Project description:One of the most detrimental hallmarks of glioblastoma multiforme (GBM) is cellular invasiveness, considered a potential cause of tumor recurrence. Infiltrated GBM cells are difficult to completely eradicate surgically and with local therapeutic modalities. Although much effort has focused on understanding the various mechanisms controlling GBM invasiveness, the nature of the invasiveness remains poorly characterized. Here, we established a highly invasive glioma cell line (U87R4 cells) and a non-invasive cell line (U87L4 cells) from U87MG glioma cells following four rounds of serial in vivo intracranial transplantation. Compared to U87L4 cells, U87R4 cells were highly invasive and had glioma stem cell-like properties. Microarray analysis showed that apoptosis-inducing genes (caspase3 and PDCD4) were downregulated, whereas several cancer stem cell-relevant genes (Wnt10A, Frizzled 4, and CD44) were upregulated in U87R4 cells compared to U87L4 cells. U87R4 cells were resistant to anticancer drug-induced cell death, which was partially due to downregulation of caspase3 and PDCD4. U87R4 cells retained activated Wnt/β-catenin signaling through Frizzled 4, which was sufficient to control neurosphere formation. In addition, Frizzled 4 promoted expression of the epithelial to mesenchymal transition regulator, SNAI1, and acquisition of a mesenchymal phenotype. Taken together, our results indicate that Frizzled 4 may be a member of the Wnt signaling family that governs both stemness and invasiveness of glioma stem cells, and may be a major cause of GBM recurrence and poor prognosis. We established a highly invasive glioma cell line (U87R4 cells) and a non-invasive cell line (U87L4 cells) from U87MG glioma cells following four rounds of serial in vivo intracranial transplantation to characterize the mRNA expression profile of highly invasive glioma cells compared to non-invasive/parental glioma cell lines.
Project description:One of the most detrimental hallmarks of glioblastoma multiforme (GBM) is cellular invasiveness, considered a potential cause of tumor recurrence. Infiltrated GBM cells are difficult to completely eradicate surgically and with local therapeutic modalities. Although much effort has focused on understanding the various mechanisms controlling GBM invasiveness, the nature of the invasiveness remains poorly characterized. Here, we established a highly invasive glioma cell line (U87MGR2/R3 cells) and a non-invasive cell line (U87MGL2/L3 cells) from U87MG glioma cells following four rounds of serial in vivo intracranial transplantation. Compared to U87MGL2/L3 cells, U87MGR2/R3 cells were highly invasive and had glioma stem cell-like properties. Microarray analysis showed that apoptosis-inducing genes (caspase3 and PDCD4) were downregulated, whereas several cancer stem cell-relevant genes (Wnt10A, Frizzled 4, and CD44) were upregulated in U87MGR2/R3 cells compared to U87MGL2/L3 cells. U87MGR2/R3 cells were resistant to anticancer drug-induced cell death, which was partially due to downregulation of caspase3 and PDCD4. U87MGR2/R3 cells retained activated Wnt/β-catenin signaling through Frizzled 4, which was sufficient to control neurosphere formation. In addition, Frizzled 4 promoted expression of the epithelial to mesenchymal transition regulator, SNAI1, and acquisition of a mesenchymal phenotype. Taken together, our results indicate that Frizzled 4 may be a member of the Wnt signaling family that governs both stemness and invasiveness of glioma stem cells, and may be a major cause of GBM recurrence and poor prognosis.
Project description:SW480 is a colorectal cancer cell line. We have shown that cancer cells are characteristic of neural stem cells and neural stemness contributes to cell tumorigenicity. We found that during serial transplantation of SW480 cells into nude mice via subcutaneous injection (in vivo passaging), the cells enhanced their neural stemness, tumorigenicity and pluripotent differentiation potential.
Project description:Glioma initiating cells (GICs) are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanism of GIC maintenance/differentiation, we established GIC clones from GBM patient tumors having the potential to differentiate into malignant gliomas in mouse intracranial xenograft, and established an in vitro glioma induction system by using serum stimulation. Upon the serum stimulation, the GIC spheres showed increased cellular proliferation, motility, filopodia/lameripodia formation and adhesion to the culture dishes. Simultaneously, the NSC marker proteins such as CD133 and Sox2 were down-regulated, and the astrocyte/glioma marker GFAP and the malignancy marker CD44 dramatically up-regulated. To identify genes/proteins whose expression changes dynamically during the differentiation of GICs into glioma cells, these GICs were subjected to DNA microarray/iTRAQ based integrated proteomics.
Project description:Glioblastomas are the most lethal tumors affecting the central nervous system in adults. Simple and inexpensive syngeneic in vivo models that closely mirror human glioblastoma, including interactions between tumor and immune cells, are urgently needed for deciphering glioma biology and developing more effective treatments. Here, we generated mouse glioblastoma cell lines by repeated in-vivo passaging of neural stem cells and tumor tissue of a neural stem cell-specific Pten/p53 double-knockout genetic mouse model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts they formed high-grade gliomas that faithfully recapitulated the histopathological characteristics, invasiveness and infiltration by myeloid cells characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioma pathomechanism and test immunotherapies in syngeneic preclinical models.
Project description:Glioblastomas are the most lethal tumors affecting the central nervous system in adults. Simple and inexpensive syngeneic in vivo models that closely mirror human glioblastoma, including interactions between tumor and immune cells, are urgently needed for deciphering glioma biology and developing more effective treatments. Here, we generated mouse glioblastoma cell lines by repeated in-vivo passaging of neural stem cells and tumor tissue of a neural stem cell-specific Pten/p53 double-knockout genetic mouse model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts they formed high-grade gliomas that faithfully recapitulated the histopathological characteristics, invasiveness and infiltration by myeloid cells characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioma pathomechanism and test immunotherapies in syngeneic preclinical models.
Project description:The goal of this experiment was to compare the gene expression of glioma cells migrating on substrates of different topographies and to determine a migration-associated gene profile. Human malignant glioma U251MG cells were cultured on highly-aligned versus randomly-oriented electrospun nanofibers of poly-caprolactone. Cells migrated actively on aligned nanofibers but motility was very restricted on randomly-oriented fibers. Results of gene expression profiling indicated upregulation of a JAK/STAT signature in actively migrating glioma cells.
Project description:The goal of this experiment was to compare the gene expression of glioma cells migrating on substrates of different topographies and to determine a migration-associated gene profile. Human malignant glioma U251MG cells were cultured on highly-aligned versus randomly-oriented electrospun nanofibers of poly-caprolactone. Cells migrated actively on aligned nanofibers but motility was very restricted on randomly-oriented fibers. Results of gene expression profiling indicated upregulation of a JAK/STAT signature in actively migrating glioma cells. Total RNA was extracted from 3 independent cultures of U251 glioma cells cultured on aligned versus random nanofibers and applied to Affymetrix HG-U133 plus 2 chips. Analysis of gene expression was used to determine a gene-signature correlated with different substrate topography
Project description:Glioma initiating cells (GICs) are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanism of GIC maintenance/differentiation, we established GIC clones from GBM patient tumors having the potential to differentiate into malignant gliomas in mouse intracranial xenograft, and established an in vitro glioma induction system by using serum stimulation. Upon the serum stimulation, the GIC spheres showed increased cellular proliferation, motility, filopodia/lameripodia formation and adhesion to the culture dishes. Simultaneously, the NSC marker proteins such as CD133 and Sox2 were down-regulated, and the astrocyte/glioma marker GFAP and the malignancy marker CD44 dramatically up-regulated. To identify genes/proteins whose expression changes dynamically during the differentiation of GICs into glioma cells, these GICs were subjected to DNA microarray/iTRAQ based integrated proteomics. Within 4 hours of tumor removal from GBM patients, tissues were subjected to GIC preparation. After successive cloning, total RNA from GIC clones (GIC03A and GIC03U) on day 2 or 7 of subculture in NSC medium with or without 10% FCS was subjected to the analysis with Affymetrix microarrays. Simultaneously, the proteins extracted from the same set of cells were subjected to LC-shot gun analyses using the 8-plex iTRAQ method. We sought to obtain the information of the common molecules that were up- or down-regulated during the GSC differentiation process, and functional targets for the early onset of GIC-associated glioma.
Project description:NSTS-11 cell line derived from a primary embryonal rhabdomyosarcoma was used for serial xenotransplantation in NSG mice in vivo. Subsequently, three cell lines, LTB1, LTB5, and LTB24, were derived from primary, secondary, and tertiary xenograft tumors, respectively. Gene expression profiling of all four cell lines was performed to identify changes in gene expression over the course of serial xenotransplantation, which was associated with a gradual increase of stemness of embryonal rhabdomyosarcoma cells. For each of the xenograft-derived cell lines, the parental NSTS-11 cell line served as a reference control.