocarditis in four patients. All patients with myocarditis exhibited extensive accompanying neutrophil infiltration, and all patients without myocarditis did not. The neutrophil infiltration group exhibited significantly higher rates of detection of interleukin-6 (100 vs. 4.6%) and tumor necrosis factor-alpha (100 vs. 31.8%) than the no-neutrophil group (both p < 0.05). On admission, four patients with neutrophil infiltration in myocardium had significantly higher baseline levels of aspartate aminotransferase, D dimer, and high-sensitivity C reactive protein than the other 22 patients (all p < 0.05). During hospitalization, patients with neutrophil infiltration had significantly higher maximum creatine kinase-MB (median 280.0 IU/L vs. 38.7 IU/L, p = 0.04) and higher troponin I (median 1.112 ng/ml vs. 0.220 ng/ml, p = 0.56) than patients without neutrophil infiltration. Conclusion: Active myocarditis was frequently associated with neutrophil infiltration in the hearts of deceased patients with severe COVID-19. Patients with neutrophil-infiltrated myocarditis had a series of severely abnormal laboratory test results on admission, and high maximum creatine kinase-MB during hospitalization. The role of neutrophils in severe heart injury and systemic conditions in patients with COVID-19 should be emphasized.

A map of the spatial distribution and tumour-associated macrophage states in glioblastoma and grade 4 IDH-mutant astrocytoma.

Tumour-associated macrophages (TAMs) abundantly infiltrate high-grade gliomas and orchestrate immune response, but their diversity in isocitrate dehydrogenase (IDH)-differential grade 4 gliomas remains largely unknown. This study aimed to dissect the transcriptional states, spatial distribution, and clinicopathological significance of distinct monocyte-derived TAM (Mo-TAM) and microglia-derived TAM (Mg-TAM) clusters across glioblastoma-IDH-wild type and astrocytoma-IDH-mutant-grade 4 (Astro-IDH-mut-G4). Single-cell RNA sequencing was performed on four cases of human glioblastoma and three cases of Astro-IDH-mut-G4. Cell clustering, single-cell regulatory network inference, and gene set enrichment analysis were performed to characterize the functional states of myeloid clusters. The spatial distribution of TAM subsets was determined in human glioma tissues using multiplex immunostaining. The prognostic value of different TAM-cluster specific gene sets was evaluated in the TCGA glioma cohort. Profiling and unbiased clustering of 24,227 myeloid cells from glioblastoma and Astro-IDH-mut-G4 identified nine myeloid cell clusters including monocytes, six Mo/Mg-TAM subsets, dendritic cells, and proliferative myeloid clusters. Different Mo/Mg-TAM clusters manifest functional and transcriptional diversity controlled by specific regulons. Multiplex immunostaining of subset-specific markers identified spatial enrichment of distinct TAM clusters at peri-vascular/necrotic areas in tumour parenchyma or at the tumour-brain interface. Glioblastoma harboured a substantially higher number of monocytes and Mo-TAM-inflammatory clusters, whereas Astro-IDH-mut-G4 had a higher proportion of TAM subsets mediating antigen presentation. Glioblastomas with a higher proportion of monocytes exhibited a mesenchymal signature, increased angiogenesis, and worse patient outcome. Our findings provide insight into myeloid cell diversity and its clinical relevance in IDH-differential grade 4 gliomas, and may serve as a resource for immunotherapy development. © 2022 The Pathological Society of Great Britain and Ireland.

Elevated Kir2.1/nuclear N2ICD defines a highly malignant subtype of non-WNT/SHH medulloblastomas.

Medulloblastoma (MB) is one of the most common childhood malignant brain tumors (WHO grade IV), traditionally divided into WNT, SHH, Group 3, and Group 4 subgroups based on the transcription profiles, somatic DNA alterations, and clinical outcomes. Unlike WNT and SHH subgroup MBs, Group 3 and Group 4 MBs have similar transcriptomes and lack clearly specific drivers and targeted therapeutic options. The recently revised WHO Classification of CNS Tumors has assigned Group 3 and 4 to a provisional non-WNT/SHH entity. In the present study, we demonstrate that Kir2.1, an inwardly-rectifying potassium channel, is highly expressed in non-WNT/SHH MBs, which promotes tumor cell invasion and metastasis by recruiting Adam10 to enhance S2 cleavage of Notch2 thereby activating the Notch2 signaling pathway. Disruption of the Notch2 pathway markedly inhibited the growth and metastasis of Kir2.1-overexpressing MB cell-derived xenograft tumors in mice. Moreover, Kir2.1high/nuclear N2ICDhigh MBs are associated with the significantly shorter lifespan of the patients. Thus, Kir2.1high/nuclear N2ICDhigh can be used as a biomarker to define a novel subtype of non-WNT/SHH MBs. Our findings are important for the modification of treatment regimens and the development of novel-targeted therapies for non-WNT/SHH MBs.

Inhibitory effects of temozolomide on glioma cells is sensitized by RSL3-induced ferroptosis but negatively correlated with expression of ferritin heavy chain 1 and ferritin light chain.

Invasive growth of glioblastoma makes residual tumor unremovable by surgery and leads to disease relapse. Temozolomide is widely used first-line chemotherapy drug to treat glioma patients, but development of temozolomide resistance is almost inevitable. Ferroptosis, an iron-dependent form of non-apoptotic cell death, is found to be related to temozolomide response of gliomas. However, whether inducing ferroptosis could affect invasive growth of glioblastoma cells and which ferroptosis-related regulators were involved in temozolomide resistance are still unclear. In this study, we treated glioblastoma cells with RSL3, a ferroptosis inducer, in vitro (cell lines) and in vivo (subcutaneous and orthotopic animal models). The treated glioblastoma cells with wild-type or mutant IDH1 were subjected to RNA sequencing for transcriptomic profiling. We then analyze data from our RNA sequencing and public TCGA glioma database to identify ferroptosis-related biomarkers for prediction of prognosis and temozolomide resistance in gliomas. Analysis of transcriptome data from RSL3-treated glioblastoma cells suggested that RSL3 could inhibit glioblastoma cell growth and suppress expression of genes involved in cell cycle. RSL3 effectively reduced mobility of glioblastoma cells through downregulation of critical genes involved in epithelial-mesenchymal transition. Moreover, RSL3 in combination with temozolomide showed suppressive efficacy on glioblastoma cell growth, providing a promising therapeutic strategy for glioblastoma treatment. Although temozolomide attenuated invasion of glioblastoma cells with mutant IDH1 more than those with wild-type IDH1, the combination of RSL3 and temozolomide similarly impaired invasive ability of glioblastoma cells in spite of IDH1 status. Finally, we noticed that both ferritin heavy chain 1 and ferritin light chain predicted unfavorable prognosis of glioma patients and were significantly correlated with mRNA levels of methylguanine methyltransferase as well as temozolomide resistance. Altogether, our study provided rationale for combination of RSL3 with temozolomide to suppress glioblastoma cells and revealed ferritin heavy chain 1 and ferritin light chain as biomarkers to predict prognosis and temozolomide resistance of glioma patients.

HOXA5 is amplified in glioblastoma stem cells and promotes tumor progression by transcriptionally activating PTPRZ1.

Although the tumorigenic potential of glioma stem cells (GSCs) is associated with multiple molecular alterations, the gene amplification status of GSCs has not been elucidated. Overexpression of HomeoboxA5 (HOXA5) is associated with increased glioma malignancy. In this study, we identify the gene amplification and protein overexpression of HOXA5 in GSCs and its function in regulating GSC maintenance and the downstream transcriptional effector, to explore the significance of HOXA5 amplification/overexpression for GSC identification and prognostic determination. The HOXA5 gene is significantly amplified in glioblastoma (GBM) and is an independent prognostic factor for predicting worse patient outcomes. Specifically, HOXA5 gene amplification and the resultant protein overexpression are correlated with increased proportions of GSCs and enhanced self-renewal/invasiveness of these cells. Disruption of HOXA5 expression impairs GSC survival and GBM tumor propagation. Mechanistically, HOXA5 directly binds to the promoter region of protein tyrosine phosphatase receptor type Z1 (PTPRZ1), thereby upregulating this gene for GSC maintenance. Suppression of PTPRZ1 largely compromises the pro-tumoral effect of HOXA5 on GSCs. In summary, HOXA5 amplification serves as a genetic biomarker for predicting worse GBM outcome, by enhancing PTPRZ1-mediated GSC survival.

Combination of p38 MAPK inhibitor with PD-L1 antibody effectively prolongs survivals of temozolomide-resistant glioma-bearing mice via reduction of infiltrating glioma-associated macrophages and PD-L1 expression on resident glioma-associated microglia.

Current conventional treatment strategies for glioblastoma (GBM) have limited efficacy due to the rapid development of resistance to temozolomide (TMZ). It is particularly urgent to develop novel therapeutic strategies that can overcome TMZ resistance and provide patients with better prognoses. Here, a TMZ-resistant GBM cell strain and a mouse model of TMZ resistance are established as valuable tools to explore novel therapeutic strategies against TMZ resistance. Experimentally, p38MAPK inhibitor reduces the accumulation of F4/80+/CD11b+ macrophages/microglia in glioma and prolongs the survivals of glioma-bearing mice. Glioma-associated macrophages/microglia have a significanct expression of PD-L1. p38MAPK inhibitor in combination with PD-L1 antibody can effectively prolongs the survivals of TMZ-resistant GBM-bearing hosts, and differentially reduces the accumulation of circulating monocytes-derived tumor-associated macrophages and PD-L1 abundances of resident glioma-associated microglia. This combination therapy could be a treatment option for patients at the recurrence or chronic TMZ maintenance stages. A clinical study to confirm the safety and effectiveness of this combination therapy is warranted.

ADP-Ribosylation Factor Like GTPase 4C (ARL4C) augments stem-like traits of glioblastoma cells by upregulating ALDH1A3.

Glioma cells with stem cell-like properties are crucial for tumor initiation, progression and therapeutic resistance. Therefore, identifying specific factors in regulating stem-like traits is critical for the design of novel glioma therapeutics. Herein, we reported that ADP-Ribosylation Factor Like GTPase 4C (ARL4C) was highly expressed in glioma stem-like cells (GSLCs). GSLCs, determined by the efficiency of sphere formation in vitro and tumor growth in vivo, was increased by overexpression of ARL4C. ARL4C induced the tumorigenesis through ALDH1A3. Analyses of 325 patient specimens showed that ARL4C was highly expressed in glioblastoma (GBM) as compared with lower grade gliomas. In addition, higher level ARL4C expression in glioma was correlated with poorer progression-free survival and overall survival of patients. Therefore, ARL4C may act as a novel prognostic marker and a therapeutic target for GBM.

Mitophagy promotes sorafenib resistance through hypoxia-inducible ATAD3A dependent Axis.

BACKGROUND: The identification of novel targets for recovering sorafenib resistance is pivotal for Hepatocellular carcinoma (HCC) patients. Mitophagy is the programmed degradation of mitochondria, and is likely involved in drug resistance of cancer cells. Here, we identified hyperactivated mitophagy is essential for sorafenib resistance, and the mitophagy core regulator gene ATAD3A (ATPase family AAA domain containing 3A) was down regulated in hypoxia induced resistant HCC cells. Blocking mitophagy may restore the sorafenib sensitivity of these cells and provide a new treatment strategy for HCC patients. METHODS: Hypoxia induced sorafenib resistant cancer cells were established by culturing under 1% O2 with increasing drug treatment. RNA sequencing was conducted in transfecting LM3 cells with sh-ATAD3A lentivirus. Subsequent mechanistic studies were performed in HCC cell lines by manipulating ATAD3A expression isogenically where we evaluated drug sensitivity, molecular signaling events. In vivo study, we investigated the combined treatment effect of sorafenib and miR-210-5P antagomir. RESULTS: We found a hyperactivated mitophagy regulating by ATAD3A-PINK1/PARKIN axis in hypoxia induced sorafenib resistant HCC cells. Gain- and loss- of ATAD3A were related to hypoxia-induced mitophagy and sorafenib resistance. In addition, ATAD3A is a functional target of miR-210-5p and its oncogenic functions are likely mediated by increased miR-210-5P expression. miR-210-5P was upregulated under hypoxia and participated in regulating sorafenib resistance. In vivo xenograft assay showed that miR-210-5P antagomir combined with sorafenib abrogated the tumorigenic effect of ATAD3A down-regulation in mice. CONCLUSIONS: Loss of ATAD3A hyperactivates mitophagy which is a core event in hypoxia induced sorafenib resistance in HCC cells. Targeting miR-210-5P-ATAD3A axis is a novel therapeutic target for sorafenib-resistant HCC.

Grincamycin B Functions as a Potent Inhibitor for Glioblastoma Stem Cell via Targeting RHOA and PI3K/AKT.

Glioblastoma multiforme (GBM) is the most malignant form of glioma, and the overall survival time of patients with GBM is usually less than 14 months. Therefore, it is urgent to find new and effective medicine for GBM. Recently, marine natural products have been shown to exhibit strong inhibitory effects on cancer cells, providing a new avenue for exploring novel drugs for GBM treatment. In this study, we investigated the inhibitory effect of the Grincamycin (GCN) B-F, newly isolated from marine-derived Streptomyces Lusitanus SCSIO LR32, on GBM cells, and evaluated the mechanism of GCN B on GBM. The results, for the first time, showed that GCN B acted as a potent inhibitor to suppress growth and invasion of two human GBM cell lines U251 and 091214 in vitro. In addition, GCN B could effectively target GSCs in GBM evidenced by attenuated formation of tumor spheres and decrease of several markers of GSCs. Furthermore, we performed gene expression microarray followed by Signal-Net analysis. The result revealed that RHOA and PI3K/AKT axis played critical roles for a GCN B-mediated inhibitory effect on GSCs. Altogether, our findings highlighted GCN B as a promising inhibitor for GSCs via targeting RHOA and PI3K/AKT.

Stromal PD-1+ tumor-associated macrophages predict poor prognosis in lung adenocarcinoma.

Immunotherapies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand (PD-L1) axis have been emerging as a promising therapeutic strategy to treat lung cancer. PD-1 is preferentially expressed by activated T lymphocytes; but whether/how its expression by tumor-associated macrophages (TAMs) in lung adenocarcinoma remains elusive. Herein, we investigate the frequency of PD-1 expression on TAMs in mouse allografts by flow cytometry analysis and evaluate the spatial distribution and clinicopathological significance of PD-1+ TAMs in 213 cases of human lung adenocarcinoma specimens by immunohistochemical staining. We find the expression of PD-1 by both mouse and human TAMs. Mouse PD-1+ TAMs possess unique transcriptional profile as compared to PD-1- TAMs. Furthermore, PD-1 is preferentially expressed by CD163+ TAMs in the tumor stroma than those in the tumor islets of lung adenocarcinoma. Stromal PD-1+ TAM infiltration is an independent predictor of reduced survival as determined by univariate (P < .001) and multivariate (P = .023) analysis. Moreover, patients with high stromal PD-1+ TAMs but low tumor cell PD-L1 expression have the shortest survival (P = .0001). Our study demonstrates that PD-1+ TAMs have unique gene expression characteristics and PD-1+ TAMs in the tumor stroma is a potential prognostic factor in lung adenocarcinoma, suggesting that a better understanding of PD-1+ TAMs will be beneficial for immunotherapy of lung adenocarcinoma patients.

CCL8 secreted by tumor-associated macrophages promotes invasion and stemness of glioblastoma cells via ERK1/2 signaling.

Tumor-associated macrophages (TAMs) constitute a large population of glioblastoma and facilitate tumor growth and invasion of tumor cells, but the underlying mechanism remains undefined. In this study, we demonstrate that chemokine (C-C motif) ligand 8 (CCL8) is highly expressed by TAMs and contributes to pseudopodia formation by GBM cells. The presence of CCL8 in the glioma microenvironment promotes progression of tumor cells. Moreover, CCL8 induces invasion and stem-like traits of GBM cells, and CCR1 and CCR5 are the main receptors that mediate CCL8-induced biological behavior. Finally, CCL8 dramatically activates ERK1/2 phosphorylation in GBM cells, and blocking TAM-secreted CCL8 by neutralized antibody significantly decreases invasion of glioma cells. Taken together, our data reveal that CCL8 is a TAM-associated factor to mediate invasion and stemness of GBM, and targeting CCL8 may provide an insight strategy for GBM treatment.

Selection of reference genes suitable for normalization of RT-qPCR data in glioma stem cells.

Considering the importance of gene expression studies for understanding the biology of glioma stem cells (GSCs), we aimed to identify the reliable reference genes in GSCs that were derived from the glioma cell lines T98G, LN229, 090116 and 091214. Quantitative real-time reverse-transcription PCR was employed using 11 reference genes identified through a PubMed literature search, and the assessment of stability through the geNorm, Normfinder and coefficient of variation methods was performed to select suitable reference genes. We found that HPRT1 and RPL13A were the most suitable reference genes, and validated the geometric mean of these genes to normalize the expression of stemness genes by GSCs. Therefore, it is necessary to select novel cell-specific reference genes with greater expression stability for gene expression studies in GSCs.

The landscape of immune microenvironment in lung adenocarcinoma and squamous cell carcinoma based on PD-L1 expression and tumor-infiltrating lymphocytes.

AIMS: The aim of this study was to investigate the tumor microenvironment immune types (TMIT) based on tumor cell programmed cell death ligand 1 (PD-L1) expression and tumor-infiltrating lymphocytes (TILs) distribution and whether distinct TMIT subtypes (TMIT I, PD-L1high /TILhigh ; TMIT II, PD-L1low /TILlow ; TMIT III, PD-L1high /TILlow ; and TMIT IV, PD-L1low /TILhigh ) differentially affect clinical outcomes of patients with lung adenocarcinoma (LAC) and squamous cell carcinoma (SCC). METHODS AND RESULTS: Immunohistochemistry (IHC) was applied to evaluate the expression of PD-L1 and the spatial distribution of programmed cell death 1 (PD-1) and CD8 TILs on the surgically resected specimens from 205 cases of LAC and 149 cases of SCC. PD-1 and CD8 TILs were more frequently distributed in SCC than those in LAC, regardless of their infiltrating in the tumor islets or stroma. The density of TILs was a poor prognostic factor in LAC but a favorable one in SCC. PD-L1 levels and its clinical prognostic significance differed in LAC vs SCC. LAC patients with TMIT III and SCC patients with TMIT I had the longest survival, respectively (P = .0197 and .0049). Moreover, TMIT stratification based on tumor cell PD-L1 expression and stromal CD8+ TILs could be considered as an independent prognostic factor of SCC patients' survival as determined by both univariate and multivariate analysis. CONCLUSION: Our study indicates that different type of TMIT provides its specific microenvironment with diverse impact on survival of LAC and SCC patients and highlights the importance of the integrative assessment of PD-L1 status and TILs' spatial distribution to predict patients' prognosis.

A four-gene signature-derived risk score for glioblastoma: prospects for prognostic and response predictive analyses.

OBJECTIVE: Glioblastoma (GBM) is the most common primary malignant brain tumor regulated by numerous genes, with poor survival outcomes and unsatisfactory response to therapy. Therefore, a robust, multi-gene signature-derived model is required to predict the prognosis and treatment response in GBM. METHODS: Gene expression data of GBM from TCGA and GEO datasets were used to identify differentially expressed genes (DEGs) through DESeq2 or LIMMA methods. The DEGs were then overlapped and used for survival analysis by univariate and multivariate COX regression. Based on the gene signature of multiple survival-associated DEGs, a risk score model was established, and its prognostic and predictive role was estimated through Kaplan-Meier analysis and log-rank test. Gene set enrichment analysis (GSEA) was conducted to explore high-risk score-associated pathways. Western blot was used for protein detection. RESULTS: Four survival-associated DEGs of GBM were identified: OSMR, HOXC10, SCARA3, and SLC39A10. The four-gene signature-derived risk score was higher in GBM than in normal brain tissues. GBM patients with a high-risk score had poor survival outcomes. The high-risk group treated with temozolomide chemotherapy or radiotherapy survived for a shorter duration than the low-risk group. GSEA showed that the high-risk score was enriched with pathways such as vasculature development and cell adhesion. Western blot confirmed that the proteins of these four genes were differentially expressed in GBM cells. CONCLUSIONS: The four-gene signature-derived risk score functions well in predicting the prognosis and treatment response in GBM and will be useful for guiding therapeutic strategies for GBM patients.

Integrated analysis identified core signal pathways and hypoxic characteristics of human glioblastoma.

As a hallmark for glioblastoma (GBM), high heterogeneity causes a variety of phenotypes and therapeutic responses among GBM patients, and it contributes to treatment failure. Moreover, hypoxia is a predominant feature of GBM and contributes greatly to its phenotype. To analyse the landscape of gene expression and hypoxic characteristics of GBM cells and their clinical significance in GBM patients, we performed transcriptome analysis of the GBM cell line U87-MG and the normal glial cell line HEB under normoxia and hypoxia conditions, with the results of which were analysed using established gene ontology databases as well as The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia. We revealed core signal pathways, including inflammation, angiogenesis and migration, and for the first time mapped the components of the toll-like receptor 6 pathway in GBM cells. Moreover, by investigating the signal pathways involved in homoeostasis, proliferation and adenosine triphosphate metabolism, the critical response of GBM to hypoxia was clarified. Experiments with cell lines, patient serum and tissue identified IL1B, CSF3 and TIMP1 as potential plasma markers and VIM, STC1, TGFB1 and HMOX1 as potential biopsy markers for GBM. In conclusion, our study provided a comprehensive understanding for signal pathways and hypoxic characteristics of GBM and identified new biomarkers for GBM patients.

Hybrids by tumor-associated macrophages × glioblastoma cells entail nuclear reprogramming and glioblastoma invasion.

Hybrid formation is a fundamental process in normal development and tissue homeostasis, while the presence and the biological role of hybrids between tumor-associated macrophages (TAMs) and glioblastoma (GBM) cells remain elusive. In this study, we observed that TAM-GBM cell hybrids existed in human GBM specimens as demonstrated by co-expression of glioma biomarkers (GFAP, IDH1R132H and PDGFRA) and macrophage biomarkers (CD68 and CD14). Furthermore, TAM-GBM cell hybrids could also be found in C57BL/6 mice orthotopically inoculated with mouse GBM cells labeled with RFP and after co-culture of bone marrow-derived macrophages from GFP-expressed mice with RFP-labeled GBM cells. The hybrids underwent nuclear reprogramming with unique gene expression profile as compared to parental cells. Moreover, glioma invasion-associated genes were enriched in the hybrids that possessed higher invasiveness, and more hybrids in the invasive margin of GBM were observed as compared to GBM core area. Our data demonstrate the presence of TAM-GBM cell hybrids that enhance GBM invasion. With a better understanding of TAM-GBM cell hybrids, new therapeutic strategies targeting GBM will be developed to treat GBM patients.

Tumour-associated macrophages secrete pleiotrophin to promote PTPRZ1 signalling in glioblastoma stem cells for tumour growth.

Intense infiltration of tumour-associated macrophages (TAMs) facilitates malignant growth of glioblastoma (GBM), but the underlying mechanisms remain undefined. Herein, we report that TAMs secrete abundant pleiotrophin (PTN) to stimulate glioma stem cells (GSCs) through its receptor PTPRZ1 thus promoting GBM malignant growth through PTN-PTPRZ1 paracrine signalling. PTN expression correlates with infiltration of CD11b+/CD163+ TAMs and poor prognosis of GBM patients. Co-implantation of M2-like macrophages (MLCs) promoted GSC-driven tumour growth, but silencing PTN expression in MLCs mitigated their pro-tumorigenic activity. The PTN receptor PTPRZ1 is preferentially expressed in GSCs and also predicts GBM poor prognosis. Disrupting PTPRZ1 abrogated GSC maintenance and tumorigenic potential. Moreover, blocking the PTN-PTPRZ1 signalling by shRNA or anti-PTPRZ1 antibody potently suppressed GBM tumour growth and prolonged animal survival. Our study uncovered a critical molecular crosstalk between TAMs and GSCs through the PTN-PTPRZ1 paracrine signalling to support GBM malignant growth, indicating that targeting this signalling axis may have therapeutic potential.