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Background: Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain.Methods: Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgamma(null) (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro.Results: Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects.Conclusions: Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain. [ABSTRACT FROM AUTHOR]

Abstract: Factor IX (FIX) plays an important role in the blood coagulation cascade. When Chinese hamster ovary (CHO) cells producing recombinant human FIX were cultivated using a serum-free medium (SFM) containing 1.12mM of Ca2+ and 0.82mM of Mg2+, a significant amount of active FIX (aFIX) was converted into undesirable activated FIX (FIXa) in the later phase of batch culture. In an effort to improve aFIX production from CHO cells, the effect of Ca2+ and Mg2+ concentrations in the culture medium on the activation of aFIX to FIXa was investigated using SFM with various concentrations of Ca2+ and Mg2+ in the range of 0–1.0mM. The highest aFIX concentration of 1.36IU/mL was obtained at 1.0mM Ca2+ and 1.0mM Mg2+, but the activation of aFIX to FIXa in the later phase of culture was rapid and significant. In contrast, at 0.5mM Ca2+ and 1.0mM Mg2+, the aFIX concentration of 1.33IU/mL was obtained and did not decrease significantly in the later phase of culture. Taken together, lowering Ca2+ concentration from 1.0 to 0.5mM inhibits the activation of aFIX to FIXa in the later phase of culture, fortifying the robustness of downstream bioprocessing. [Copyright &y& Elsevier]

Background/aim: The aim of our study was to investigate the pharmacokinetics (PK), tissue distribution and toxicity of F11 antibody to semaphorin 3A in mouse models and explore its anti-angiogenic and tumor-inhibitory effect., Materials and Methods: Patient-derived xenograft (PDX) models were established via subcutaneous implantation of glioblastoma multiforme (GBM) cells and treated with F11., Results: F11 significantly attenuated tumor growth and angiogenesis in the GBM PDX model. Within the range of administered doses, the PK of F11 in serum demonstrated a linear fashion, consistent with general PK profiles of soluble antigen-targeting antibodies. Additionally, the clearance level was detected at between 4.63 and 7.12 ml/d/kg, while the biological half-life was measured at 6.9 and 9.4 days. Tissue distribution of F11 in kidney, liver and heart was consistent with previously reported antibody patterns. However, the presence of F11 in the brain was an interesting finding., Conclusion: Collectively, our results revealed angiogenic and tumor-inhibitory effect of F11 antibody and its potential therapeutic use within a clinical framework based on PK, biodistribution and toxicity evaluation in mouse models., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed., Methods: We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells., Results: USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice., Conclusion: USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glioblastoma (GBM) is the most lethal brain cancer with profound genomic alterations. While the bona fide tumor suppressor genes such as PTEN, NF1, and TP53 have high frequency of inactivating mutations, there may be the genes with GBM-suppressive roles for which genomic mutation is not a primary cause for inactivation. To identify such genes, we employed in vivo RNAi screening approach using the patient-derived GBM xenograft models. We found that Nemo-Like Kinase (NLK) negatively regulates mesenchymal activities, a characteristic of aggressive GBM, in part via inhibition of WNT/β-catenin signaling. Consistent with this, we found that NLK expression is especially low in a subset of GBMs that harbors high WNT/mesenchymal activities. Restoration of NLK inhibited WNT and mesenchymal activities, decreased clonogenic growth and survival, and impeded tumor growth in vivo. These data unravel a tumor suppressive role of NLK and support the feasibility of combining oncogenomics with in vivo RNAi screen.

Glioblastoma multiforme (GBM) is the most common human primary brain malignancy and has a dismal prognosis. Aggressive treatments using maximal surgical resection, radiotherapy, and temozolomide result in median survival of only 14.6 months in patients with GBM. Numerous clinical approaches using small molecule inhibitors have shown disappointing results because of the genetic heterogeneity of GBM. The epithelial to mesenchymal transition (EMT) is a crucial biological process occurring in the early development stages of many species. However, cancer cells often obtain the ability to invade and metastasize through the EMT, which triggers the scattering of cells. The hepatocyte growth factor (HGF)/MET signaling pathway is indicative of the EMT during both embryogenesis and the invasive growth of tumors, because HGF potently induces mesenchymal transition in epithelial-driven cells. Activation of MET signaling or co-overexpression of HGF and MET frequently represents aggressive growth and poor prognosis of various cancers, including GBM. Thus, efforts to treat cancers by inhibiting MET signaling using neutralizing antibodies or small molecule inhibitors have progressed during the last decade. In this review, we discuss HGF/MET signaling in the development of diseases, including cancers, as well as updates on MET inhibition therapy.

Purpose: Circulating endothelial cells (CEC) have been widely used as a prognostic biomarker and regarded as a promising strategy for monitoring the response to treatment in several cancers. However, the presence and biologic roles of CECs have remained controversial for decades because technical standards for the identification and quantification of CECs have not been established. Here, we hypothesized that CECs detected by flow cytometry might be monocytes rather than endothelial cells., Experimental Design: The frequency of representative CEC subsets (i.e., CD45(-)/CD31(+), CD45(-)/CD31(+)/CD146(+), CD45(-)/CD31(+)/CD105(+)) was analyzed in the peripheral blood of patients with gynecologic cancer (n = 56) and healthy volunteers (n = 44). CD45(-)/CD31(+) cells, which are components of CECs, were isolated and the expression of various markers (CD146, CD105, vWF, and CD144 for endothelial cells; CD68 and CD14 for monocytes) was examined by immunocytochemistry., Results: CD45(-)/CD31(+)/CD105(+) cells were significantly increased in the peripheral blood of patients with cancer, whereas evaluation of CD45(-)/CD31(+)/CD146(+) cells was not possible both in patients with cancer and healthy controls due to the limited resolution of the flow cytometry. Immunocytochemistry analyses showed that these CD45(-)/CD31(+)/CD105(+) cells did not express vWF and CD146 but rather CD144. Furthermore, CD45(-)/CD31(+)/CD105(+) cells uniformly expressed the monocyte-specific markers CD14 and CD68. These results suggest that CD45(-)/CD31(+)/CD105(+) cells carry the characteristics of monocytes rather than endothelial cells., Conclusions: Our data indicate that CD45(-)/CD31(+)/CD105(+) circulating cells, which are significantly increased in the peripheral blood of patients with gynecologic cancer, are monocytes rather than endothelial cells. Further investigation is required to determine the biologic significance of their presence and function in relation with angiogenesis., (©2013 AACR.)

Lipocalin 2 (Lcn2) has been reported to induce cellular proliferation based on its expression in a variety of proliferative cells. Consistent with these findings, the present study demonstrates a significant increase in Lcn2 levels in human hepatocellular carcinoma (HCC) tissues compared with non-tumor liver tissues. However, the role of Lcn2 in hepatocarcinogenesis is far from clear. To investigate the effects of Lcn2 expression on hepatocarcinogenesis, Chang liver and SK-Hep1 HCC cell lines were genetically manipulated to express Lcn2, and the effects on the proliferation and invasion of HCC cells were analyzed. Ectopic expression of Lcn2 in HCC cells significantly inhibited the growth of HCC cells in vitro and in vivo, reduced the invasive potential of cells, and inhibited the expression of matrix metalloproteinase 2 (MMP-2). Lcn2 may exert its function partly through the inhibition of the c-Jun N-terminal kinase (JNK) and phospha-tidyl inositol 3'-kinase (PI3K)/Akt signaling pathways in HCC cells. The selective inhibition of these pathways using pharmacological inhibitors significantly inhibited proliferation, invasion and MMP-2 expression, whereas Lcn2 expression suppressed the JNK and PI3K/Akt pathways. Collectively, these results clearly indicate that Lcn2 may play a protective role against the progression of HCCs by suppressing cell proliferation and invasion. The clinical significance of the present findings should be evaluated further.

We describe the development of an efficient expression system suitable for the stable expression of recombinant genes in Chinese hamster ovary (CHO) cells using the human interferon beta SAR element. The insertion of two copies of the human interferon beta SAR element at the 5' and 3' flanking regions of the beta-galactosidase reporter gene increased the frequency of beta-galactosidase positive colonies by up to 75% and enhanced beta-galactosidase expression by 15- to 20-fold after G418 selection or 30- to 40-fold at the initial stage of the MTX selection procedure. Deletion analysis showed that the whole DNA regions of the human interferon beta SAR element are required for beta-galactosidase expression enhancement. The developed expression system was also highly effective at enhancing the stable expression of two therapeutically important proteins, namely, erythropoietin (EPO) and hepatocyte growth factor (HGF). We isolated stable colonies with expression levels of 47 microg/10(6) cells/day for EPO and 13 microg/10(6) cells/day for HGF, suggesting that the developed expression system based on the human beta SAR element is suitable for expressing high levels of recombinant proteins in CHO cells.