Eiichi Nakai

Enhanced MDR1 Expression and Chemoresistance of Cancer Stem Cells Derived from Glioblastoma

ABSTRACT We established a cancer stem (CS) cell line, U87CS, by means of spheroid culture of U87MG cells derived from glioblastoma (GBM) in neuronal stem cell medium. U87CS cells presented positive immunohistochemical staining for multidrug resistance (MDR)1 and CD133, a marker for a subset of leukemia and GBM CS cells. The gene expression of MDR1 and CD133 on U87CS cells increased by an average of 8.51 and 47.18 times, respectively, compared to the levels on U87MG cells by real-time quantitative RT-PCR. U87CS cells possessed stronger drug-resistance to conventional anti-cancer drugs, such as doxorubicin (Dox), etoposide (VP-16), carboplastin, and BCNU than U87MG cells. Double immunofluoresence staining showed co-expression of MDR1 and CD133 on U87CS cells transplanted into nude mice brains. In addition, we identified the crossreactivity of CD133 and MDR1 in a surgical specimen of GBM. Our results suggest that CS cells may be resistant to current chemotherapy and represent a novel target for GBM therapeutics.

Characterization of microglia induced from mouse embryonic stem cells and their migration into the brain parenchyma.

We derived microglia from mouse embryonic stem cells (ES cells) at very high density. Using the markers Mac1(+)/CD45(low) and Mac1(+)/CD45(high) to define microglia and macrophages, respectively, we show that Mac1(+) cells are induced by GM-CSF stimulation following neuronal differentiation of mouse ES cells using a five-step method. CD45(low) expression was high and CD45(high) expression was low on induced cells. We used a density gradient method to obtain a large amount of microglia-like cells, approximately 90% of Mac1(+) cells. Microglia-like cells expressed MHC class I, class II, CD40, CD80, CD86, and IFN-gammaR. The expression level of these molecules on microglia-like cells was barely enhanced by IFN-gamma. Intravenously transferred GFP(+) microglia derived from GFP(+) ES cells selectively accumulated in brain but not in peripheral tissues such as spleen and lymph node. GFP(+) cells were detected mainly in corpus callosum and hippocampus but were rarely seen in cerebral cortex, where Iba1, another marker of microglia, is primarily expressed. Furthermore, both GFP(+) and Iba1(+) cells exhibited a ramified morphology characteristic of mature microglia. These studies suggest that ES cell-derived microglia-like cells obtained using our protocol are functional and migrate selectively into the brain but not into peripheral tissues after intravenous transplantation.

Enhanced expression of cancer testis antigen genes in glioma stem cells

Cancer stem cells are an important target for effective therapy, since they show tumorigenicity, chemoresistance, and radioresistance. We isolated cancer stem cells from glioma cell lines and tissues and examined the expression of cancer testis antigen (CTA) genes as potential target molecules for cancer vaccine therapy. CTA genes were highly and frequently expressed in cancer stem cells compared with differentiated cells. In addition, histone acetylation levels in the promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, while DNA methylation analysis of the promoter regions revealed hypomethylation in cancer stem cells. This epigenetic difference between cells leads to heterogeneous expression of CTA genes in the tumor mass, which consists of cells at various levels of differentiation. Moreover, the expression level of HLA class I antigens was not affected by the differentiation status, suggesting that CTA genes may present as surface antigens in cancer stem cells. Taken together, these findings suggest that CTA genes may be attractive candidates for targeted vaccine therapy against cancer stem cells in glioma patients.

Evaluation of two- and three-dimensional visualization for endoscopic endonasal surgery using a novel stereoendoscopic system in a novice: a comparison on a dry laboratory model

BackgroundThree-dimensional (3-D) stereoscopic vision is theoretically superior to two-dimensional (2-D) vision in endoscopic endonasal surgery. However, only few reports have quantitatively compared endoscopic performance under the two visual conditions. We introduced a newly designed stereoendoscopic system with a “dual-lens and single camera” for endoscopic endonasal surgery and objectively compared the performances under 3-D and high-definition 2-D visualizations on a dry laboratory model.MethodsThirty subjects without experience performing endoscopic surgery, computer-simulated training or any 3-D video system were recruited and divided into two groups (Group A and Group B) for performing two different tasks. The novel 4.7-mm-diameter stereoendoscope provided high-definition (HD) images. In Task 1, Group A started the task under the 3-D condition followed by the 2-D condition, and Group B vice versa. In Task 2, Group A started the task under the 2-D condition followed by the 3-D condition, and Group B vice versa. The performance accuracy and speed under the two visual conditions were analyzed.ResultsSignificant improvement in performance accuracy and speed was seen under 3-D conditions in the both “3-D first” and “2-D first” subgroups during both tasks (P < .001). Regardless of order, the inaccuracy rate and performance time under 3-D conditions was significantly lower than that under 2-D conditions in each subject.ConclusionsWe demonstrated the advantage of 3-D visualization over 2-D visualization for inexperienced subjects. Further quantitative clinical studies are required to confirm whether stereoendoscopy actually provides benefits in clinical settings.

Elevated cell invasion in a tumor sphere culture of RSV-M mouse glioma cells.

Cancer stem cells (CSCs) are the sole population possessing high self-renewal activity in tumors, with their existence affecting tumor recurrence. However, the invasive activity of CSCs has yet to be fully understood. In this article, we established a tumor sphere culture of RSV-M mouse glioma cells (RSV-M-TS) and evaluated their migration and invasion activities. Histological analysis of a tumor formed by cranial injection of the RSV-M-TS cells showed highly invasive properties and similarities with human malignant glioma tissues. When the migration activity of both RSV-M and RSV-M-TS cells were compared by intracranial injection, rapid migration of RSV-M-TS cells was observed. To confirm the invasive capabilities of RSV-M-TS cells, a three-dimensional collagen invasion assay was performed in vitro using RSV-M, RSV-M-TS, and RSV-M-TS cells cultured with medium containing serum. RSV-M and RSV-M-TS cultured with medium containing serum for 8 days indicated low migration activity, while moderate invasion activity was observed in RSV-M-TS cells. This activity was further enhanced by incubation with medium containing serum overnight. To identify the genes involved in this invasion activity, we performed quantitative polymerase chain reaction (PCR) array analysis of RSV-M and RSV-M-TS cells. Of 84 cancer metastasis-related genes, up-regulation was observed in 24 genes, while 4 genes appeared to be down-regulated in RSV-M-TS cells. These results suggest that the enhanced invasive activity of glioma sphere cells correlates with a number of tumor metastasis-related genes and plays a role in the dissemination and invasion of glioma cells.

Use of fat-suppressed T2 -weighted sagittal images after infusion of excess saline into the subarachnoid space as a new diagnostic modality for cerebrospinal fluid hypovolemia: technical note

The diagnosis of CSF hypovolemia remains controversial. The primary diagnostic factor relies on confirmation of leakage of the CSF based on reduced spinal fluid pressure. Determining the specific leakage site is the most important issue for effective treatment but remains a difficult task. Although CT myelography, radioisotope cisternography, and MRI are commonly performed in the diagnosis of CSF hypovolemia, these techniques can rarely identify the precise leakage site. Therefore, an epidural blood patch is performed in the lumbar spine in many cases. This study reports a new diagnostic modality that can help to confirm the leakage site. Fat-suppressed T2-weighted sagittal images were compared before and after the infusion of 20 ml of saline into the subarachnoid space of the lumbar region to detect the specific leakage site with high probability. Three patients were successfully treated by the epidural blood patch based on data obtained with the new diagnostic modality. Two patients were treated in the cervical region and 1 in the lumbar region. The use of fat-suppressed T2-weighted sagittal images after saline infusion could be a relevant diagnostic modality compared with images obtained by CT myelography, radioisotope cisternography, and ordinary MRI to achieve accurate diagnosis and effective treatment of patients with CSF hypovolemia.

P3-p22 Role of diffusion tensor imaging and visual evoked potentials in brain tumor surgery adjacent to the optic pathway

It has been reported that low-power laser irradiation (LLI) can modulate various biological processes including cell proliferation. Some reports suggest LLI interferes with cell cycle and inhibits cell proliferation, while others suggest LLI has a stimulatory effect. Mechanism underlying the effect of LLI remains unclear. We have reported that 808 nm (60 mW) LLI has a potential of suppressive effect on the cell proliferation of human-derived glioblastoma A-172. To reveal a wavelength dependency of such an effect, the present studies using 2 other kinds of diode laser generators (405 nm with 27 mW and 532 nm with 60 mW) were designed. The A-172 cells were cultivated in culture dishes or a 96-well plate. No irradiation was applied in control-group, whereas in experimental group, the center of dish or the selected well was irradiated for 20, 40 and 60 min, respectively. Cells were cultivated in CO2 incubator for 1 or 2 days. The dishes or wells were photographed at pre-, just post-irradiation, 24 and 48 hrs after irradiation with a digital camera. Cell countings were performed on the PC screen and the ratio of cell proliferation was measured. MTT colorimetric analysis was also applied at 48 hrs after irradiation and viable cells and cell proliferation were estimated. These analyses showed that 405 nm LLI provided a significant suppressive effect on the proliferation of A-172 cells (p < 0.05 or p < 0.01), and the effect of LLI had a dose-dependency, whereas 532 nm LLI showed a significant stimulatory effect on them (p < 0.05 or p < 0.01), and also had a dose-dependency. Taking the results of 808 nm (60 mW) application into consideration, it is concluded that LLI effects on A-172 cell proliferation have a wavelength dependency.