Recomendados
Mais conteúdo relacionado
Mais procurados
Mais procurados (20)
Destaque
Destaque (17)
Semelhante a Biotechpres 040513.ptx
Semelhante a Biotechpres 040513.ptx (20)
Biotechpres 040513.ptx
- 1. A restricted cell population propagates glioblastoma growth after chemotherapy J Chen et al. Nature 2012 Ana Gomez Biotech Presentation April 5th, 2013
- 2. Glioblastoma Multiforme • Most common primary malignant brain tumor • 15 months median survival if treated with temozolomide (TMZ) – 4.5 months without treatment • High recurrence rate – Mechanism is unknown – Cancer stem cell theory Glioblastoma Multiforme
- 3. SVZ and RMS • Subventricular Zone (SVZ) is a paired brain structure in the lateral ventricles • SVZ is a site of neurogenesis • Rostral Migratory System (RMS) – Neuroblasts migrate from SVZ into the olfactory bulb (OB) – Differentiate when they reach OB
- 4. Transgenic Murine Model • Mut7 mice have conditional alleles of Nf1, p53, and Pten – Tumor suppressor genes – Mice develop malignant gliomas • Nestin is expressed by adult NSCs and glial cells • Nestin drives Nes-ΔTK-GFP transgene expression • Herpes Simplex Virus (HSV) thymidine kinase (ΔTK) – Ablate dividing neural progenitors with ganciclovir (GCV) • IRES-GFP to mark Nes-ΔTK expressing cells – In absence of GCV
- 5. Characterization of Nes-ΔTK-GFP Transgene • 1 month old transgenic mice treated with GCV for 2 weeks • RMS is visualized by Nissl staining – RMS visible in wild type mice and control transgenic mice – RMS is diminished in transgenic mice treated with GCV • Control mice had DCX+ NPCs and GFAP+ NSCs • After transgenic mice treated with GCV: – DCX+ NPCs diminished – Quiescent NSCs remained unaffected DCX: committed neural progenitors GFAP: quiescent NSCs GFP: transgene
- 6. GFP Immunostaining of Untreated Tumors • TOPO-3 stains nuclei • Varying numbers of GFP+ cells • Ki67 stains proliferating cells • In tumors 3 and 4, many transgene GFP+ cells are KI67- – These transgene cells are quiescent
- 7. Mut7 Mice vs. Mut7; Nes-ΔTK Mice • No difference in percent survival • Infiltrative malignant gliomas are present in the cortex of both genotypes
- 8. TMZ Treatment of Mice Gliomas • TMZ administered over 5 days to tumor-bearing mice • BrdU injected 2 hours after final treatment – Synthetic nucleotide that substitutes thymidine during DNA replication – Detects proliferating cells • Reduction of BrdU+ in treated cells
- 9. Pulse Chase Analysis of Tumor Proliferation • Mice were treated with TMZ for 5 days • Injected with BrdU analogues: – CldU (1 day after end of treatment) – IdU (3 days after end of treatment) • Cells repopulated after TMZ treatment • CldU+ cells • IdU+ cells • Majority of CldU+ cells and IdU+ cells are GFP+ • Majority of IdU+ cells are also CldU+
- 10. GCV Treatment Prolongs Survival • Elimination of Nestin+ and GFP+ cells in Mu7;Nes-ΔTK mice treated with GCV
- 11. GCV Treatment: Less Infiltrative Tumors • GCV-treated Mut7;Nes-ΔTK mice have defined boundary – Lack infiltrative Sox2+ cells • GCV treatment reduces Ki67 proliferation index
- 12. TMZ & GCV Treatment Inhibits Glioma Growth
- 13. Conclusions • Nes-ΔTK transgene labels SVZ stem cells and a specific subset of glioblastoma multiforme cells that possess many features of CSCs – Nestin expression and relative quiescence • CSC hypothesis: Only a subset retains self-renewal and differentiation capacity • Study identified an endogenous glioma stem cell responsible for tumor maintenance and recurrence after chemotherapy
- 15. Acknowledgements • Dr. Charlie Roth • Melissa Przyborowski • Max Balter • Rutgers UMDNJ Biotechnology Training Program
- 16. Questions?
- 17. References • Chen et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature (2012) • Lennington et al. Neural stem cells and the regulation of adult neurogenesis. Reproductive Biology and Endocrinology (2003) • Chen et al. Malignant astrocytomas originate from neural stem/progenitor cells in a somatic tumor suppressor mouse model. Cancer Cell (2009) • “Glioblastoma Multiforme.” AANS. Web. <http://www.aans.org/Patient%20Information/Conditions%20and%20Treatm ents/Glioblastoma%20Multiforme.aspx>
Notas do Editor
- Paper shows how a subset of quiescent cells in a tumor can give rise to highly proliferative cancer cells. Found a population of “cancer stem”-like cells that could repopulate cancer cells even after chemotherapy treatment.
- 80% of malignant tumors in the CNS are malignant gliomas.
- First a little anatomy background: The subventricular zone is a paired brain structure located in the lateral ventricles. It has been found to be one of the sites of neurogenesis. The newly formed neuroblasts in the SVZ migrate to the olfactory bulb, through the rostral migratory system which can be seen in this image as the red line. Once in the OB, they differentiate into interneurons (periglomerular cells and granule cells).This paper will focus on the RMS system, to understand if neural progenitor cells are successfully being targeted and killed in their transgenic model.*hippocampus is another neurogenetic site*neuroblasts differentiate from NSCs but are committed to neuronal fate
- To understand what cell population was involved in the repopulation of cancer cells after chemotherapy, they decided to use a transgenic murine model. They used Mut7 mice, which have conditional alleles of: Nf1, p53, and Pten. These are the most common tumor suppressor genes, and by turning them off the mice are unable to suppress tumors and therefore develop malignant gliomas with 100% penetrance. In this paper, they take advantage of the fact that nestin is expressed by adult neural stem cells and glial cells. So they injected the Mut7 mice with a transgene whose expression can be driven by nestin. The transgene contains a cassette for herpes virus thymidine kinase. Since ganciclovir is an antiviral medication. They can inject ganciclovir systemically to ablate dividing neural progenitor cells that will express the transgene.If they don’t inject ganciclovir, the transgene will mark cells that express it with Green fluorescent protein. This way they can monitor what populations of cells express the transgene..
- To characterize their transgene, they treated 1 month old transgenic mice with ganciclovir for 2 weeks before they sacrificed the animals and analyzed SVZ sections using Nissl staining on the left column (which is just a basic dye that binds to the negatively charged RNA or DNA). They were able to visualize the RMS in wild type mice that were treated with GCV and in transgenic mice that were treated with PBS. However, the transgenic mice that were treated with GCV, had a greatly diminished RMS. This means that the transgene was being expressed in the neuroprogenitor cells in the RMS, since they can be effectively ablated by GCV injection. They further characterized their transgene on the right column, by determining which cells are committed neural progenitors, quiescent neural stem cells or transgene expressing cells. As can be seen from the images, control mice had DCX+ neural progenitor cells and GFAP+ neural stem cells. However, the transgenic mice that were treated with GCV, saw a diminised number of DCX+ neural progenitor cells, while the number of quiescent neural stem cells remained the same. This shows that adding GCV to the transgenic mice effectively ablates neural progenitor cells. *The white arrows in middle panel show that DCX+ GFP- cells are more distal in RMS. In bottom panel , show GFP+/GFAP+ but DCX- quiescent cells. *The committed neural progenitors are marked by doublecortin (DCX) expression, since NPCs begin to express DCX while actively dividing (great marker for neurogenesis). Quiescent cells are marked by glial fibrillary acidic protein and transgene expressing cells are marked by green fluorescent protein.
- The next step to understand where this transgene was being expressed, was to observe untreated tumors. These are the images for the immunostaining of 4 tumors: as you can see there are varying levels of GFP+ cells (which are the cells expressing the transgene). In the two tumors on the bottom panel, they simultaneously stained for Ki67 protein which is expressed in proliferating cells. And it was found that there are many cells that express the transgene because they are GFP+ but that remain quiescent since they did not express Ki67. Graph f quantifies these results. And it shows that the number of cells that express Ki67 and are GFP- is statistically significant from those that express Ki67 but are GFP+. *** P < 0.001
- Include % numbers of CldU+ IdU+ cells and GFP+
- *other tumors ended up killing the animal
- WT with PBS, is GCV drug affecting RMS