Raita Fukaya

Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line, SK-MG-1

Accumulating evidence suggests that in several types of brain tumors, including glioma, only a phenotypic subset of tumor cells called brain cancer stem cells (BCSCs) may be capable of initiating tumor growth. Recently, the isolation of side population (SP) cells using Hoechst dye has become a useful method for obtaining cancer stem cells in various tumors. In this study, we isolated cancer stem-like cells from human glioma cell lines using the SP technique. Flow cytometry analysis revealed that SK-MG-1, a human glioblastoma cell line, contained the largest number of SP cells among the five glioma cell lines that were analyzed. The SP cells had a self-renewal ability and were capable of forming spheres in a neurosphere culture medium containing EGF and FGF2. Spheres derived from the SP cells differentiated into three different lineage cells: neurons, astrocytes and oligodendrocytes. RT-PCR analysis revealed that the SP cells expressed a neural stem cell marker, Nestin. The SP cells generated tumors in the brains of NOD/SCID mice at 8weeks after implantation, whereas the non-SP cells did not generate any tumors in the brain. These results indicate that SP cells isolated from SK-MG-1 possess the properties of cancer stem cells, including their self-renewal ability, multi-lineage differentiation, and tumorigenicity. Therefore, the SP cells from SK-MG-1 may be useful for analyzing BCSCs because of the ease with which they can be handled and their yield.

Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells.

BackgroundHOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies.ResultsIn this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs).ConclusionsOur results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.

Povidone-iodine ointment and gauze dressings associated with reduced catheter-related infection in seriously ill neurosurgical patients.

Povidone-iodine ointment and gauze covered by transparent dressings were compared with transparent dressings alone in historical controls (both changed twice weekly) in neurosurgical patients needing catheter placement for prolonged periods. Colonization and bloodstream infection were both reduced with the new method (P < .01 and P = .062, respectively).

MIF maintains the tumorigenic capacity of brain tumor-initiating cells by directly inhibiting p53

Tumor-initiating cells thought to drive brain cancer are embedded in a complex heterogeneous histology. In this study, we isolated primary cells from 21 human brain tumor specimens to establish cell lines with high tumorigenic potential and to identify the molecules enabling this capability. The morphology, sphere-forming ability upon expansion, and differentiation potential of all cell lines were indistinguishable in vitro However, testing for tumorigenicity revealed two distinct cell types, brain tumor-initiating cells (BTIC) and non-BTIC. We found that macrophage migration inhibitory factor (MIF) was highly expressed in BTIC compared with non-BTIC. MIF bound directly to both wild-type and mutant p53 but regulated p53-dependent cell growth by different mechanisms, depending on glioma cell line and p53 status. MIF physically interacted with wild-type p53 in the nucleus and inhibited its transcription-dependent functions. In contrast, MIF bound to mutant p53 in the cytoplasm and abrogated transcription-independent induction of apoptosis. Furthermore, MIF knockdown inhibited BTIC-induced tumor formation in a mouse xenograft model, leading to increased overall survival. Collectively, our findings suggest that MIF regulates BTIC function through direct, intracellular inhibition of p53, shedding light on the molecular mechanisms underlying the tumorigenicity of certain malignant brain cells. Cancer Res; 76(9); 2813-23. ©2016 AACR.

Our Method of Povidone-Iodine Ointment and Gauze Dressings Reduced Catheter-Related Infection in Serious Cases

In experiment 1, we evaluated our method of catheter care at subclavian vein insertion sites for the control of catheter-related infections in seriously ill neurosurgical patients who needed prolonged catheter placement, compared with an older method. In our method, the insertion site was prepared with 10% povidone-iodine solution, followed by application of 10% povidone-iodine ointment, and covered with sterile gauze and a transparent polyurethane dressing. The older method was based on 1996 guidelines for the prevention of intravascular device-related infections. Catheter colonization and mortality were both found to be significantly reduced with our method (p = 0.0214, p = 0.0379, respectively). In experiment 2, we evaluated whether a regimen of catheter care with 10% povidone-iodine ointment was more effective than that without povidone-iodine ointment for the prevention of infections. This suggested effectiveness of 10% povidone-iodine ointment for reduction of infection. Our method of catheter care was useful even in seriously ill neurosurgical patients.