Isako Saga

IGF1 Receptor Signaling Regulates Adaptive Radioprotection in Glioma Stem Cells

Cancer stem cells (CSCs) play an important role in disease recurrence after radiation treatment as a result of intrinsic properties such as high DNA repair capability and antioxidative capacity. It is unclear, however, how CSCs further adapt to escape the toxicity of the repeated irradiation regimens used in clinical practice. Here, we have exposed a population of murine glioma stem cells (GSCs) to fractionated radiation in order to investigate the associated adaptive changes, with the ultimate goal of identifying a targetable factor that regulates acquired radioresistance. We have shown that fractionated radiation induces an increase in IGF1 secretion and a gradual upregulation of the IGF type 1 receptor (IGF1R) in GSCs. Interestingly, IGF1R upregulation exerts a dual radioprotective effect. In the resting state, continuous IGF1 stimulation ultimately induces downregulation of Akt/extracellular‐signal‐regulated kinases (ERK) and FoxO3a activation, which results in slower proliferation and enhanced self‐renewal. In contrast, after acute radiation, the abundance of IGF1R and increased secretion of IGF1 promote a rapid shift from a latent state toward activation of Akt survival signaling, protecting GSCs from radiation toxicity. Treatment of tumors formed by the radioresistant GSCs with an IGF1R inhibitor resulted in a marked increase in radiosensitivity, suggesting that blockade of IGF1R signaling is an effective strategy to reverse radioresistance. Together, our results show that GSCs evade the damage of repeated radiation not only through innate properties but also through gradual inducement of resistance pathways and identify the dynamic regulation of GSCs by IGF1R signaling as a novel mechanism of adaptive radioprotection. STEM CELLS 2013;31:627–640

Integrated analysis identifies different metabolic signatures for tumor-initiating cells in a murine glioblastoma model

BACKGROUND The metabolic preference of malignant glioma for glycolysis as an energy source is a potential therapeutic target. As a result of the cellular heterogeneity of these tumors, however, the relation between glycolytic preference, tumor formation, and tumor cell clonogenicity has remained unknown. To address this issue, we analyzed the metabolic profiles of isogenic glioma-initiating cells (GICs) in a mouse model. METHODS GICs were established by overexpression of H-Ras(V12) in Ink4a/Arf-null neural stem cells. Subpopulations of these cells were obtained by single-cell cloning, and clones differing in extracellular acidification potential were assessed for metabolic characteristics. Tumors formed after intracranial implantation of these clones in mice were examined for pathological features of glioma and expression of glycolytic enzymes. RESULTS Malignant transformation of neural stem cells resulted in a shift in metabolism characterized by an increase in lactic acid production. However, isogenic clonal populations of GICs manifested pronounced differences in glucose and oxygen consumption, lactate production, and nucleoside levels. These differences were paralleled by differential expression of glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2, with this differential expression also being evident in tumors formed by these clones in vivo. CONCLUSIONS The metabolic characteristics of glioma cells appear early during malignant transformation and persist until the late stages of tumor formation. Even isogenic clones may be heterogeneous in terms of metabolic features, however, suggesting that a more detailed understanding of the metabolic profile of glioma is imperative for effective therapeutic targeting.

Epidural anterior petrosectomy with subdural visualization of sphenobasal vein via the anterior transpetrosal approach—technical case report

The drainage of the superficial middle cerebral vein (SMCV) is classified into four subtypes. The sphenobasal vein (SBV) drains from the SMCV to the pterygoid venous plexus at the temporal skull base. Epidural procedures in the standard anterior transpetrosal approach (ATPA) may damage the route of the SBV. We report a case in which modified surgical procedures via the ATPA were used to preserve the SBV. A 45-year-old man complained of right facial pain. Magnetic resonance images revealed a right cerebellopontine tumor suggestive of an epidermoid cyst. Right carotid angiography revealed that the SMCV drained into the pterygoid venous plexus via the SBV. The convexity dura mater of the temporal lobe was cut and the anterior part of the temporal lobe was retracted subdurally. The SBV was visualized from the subdural side. The basal dura mater of the temporal lobe posterior to the SBV was cut and the posterior part of the temporal lobe was retracted epidurally. After dissecting the dura mater medial to the greater petrosal nerve and to the edge of the petrous apex, the petrous apex was exposed and drilled out without injuring the SBV. The superior petrous sinus and the tentorium were cut. The tumor compressed the root exit zone of the trigeminal nerve. The tumor was grossly totally removed. The modified ATPA (epidural anterior petrosectomy with subdural visualization of the SBV) is effective in preserving the SBV.

Abstract 4342: Crosstalk between initiating cells with different metabolism in a murine model of malignant glioma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background The metabolic preference of malignant glioma for glycolysis as an energy source is a potential therapeutic target. As a result of the cellular heterogeneity of these tumors, however, the relation between glycolytic preference, tumor formation, and tumor cell clonogenicity has remained unknown. To address this issue, we analyzed the metabolic profiles of isogenic glioma-initiating cells (GICs) in a mouse model. Methods GICs were established by overexpression of H-RasV12 in Ink4a/Arf-null neural stem cells. Subpopulations of these cells were obtained by single-cell cloning, and clones differing in extracellular acidification potential were assessed for metabolic characteristics by quantification of intra- and extracellular metabolites. Tumorigenicity was assessed by implantation of 100 cells of each subpopulation into the forebrain of wild-type mice. Tumors were examined for pathological features of glioma and expression of glycolytic enzymes. Results Malignant transformation of neural stem cells resulted in a shift in metabolism characterized by a significant increase in glucose uptake and lactic acid production. Clonal populations of GICs also manifested pronounced differences in their metabolic profiles. Certain GICs consumed more glucose and produced more lactate, while others had higher oxygen consumption. These differences were reflected in the levels of intracellular metabolites and they were paralleled by a differential expression of glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2. GIC clones with different metabolic profiles had the same level of tumorigenic ability and the tumors formed by all types of GICs displayed the histopathological features of glioblastoma. However, the differential expression of the glycolytic enzymes was also evident in the tumors formed by each of the clones. Implantation of a mix of GICs with different metabolic profiles showed that clones with higher glycolytic ability were the major component of the tumor mass and that they provided a scaffold for the less glycolytic clones, supporting their expansion. Conclusions The metabolic characteristics of glioma cells appear early during malignant transformation and persist until the late stages of tumor formation. Even isogenic clones may be heterogeneous in terms of metabolic features, however, and this heterogeneity may play a role in tumor cell proliferation and survival. Our results suggest that a more detailed understanding of the metabolic profile of malignant gliomas is imperative for their effective therapeutic targeting. Citation Format: Oltea Sampetrean, Isako Saga, Shunsuke Shibao, Jun Okubo, Satoru Osuka, Nobuyuki Onishi, Hideyuki Saya. Crosstalk between initiating cells with different metabolism in a murine model of malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4342. doi:10.1158/1538-7445.AM2014-4342

Brain Tumor Stem Cells and Immunotherapy

Glioblastoma multiforme (GBM) is one of the most common aggressive primary brain tumors, and it responds poorly to the current treatment combination of surgery, radio-, and chemotherapy. The hypothesis that cancer stem cells may account for the pathogenesis underlying various tumors, including GBM, has been accepted widely in recent years. Brain tumor stem cells (BTSCs) have been shown to contribute to therapeutic resistance and the presence of BTSCs may explain the recurrence of GBM following conventional treatment, as just a few BTSCs are sufficient to give rise to a new tumor. Therefore, the therapeutic targeting of BTSCs is of utmost importance. Among emerging treatment modalities, immunotherapy is a strategy that has the potential to target BTSCs that are resistant to conventional therapies. This review describes recent advancements in the study of BTSCs and immunotherapy.

Abstract 487: Invasion characteristics of tumor cells in a glioblastoma model of genetically modified neural stem cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Although several types of cells in the brain are able to migrate during development or even in adulthood, only cells of high grade malignancies exhibit an invasive phenotype. The present study aims to characterize the differences between infiltrating tumor cells and their cells of origin using a model of genetically-induced glioblastoma-initiating cells (GICs) in the syngeneic mouse adult brain. Methods: Neural stem cells/ multipotent progenitor cells (NSCs/NPCs) were isolated from the subventricular zone of adult C57BL/6 mice with a homozygous deletion of the Ink4a/ARF locus and cultured as neurospheres. GICs were established by overexpression of H-RasV12 in the NSCs/NPCs. Tumor cells were isolated from the tumors formed by orthotopic implantation of the GICs into 6-week-old wild type mice and propagated as tumorspheres (TS). The motility of each type of cells and their response to various chemoattractants was assessed using in vitro migration assays. Their preference for anatomical structures and their ability to infiltrate the surrounding normal brain was analyzed by tracking the cells in cultured coronal brain slices established from stereotactically-implanted mice. Results: In vitro, both tumor cells and NSCs exhibited a similar degree of motility, but consistently varied in their response to a panel of chemoattractants. Compared to NSCs, GICs and TS responded only slightly to single growth factors or cytokines, but showed significant responses to combinations of chemoattractants. The most marked increase in migration was triggered by the supernatant of cultured tumor cells. Supernatants obtained from GICs, TS or human glioma cell lines induced migration to a similar degree. Timelapse microscopy confirmed motility of both normal and tumor cells, with all three groups being able to move both within the cortex and within the striatum, and both along blood vessels and fiber tracts. However, NSCs/NPCs did not leave the injection site as single cells, while GICs and TS entered and infiltrated the normal brain as solitary cells. This pattern of migration was greatly reduced by the administration of epithelial-to-mesenchymal transition (EMT) inhibitors, both in vitro and in vivo. Conclusion: Our results show that tumor invasion is multifactorial, with both autocrine secretion of several factors, as well as a mesenchymal phenotype being key regulators. Moreover, the mesenchymal phenotype in itself is a targetable attribute and should be assessed in patient samples in order to adjust anti-invasive therapy when warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 487. doi:1538-7445.AM2012-487

Abstract 409: Analysis of invasion patterns in an induced cancer stem cell model of malignant brain tumor

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: The invasive phenotype of malignant brain tumors is a major cause for their recurrence and resistance to therapy. It has been suggested that tumor cells recapitulate the migration patterns of glial progenitors. However, it is still unclear which cells acquire a migratory potential, at which stage they acquire it and whether and how invasion patterns change during tumorigenesis and treatment. The present study aims to define the characteristics of infiltrating cells and the patterns of invasion of genetically-induced brain tumor-initiating cells (BTICs) in the syngeneic mouse adult brain. Methods: We have established a mouse malignant brain tumor model by overexpressing RASV12 in neural stem cells/multipotent progenitor stem cells derived from the subventricular zone of mice with a homozygous deletion of the Ink4a/ARF locus. Orthotopic implantation of these BTICs into 6-week-old wild type mice resulted in formation of highly invasive, hypervascular, serially transplantable glioblastoma-like tumors with a 100% penetrance and a 5-week median survival. Fifteen mice were sacrificed at one-week intervals (n=3) and fixed brain sections were analyzed for onset and direction of cell migration. A second series of 10 mice were sacrificed at two-day intervals (n=2), and the live brains were sliced, cultured and motility and infiltration patterns were analyzed by timelapse microscopy, cell tracking and 3D reconstruction. Results: Pathological analysis revealed that cellular migration was detectable at one week post-injection, with both movement along fiber tracts, as well as perivascular trajectories. During the later stages of tumorigenesis, invasive foci were located mostly around blood vessels and the invasive front coincided with VEGFR and HO-1 expression. Timelapse microscopy confirmed motility of tumor cells as early as two days post- injection. Movement along blood vessels was quick and directed away from the tumor, while intraparenchymal movement was more saltatory, with repeated extension and retraction of leading processes, pausing and turning. Cells which exited the tumor early sometimes tested several routes before taking one and then exhibited a to-and-fro movement, creating paths for other cells. Motility was not affected by cell number and cells from secondary and tertiary tumors retained the infiltrative characteristics. Furthermore, co-injection of dsRed-labeled nestin-positive and GFP-labeled GFAP-positive tumor cells showed that differentiation status seems to affect infiltration patterns more than motility in itself. Conclusion: Our results show that invasion is one of the earliest events in tumorigenesis. Moreover, the once established infiltration paths might facilitate further invasion at later stages. We therefore suggest that, to efficiently prevent recurrence, migration should be considered as a therapeutic target from the time of diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 409.