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Brain tumor initiating cells: Implications for neural stem cell biology and cancer
488
Symposia & Short Talk Abstracts / Int. J. Devl Neuroscience 24 (2006) 471–493
that readily progresses to malignancy. Neurofibromin, the tumor suppressor protein encoded by NF1 has rasGap activity thus, implicating constitutive activation of the ras pathway as a major consequence of NF1 loss of function. Patients with NF1 also have increased incidence of glioblastoma formation. We have sought to model these tumors for which no effective therapies have been developed. The prognosis remains unchanged over the past three decades. According to the WHO classification, four grades of astrocytoma exist. Grade 1 is benign and also referred to as pilocytic astrocytoma. Grade II or low grade astrocytoma is characterized by infiltrative cells that home on neuronal bodies (perineural satellosis). Grade III or anaplastic astrocytoma is cell dense and highly proliferative. Grade IV or glioblastoma multiforme is characterized by pseudopalisading, necrotic foci, and intense microvascularization. All forms of astrocytoma express primitive cell markers such as nestin. In addition, all forms of astrocytoma appear throughout the brain but do not leave the CNS. These observations have led to the suggestion that the CNS provides a niche that is required for tumor growth and that spontaneous tumorigenesis occurs throughout. Historically, the prevalent model for astrocytoma formation invoked mechanisms of dedifferentiation of glial cells, followed by genetic and epigenetic signals that drive neoplastic transformation. The more recent appreciation of the existence of stem cells in the lateral ventricles and dentate gyrus has raised the question of a potential role for stem cells in tumor formation. We have undertaken to model NF1 in mice through conventional knock out and conditional knock out technologies and have developed tumor suppressor based mouse models of glioblastoma. The resultant tumors histologically and molecularly resemble human glioblastoma, including progression from Grades II through IV. Through analysis of a variety of criteria, our data have provided evidence that stem cells could account for the origin of glioblastoma. Through the use of virally transduced cre delivery to stem cell compartments as well as use of tamoxifen inducible stem cell specific cre transgenes, we have developed direct evidence for precursor cells as the origin of tumor cell. doi:10.1016/j.ijdevneu.2006.09.044 [S38] Brain tumor initiating cells: Implications for neural stem cell biology and cancer P. Dirks Hospital for Sick Children, University of Toronto, Canada Human brain tumor tissues have been long recognized to be heterogeneous, comprised of cells of much different morphology and also identified by their expression of different markers. Indeed, in the glioblastoma multiforme, the diverse cellular heterogeneity is a salient feature in the
very name of this tumor. Recently, using experimental approaches developed to study normal neural stem cells and in vivo functional assays developed for the study of leukemic stem cells, our laboratory has identified a subpopulation of brain tumor cells that has potent tumor initiating ability in vivo. The tumor initiating fraction can be enriched for by sorting for the normal neural precursor marker CD133/ AC133. CD133 represents a minority of the cells in human brain tumors, but the clonogenic activity in vitro as neurospheres and tumor initiating ability in vivo is uniquely contained within this fraction. CD133+ human brain tumor cells can be induced to differentiate in vitro, supporting the idea that brain tumors represent a caricature of normal development. The implications for the cancer stem cell hypothesis on the understanding of brain tumor cell of origin, pathogenesis, and treatment will be discussed. doi:10.1016/j.ijdevneu.2006.09.045 [S39] EGFR signalling identifies an embryonic peripheral nerve progenitor cell amplified by mutation in the Nf1 gene N. Ratner *, J.P. Williams, J. Wu, J.J. Kordich, S.C. Miller, M.C. Colbert, J.A. Cancelas Cincinnati Children’s Hospital, University of Cincinnati, USA Peripheral nerve stem and progenitor cells that retain multilineage differentiation potential have been identified. Here, we show that embryonic dorsal root ganglia (DRG) progenitors can be propagated as self-renewing spheres in vitro. The growth factor requirements for nerve progenitor cells have not been defined. We identified EGF-dependent self-renewing spheres. Growth and self-renewal are blocked by exposure to EGFR tyrosine kinase inhibitor, indicating a requirement for EGFR activity. Cells mutant for the Nf1 Ras-GAP show an increase in sphere formation. Spheres from both wild type and Nf1 mutant DRG contained cells capable of glial differentiation and differentiation into SMA+ cells. Spheres from Nf1 mutants showed increased propensity to form neurons in vitro. An EGFR+; p75+ cell population was previously isolated from Nf1 / DRG cultures, here we show that these cells (Nf1 / TXF) can be propagated as spheres at greatly (20) increased efficiency. Consistent with a progenitor phenotype, Nf1 / TXF cells showed migratory characteristics of neural crest stem cells in a chicken xenograft model and expressed markers (as assessed using Affymetrix GeneChips and QRTPCR) of neural crest and at least three crest derivatives: neurons; Schwann cells; and melanocytes. Knockout of Nf1 at the embryonic Schwann cell stage of development, but not earlier or later, generated this progenitor like population and self-renewing spheres, implying that loss of Nf1 at a critical stage in development causes an expansion of progenitor cells. Nf1+/ but not wild type perinatal mouse nerves contained
Symposia & Short Talk Abstracts / Int. J. Devl Neuroscience 24 (2006) 471–493
that readily progresses to malignancy. Neurofibromin, the tumor suppressor protein encoded by NF1 has rasGap activity thus, implicating constitutive activation of the ras pathway as a major consequence of NF1 loss of function. Patients with NF1 also have increased incidence of glioblastoma formation. We have sought to model these tumors for which no effective therapies have been developed. The prognosis remains unchanged over the past three decades. According to the WHO classification, four grades of astrocytoma exist. Grade 1 is benign and also referred to as pilocytic astrocytoma. Grade II or low grade astrocytoma is characterized by infiltrative cells that home on neuronal bodies (perineural satellosis). Grade III or anaplastic astrocytoma is cell dense and highly proliferative. Grade IV or glioblastoma multiforme is characterized by pseudopalisading, necrotic foci, and intense microvascularization. All forms of astrocytoma express primitive cell markers such as nestin. In addition, all forms of astrocytoma appear throughout the brain but do not leave the CNS. These observations have led to the suggestion that the CNS provides a niche that is required for tumor growth and that spontaneous tumorigenesis occurs throughout. Historically, the prevalent model for astrocytoma formation invoked mechanisms of dedifferentiation of glial cells, followed by genetic and epigenetic signals that drive neoplastic transformation. The more recent appreciation of the existence of stem cells in the lateral ventricles and dentate gyrus has raised the question of a potential role for stem cells in tumor formation. We have undertaken to model NF1 in mice through conventional knock out and conditional knock out technologies and have developed tumor suppressor based mouse models of glioblastoma. The resultant tumors histologically and molecularly resemble human glioblastoma, including progression from Grades II through IV. Through analysis of a variety of criteria, our data have provided evidence that stem cells could account for the origin of glioblastoma. Through the use of virally transduced cre delivery to stem cell compartments as well as use of tamoxifen inducible stem cell specific cre transgenes, we have developed direct evidence for precursor cells as the origin of tumor cell. doi:10.1016/j.ijdevneu.2006.09.044 [S38] Brain tumor initiating cells: Implications for neural stem cell biology and cancer P. Dirks Hospital for Sick Children, University of Toronto, Canada Human brain tumor tissues have been long recognized to be heterogeneous, comprised of cells of much different morphology and also identified by their expression of different markers. Indeed, in the glioblastoma multiforme, the diverse cellular heterogeneity is a salient feature in the
very name of this tumor. Recently, using experimental approaches developed to study normal neural stem cells and in vivo functional assays developed for the study of leukemic stem cells, our laboratory has identified a subpopulation of brain tumor cells that has potent tumor initiating ability in vivo. The tumor initiating fraction can be enriched for by sorting for the normal neural precursor marker CD133/ AC133. CD133 represents a minority of the cells in human brain tumors, but the clonogenic activity in vitro as neurospheres and tumor initiating ability in vivo is uniquely contained within this fraction. CD133+ human brain tumor cells can be induced to differentiate in vitro, supporting the idea that brain tumors represent a caricature of normal development. The implications for the cancer stem cell hypothesis on the understanding of brain tumor cell of origin, pathogenesis, and treatment will be discussed. doi:10.1016/j.ijdevneu.2006.09.045 [S39] EGFR signalling identifies an embryonic peripheral nerve progenitor cell amplified by mutation in the Nf1 gene N. Ratner *, J.P. Williams, J. Wu, J.J. Kordich, S.C. Miller, M.C. Colbert, J.A. Cancelas Cincinnati Children’s Hospital, University of Cincinnati, USA Peripheral nerve stem and progenitor cells that retain multilineage differentiation potential have been identified. Here, we show that embryonic dorsal root ganglia (DRG) progenitors can be propagated as self-renewing spheres in vitro. The growth factor requirements for nerve progenitor cells have not been defined. We identified EGF-dependent self-renewing spheres. Growth and self-renewal are blocked by exposure to EGFR tyrosine kinase inhibitor, indicating a requirement for EGFR activity. Cells mutant for the Nf1 Ras-GAP show an increase in sphere formation. Spheres from both wild type and Nf1 mutant DRG contained cells capable of glial differentiation and differentiation into SMA+ cells. Spheres from Nf1 mutants showed increased propensity to form neurons in vitro. An EGFR+; p75+ cell population was previously isolated from Nf1 / DRG cultures, here we show that these cells (Nf1 / TXF) can be propagated as spheres at greatly (20) increased efficiency. Consistent with a progenitor phenotype, Nf1 / TXF cells showed migratory characteristics of neural crest stem cells in a chicken xenograft model and expressed markers (as assessed using Affymetrix GeneChips and QRTPCR) of neural crest and at least three crest derivatives: neurons; Schwann cells; and melanocytes. Knockout of Nf1 at the embryonic Schwann cell stage of development, but not earlier or later, generated this progenitor like population and self-renewing spheres, implying that loss of Nf1 at a critical stage in development causes an expansion of progenitor cells. Nf1+/ but not wild type perinatal mouse nerves contained