Vasorin promotes endothelial differentiation of glioma stem cells via stimulating the transcription of VEGFR2.

Gliomas are highly vascularized malignancies, but current anti-angiogenic treatments have not demonstrated practical improvements in patient survival. Studies have suggested that glioma-derived endothelial cell (GdEC) formed by glioma stem cell (GSC) differentiation may contribute to the failure of this treatment. However, the molecular mechanisms involved in GSC endothelial differentiation remain poorly understood. We previously reported that vasorin (VASN) is highly expressed in glioma and promotes angiogenesis. Here, we show that VASN expression positively correlates with GdEC signatures in glioma patients. VASN promotes the endothelial differentiation capacity of GSC in vitro and participates in the formation of GSC-derived vessels in vivo. Mechanistically, vascular endothelial growth factor receptor 2 (VEGFR2) is a critical factor that mediates the regulation of VASN on GSC endothelial differentiation. Separation of cell chromatin fractionation and chromatin immunoprecipitation-sequencing analysis show that VASN interacts with Notch1 and co-translocates into the cell nuclei, where VASN binds to the VEGFR2 gene promoter to stimulate its transcription during the progression of GSC differentiation into GdEC. Together, these findings elucidate the role and mechanisms of VASN in promoting the endothelial differentiation of GSC and suggest VASN as a potential target for anti-angiogenic therapy based on intervention in GdEC formation in gliomas.

Vasorin exocytosed from glioma cells facilitates angiogenesis via VEGFR2/AKT signaling pathway.

Glioma is a highly vascularized tumor of the central nervous system. Angiogenesis plays a predominant role in glioma progression and is considered an important therapeutic target. Our previous study showed that vasorin (VASN), a transmembrane protein, is overexpressed in glioma and promotes angiogenesis; however, the potential mechanism remains unclear. In this study, we found that human vascular endothelial cells (hECs) co-cultured with VASN-overexpressing glioma cells exhibited accelerated migration ability and increased expression of VASN originated from glioma cells. VASN was found in exosomes secreted by glioma cells and could be taken up by hECs. hECs showed more edge filopodia and significantly upregulated expression of endothelial tip cell marker gene and protein levels after co-culture with VASN-overexpressing glioma cells. In clinical glioma tissue and orthotopic transplantation glioma tissue, the vascular density and the number of vascular endothelial cells with a tip cell phenotype in VASN-overexpressed tissues were significantly higher than in tissues with low expression. At the molecular level, VASN interacted with VEGFR2 and caused internalization and autophosphorylation of VEGFR2 protein, and then activated the AKT signaling pathway. Our study collectively reveals the function and mechanism of VASN in facilitating angiogenesis in glioma, providing a new therapeutic target for glioma. Implications: These findings demonstrate that VASN exocytosed from glioma cells enhanced the migration of vascular endothelial cells by VEGFR2/AKT signaling pathway.

Nuclear VCP drives colorectal cancer progression by promoting fatty acid oxidation.

Fatty acid oxidation (FAO) fuels many cancers. However, knowledge of pathways that drive FAO in cancer remains unclear. Here, we revealed that valosin-containing protein (VCP) upregulates FAO to promote colorectal cancer growth. Mechanistically, nuclear VCP binds to histone deacetylase 1 (HDAC1) and facilitates its degradation, thus promoting the transcription of FAO genes, including the rate-limiting enzyme carnitine palmitoyltransferase 1A (CPT1A). FAO is an alternative fuel for cancer cells in environments exhibiting limited glucose availability. We observed that a VCP inhibitor blocked the upregulation of FAO activity and CPT1A expression triggered by metformin in colorectal cancer (CRC) cells. Combined VCP inhibitor and metformin prove more effective than either agent alone in culture and in vivo. Our study illustrates the molecular mechanism underlying the regulation of FAO by nuclear VCP and demonstrates the potential therapeutic utility of VCP inhibitor and metformin combination treatment for colorectal cancer.

Targeting VCP potentiates immune checkpoint therapy for colorectal cancer.

Immune checkpoint blockade therapies are still ineffective for most patients with colorectal cancer (CRC). Immunogenic cell death (ICD) enables the release of key immunostimulatory signals to drive efficient anti-tumor immunity, which could be used to potentiate the effects of immune checkpoint inhibitors. Here, we showed that inhibition of valosin-containing protein (VCP) elicits ICD in CRC. Meanwhile, VCP inhibitor upregulates PD-L1 expression and compromises anti-tumor immunity in vivo. Mechanistically, VCP transcriptionally regulates PD-L1 expression in a JAK1-dependent manner. Combining VCP inhibitor with anti-PD1 remodels tumor immune microenvironment and reduces tumor growth in mouse models of CRC. Addition of oncolytic virus further augments the therapeutic activity of the combination regimen. Our study shows the molecular mechanism for regulating PD-L1 expression by VCP and suggests that inhibition of VCP has the potential to increase the efficacy of immunotherapy in CRC.

IDH1 mutation impairs antiviral response and potentiates oncolytic virotherapy in glioma.

IDH1 mutations frequently occur early in human glioma. While IDH1 mutation has been shown to promote gliomagenesis via DNA and histone methylation, little is known regarding its regulation in antiviral immunity. Here, we discover that IDH1 mutation inhibits virus-induced interferon (IFN) antiviral responses in glioma cells. Mechanistically, D2HG produced by mutant IDH1 enhances the binding of DNMT1 to IRF3/7 promoters such that IRF3/7 are downregulated, leading to impaired type I IFN response in glioma cells, which enhances the susceptibility of gliomas to viral infection. Furthermore, we identify DNMT1 as a potential biomarker predicting which IDH1mut gliomas are most likely to respond to oncolytic virus. Finally, both D2HG and ectopic mutant IDH1 can potentiate the replication and oncolytic efficacy of VSVΔ51 in female mouse models. These findings reveal a pivotal role for IDH1 mutation in regulating antiviral response and demonstrate that IDH1 mutation confers sensitivity to oncolytic virotherapy.

Glioblastoma Vascular Plasticity Limits Effector T-cell Infiltration and Is Blocked by cAMP Activation.

Glioblastoma (GBM) is the deadliest form of brain cancer. It is a highly angiogenic and immunosuppressive malignancy. Although immune checkpoint blockade therapies have revolutionized treatment for many types of cancer, their therapeutic efficacy in GBM has been far less than expected or even ineffective. In this study, we found that the genomic signature of glioma-derived endothelial cells (GdEC) correlates with an immunosuppressive state and poor prognosis of patients with glioma. We established an in vitro model of GdEC differentiation for drug screening and used this to determine that cyclic adenosine monophosphate (cAMP) activators could effectively block GdEC formation by inducing oxidative stress. Furthermore, cAMP activators impaired GdEC differentiation in vivo, normalized the tumor vessels, and altered the tumor immune profile, especially increasing the influx and function of CD8+ effector T cells. Dual blockade of GdECs and PD-1 induced tumor regression and established antitumor immune memory. Thus, our study reveals that endothelial transdifferentiation of GBM shapes an endothelial immune cell barrier and supports the clinical development of combining GdEC blockade and immunotherapy for GBM. See related Spotlight by Lee et al., p. 1300.

Activated KRAS reprograms neural progenitor cells to glioma stem cell­like phenotype.

Glioma is the most common primary brain tumor. Glioma stem cells (GSCs) are the origin of gliomagenesis and may develop from normal neural progenitor cells (NPCs). However, how neoplastic transformation occurs in normal NPCs and the role of the Ras/Raf/MAPK pathway in NPC transformation is unclear. The present study generated NPCs from human embryonic stem cells (ESCs) carrying gene alterations in the Ras/Raf/MAPK pathway. The CCK­8 proliferation, single­cell clonal expansion, cell migration, RT­qPCR, immunofluorescence staining, western blotting, transcriptome and Seahorse analyses, and intracranial implantation assay were performed to identify the characterization of transformed NPCs in vitro and in vivo. Brain organoids were used to verify the phenotypes transforming in NPCs. KRAS­activated NPCs exhibited increased proliferation and migration in vitro. KRAS­activated NPCs showed atypical morphology and formed aggressive tumors in immunodeficient mice. At the molecular level, KRAS­activated NPCs displayed neoplasm­associated metabolic and gene expression profiles. Moreover, activation of KRAS led to substantial cell proliferation and abnormal structure in ESC­derived brain organoids. The present study showed that activated KRAS transformed normal NPCs to GSC­like cells and established a simple cellular model to investigate gliomagenesis.

MELK predicts poor prognosis and promotes metastasis in esophageal squamous cell carcinoma via activating the NF­κB pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide with a low 5­year survival rate due to the lack of effective therapeutic strategies. Accumulating evidence has indicated that maternal embryonic leucine zipper kinase (MELK) is highly expressed in several tumors and associated with tumor development. However, the biological effects of MELK in ESCC remain unknown. In the present study, cell phenotypical experiments and animal metastasis assays were performed to detect the influence of MELK knockdown in vitro and in vivo. The potential molecular mechanism of MELK­mediated ESCC metastasis was further investigated by western blotting and immunofluorescence staining. The results revealed that the expression of MELK in human ESCC tissues was higher than that in adjacent normal tissues and was positively associated with the poor prognosis of patients. Reducing MELK expression resulted in growth inhibition and suppression of the invasive ability of ESCC cells in vitro and in vivo. MELK inhibition induced alterations of epithelial­mesenchymal transition­associated proteins. Mechanistically, MELK interacted with IκB kinase (IKK) and promoted the phosphorylation of IKK, by which MELK regulated activation of the NF­κB pathway. Collectively, the present study revealed the function and mechanism of MELK in the cell metastasis of ESCC, which may be a potential therapeutic target for ESCC.

Selective Single-Cell Expansion on a Microfluidic Chip for Studying Heterogeneity of Glioma Stem Cells.

Accumulating evidence suggests that a subpopulation of stem-cell-like tumor cells in glioma (GSCs) is the major factor accounting for intratumoral heterogeneity and acquired chemotherapeutic resistance. Therefore, understanding intratumoral heterogeneity of GSCs may help develop more effective treatments against this malignancy. However, the study of GSCs' heterogeneity is highly challenging because tumor stem cells are rare. To overcome the limitation, we employed a microfluidic single-cell culture approach to expand GSCs by taking advantage of the self-renewal property of stem cells. Stemness of the recovered cells was confirmed by immunofluorescence, RT-PCR, RNA-sequencing, and cell function assays. The recovered cells were classified into three groups based on their morphological characteristics, namely, the tight-format (TF), the loose-format (LF), and the limited-size group (LS). The serial passage assay showed that the LS group has a lower sphere-forming rate than the LF and TF group, and the invasion assay showed that the LF and TF cells migrated longer distances in Matrigel. The transcriptomic analysis also revealed differences in gene expression profiling among these GSC subtypes. The abovementioned results suggest that GSCs have transcriptional and functional heterogeneities that correlate with morphological differences. The presented microfluidic single-cell approach links morphology with function and thus can provide an enabling tool for studying tumor heterogeneity.

Tetrahydropalmatine has a therapeutic effect in a lipopolysaccharide-induced disseminated intravascular coagulation model.

OBJECTIVES: The aim of this study was to determine the therapeutic effects of tetrahydropalmatine (Tet) on disseminated intravascular coagulation (DIC) by exploring the role of Tet using a lipopolysaccharide (LPS)-induced DIC model. Methods/Materials: We established a mouse DIC model by injecting LPS. Hematoxylin-eosin (HE) staining was performed to detect liver and kidney damage. Blood samples were obtained to determine liver and kidney injury indexes, coagulation indexes, and inflammatory cytokines. An in vitro cell inflammation model was also established. Tumor necrosis factor-α (TNF-α) levels and nuclear factor kappa B (NF-κB) signaling pathway activation were determined by western blot. RESULT: Tet ameliorated the damage to organ tissues, improved coagulation indexes, and reduced the inflammatory cytokine production in LPS-induced mouse DIC. Tet also inhibited TNF-α expression by suppressing NF-κB signaling pathway activation in an in vitro LPS model using RAW 264.7 macrophages. CONCLUSIONS: Tet has a mitigating and therapeutic effect on the LPS-induced DIC model via anticoagulant and anti-inflammatory effects, showing its potential as an adjunct to DIC treatment.

DNA-PK inhibition synergizes with oncolytic virus M1 by inhibiting antiviral response and potentiating DNA damage.

Oncolytic virotherapy is a promising therapeutic strategy that uses replication-competent viruses to selectively destroy malignancies. However, the therapeutic effect of certain oncolytic viruses (OVs) varies among cancer patients. Thus, it is necessary to overcome resistance to OVs through rationally designed combination strategies. Here, through an anticancer drug screening, we show that DNA-dependent protein kinase (DNA-PK) inhibition sensitizes cancer cells to OV M1 and improves therapeutic effects in refractory cancer models in vivo and in patient tumour samples. Infection of M1 virus triggers the transcription of interferons (IFNs) and the activation of the antiviral response, which can be abolished by pretreatment of DNA-PK inhibitor (DNA-PKI), resulting in selectively enhanced replication of OV M1 within malignancies. Furthermore, DNA-PK inhibition promotes the DNA damage response induced by M1 virus, leading to increased tumour cell apoptosis. Together, our study identifies the combination of DNA-PKI and OV M1 as a potential treatment for cancers.

Pericardial application as a new route for implanting stem-cell cardiospheres to treat myocardial infarction.

KEY POINTS: Cardiospheres (CSps) are a promising new form of cardiac stem cells with advantage over other stem cells for myocardial regeneration, but direct implantation of CSps by conventional routes has been limited due to potential embolism. We have implanted CSps into the pericardial cavity and systematically demonstrated its efficacy regarding myocardial infarction. Stem cell potency and cell viability can be optimized in vitro prior to implantation by pre-conditioning CSps with pericardial fluid and hydrogel packing. Transplantation of optimized CSps into the pericardial cavity improved cardiac function and alleviated myocardial fibrosis, increased myocardial cell survival and promoted angiogenesis. Mechanistically, CSps are able to directly differentiate into cardiomyocytes in vivo and promote regeneration of myocardial cells and blood vessels through a paracrine effect with released growth factors as potential paracrine mediators. These findings establish a new strategy for therapeutic myocardial regeneration to treat myocardial infarction. ABSTRACT: Cardiospheres (CSps) are a new form of cardiac stem cells with an advantage over other stem cells for myocardial regeneration. However, direct implantation of CSps by conventional routes to treat myocardial infarction has been limited due to potential embolism. We have implanted CSps into the pericardial cavity and systematically assessed its efficacy on myocardial infarction. Preconditioning with pericardial fluid enhanced the activity of CSps and matrix hydrogel prolonged their viability. This shows that pretransplant optimization of stem cell potency and maintenance of cell viability can be achieved with CSps. Transplantation of optimized CSps into the pericardial cavity improved cardiac function and alleviated myocardial fibrosis in the non-infarcted area, and increased myocardial cell survival and promoted angiogenesis in the infarcted area. Mechanistically, CSps were able to directly differentiate into cardiomyocytes in vivo and promoted regeneration of myocardial cells and blood vessels in the infarcted area through a paracrine effect with released growth factors in pericardial cavity serving as possible paracrine mediators. This is the first demonstration of direct pericardial administration of pre-optimized CSps, and its effectiveness on myocardial infarction by functional and morphological outcomes with distinct mechanisms. These findings establish a new strategy for therapeutic myocardial regeneration to treat myocardial infarction.

ClC-3 chloride channel protein induces G1 arrest in hepatocellular carcinoma Hep3B cells.

ClC-3 is a type of chloride channel that has multiple functions in tumorigenesis and tumor growth, and can be blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). In the present study, we found that DIDS inhibited the proliferation of Hep3B hepatocellular carcinoma (HCC) cells in a concentration-dependent manner. More in-depth research demonstrated that DIDS downregulated the protein expression levels of cyclin D1 and cyclin E, which are key proteins of the G1 phase. Additionally, we found that ClC-3 siRNA transfection induced G1 arrest in the Hep3B cells, confirming that ClC-3 is involved in the DIDS-induced inhibition of Hep3B cells. Moreover, the level of α-fetoprotein (AFP), a negative prognostic indicator of HCC, was decreased after treatment with DIDS and ClC-3 siRNA. In conclusion, we demonstrated that ClC-3 can arrest the cell cycle at the G1 phase to inhibit cell proliferation, suggesting that ClC-3 has the potential to be a novel target for HCC therapy and potentially improve the prognosis of HCC patients.

Preparation of high bioactivity multilayered bone-marrow mesenchymal stem cell sheets for myocardial infarction using a 3D-dynamic system.

Cell sheet techniques offer a promising future for myocardial infarction (MI) therapy; however, insufficient nutrition supply remains the major limitation in maintaining stem cell bioactivity in vitro. In order to enhance cell sheet mechanical strength and bioactivity, a decellularized porcine pericardium (DPP) scaffold was prepared by the phospholipase A2 method, and aspartic acid was used as a spacer arm to improve the vascular endothelial growth factor crosslink efficiency on the DPP scaffold. Based on this scaffold, multilayered bone marrow mesenchymal stem cell sheets were rapidly constructed, using RAD16-I peptide hydrogel as a temporary 3D scaffold, and cell sheets were cultured in either the 3D-dynamic system (DCcs) or the traditional static condition (SCcs). The multilayered structure, stem cell bioactivity, and ultrastructure of DCcs and SCcs were assessed. The DCcs exhibited lower apoptosis, lower differentiation, and an improved paracrine effect after a 48 h culture in vitro compared to the SCcs. Four groups were set to evaluate the cell sheet effect in rat MI model: sham group, MI control group, DCcs group, and SCcs group. The DCcs group improved cardiac function and decreased the infarcted area compared to the MI control group, while no significant improvements were observed in the SCcs group. Improved cell survival, angiogenesis, and Sca-1+ cell and c-kit+ cell amounts were observed in the DCcs group. In conclusion, the DCcs maintained higher stem cell bioactivity by using the 3D-dynamic system to provide sufficient nutrition, and transplanting DCcs significantly improved the cardiac function and angiogenesis. STATEMENT OF SIGNIFICANCE: This study provides an efficient method to prepare vascular endothelial growth factor covalent decellularized pericardium scaffold with aspartic acid, and a multilayered bone marrow mesenchymal stem cell (BMSC) sheet is constructed on it using a 3D-dynamic system. The dynamic nutrition supply showed a significant benefit on BMSC bioactivity in vitro, including decreasing cell apoptosis, reducing stem cell differentiation, and improving growth factor secretion. These favorable bioactivity improved BMSC survival, angiogenesis, and cardiac function of the infarcted myocardium. The study highlights the importance of dynamic nutrition supply on maintaining stem cell bioactivity within cell sheet, and it stresses the necessity and significance of setting a standard for assessing cell sheet products before transplantation in the future application.

A novel fibrinogenase from Agkistrodon acutus venom protects against LPS-induced endotoxemia via regulating NF-κB pathway.

CONTEXT: Endotoxins including lipopolysaccharide (LPS) could cause endotoxemia which often results in excessive inflammation, organ dysfunction, sepsis, disseminated intravascular coagulation (DIC) or even death. Previously, a novel fibrinogenase (FII) showed protective effects on LPS-induced DIC via activating protein C and suppressing inflammatory cytokines. OBJECTIVE: To evaluate whether FII has protective effect on LPS-induced endotoxemia in mice and learn about the role of NF-κB pathway in TNF-α producing process. METHODS: BALB/C mice were intraperitoneally injected (i.p.) with (a) 30 mg/kg LPS, (b) LPS + 0.3 mg/kg FII, (c) LPS + 1.0 mg/kg FII, (d) LPS + 3.0 mg/kg FII or (e) saline. Both survival rate and organ function were tested, including alanine aminotransferase (ALT), blood urine nitrogen (BUN) and tissue section, and TNF-α was examined by ELISA. RAW 264.7 macrophage was administered with (a) LPS, (b) LPS + FII, (c) FII alone or (d) saline, and TNF-α and phosphorylation (P)-NF-κB (P65) were determined by Western blot. RESULTS: The administration of LPS led to 65% mortality rate, a rise of serum TNF-α, BUN and ALT levels, and both liver and renal tissue damage were observed. While FII treatment significantly increased the survival rate of LPS-induced endotoxemia mice model, histopathology and protein analysis results also revealed that FII remarkably protected liver and renal from LPS damage as well as decreasing TNF-α level. In vitro, FII significantly decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65). CONCLUSIONS: Our findings suggested that FII had protective effect on LPS-induced endotoxemia and organ injuries by suppressing the activation of NF-κB which decreased TNF-α level.