1. THE PROMISE OF STEM CELL RESEARCH Marisa Bowers Donghong Zhao Karen S. Aboody, M.D. et al. Assistant Professor Divisions of Hematology/HCT & Neurosciences City of Hope National Cancer Center & Beckman Research Institute COH Summer Student Program June 26, 2005

2. MBowers/Aboody Lab 2005 Outline Background Information/Definitions Neural Stem Cell Migration Migration Assays Migration Data and Results Future Experiments

3. MBowers/Aboody Lab 2005 Background – Stem Cells What is a Stem Cell? -undifferentiated cell has potential to differentiate into some/all cell types in body What is a Neural Stem Cell? -differentiate into neuron, astrocyte or oligdendrocyte What are the potential therapeutic benefits of stem cells? -exogenous stem cells repair damaged/diseased tissue -deliver therapeutic agents to tumors -prevent/treat birth defects in womb

4. MBowers/Aboody Lab 2005 Background - Glioblastoma What is Glioblastoma? -Type of human brain cancer -Highly invasive: metastasizes quickly throughout brain, surgeons remove main tumor mass, missing metastases -Expected life-span of a patient with glioma is 3-6 months -No cure

5. MBowers/Aboody Lab 2005 Past and Present Glioma Research Previous research: drug delivery -through gene therapy -problem with drug delivery: no movement of cells within tissue, does not target metastases -blood-brain barrier prohibits entry of some therapeutic agents into brain Potential Solution: neural stem cells as vehicles -targeted delivery of therapeutic agents

6. MBowers/Aboody Lab 2005 NSC’s Directed Migration to Tumor Cells Cancer cell Stem cell

7. MBowers/Aboody Lab 2005 Why does Directed Migration of NSC’s to Tumor Cells Occur? Theories: -tumor cells excrete cytokines, attract stem cells -pockets of hypoxia exist within tumor mass, draws stem cells -genes expressed in cells different in hypoxic (1%O 2 ) and normoxic (16%O 2 ) conditions My Project: Determine factors involved in NSC migration to tumor Hypothesis: one or more of the following cytokines effects NSC migration under hypoxic conditions -SDF-1/CXCR-4 -HGF/C-Met -SCF/CKit -VEGF/VEGFR -EGF/EGFR -IL8/IL8R signaling pathways Stat3, RaS and Wnt are downstream of genes code for cytokines, pathway effected by cytokine expression Tumor Cell NSC

8. MBowers/Aboody Lab 2005 Initial Tests: Boyden Chamber Migration Assays

9. MBowers/Aboody Lab 2005 Variable Conditions: -Upper Chamber: -NSC grown at 16%O 2 -NSC grown at 16% then incubated 24 hours at 1%O 2 -Lower Chamber: -tumor CM from tumor cells incubated 48 hours under 16%O 2 -tumor CM from tumor cells incubated 48 hours under 1%O 2 -media containing different levels of serum Boyden Chamber Migration Set-up

10. MBowers/Aboody Lab 2005 Controls: -DMEM/RPMI -5%BSA -10%FBS Tumor CM -16%O 2 U251 -1%O 2 U251 -16%O 2 U251 +SDF-1 -16%O2 SKNAS -1%O 2 SKNAS -16%O 2 SKNAS +SDF-1 Lower Wells Boyden Chamber Set-Up 16%O 2 F3.C11%O 2 F3.C1 Upper Wells

11. MBowers/Aboody Lab 2005 U251 Graph 16%O 2 Plate 1%O 2 Plate

12. MBowers/Aboody Lab 2005 SKNAS Graph 16%O 2 Plate 1%O 2 Plate

13. MBowers/Aboody Lab 2005 Migration Results Effect of SDF-1 on Migration -no effect on migration -seen repeatedly in assays -either not involved in migration or levels of SDF-1 high prior to migration, additional SDF-1 no effect Effect of VEGFR on Migration -normoxic and hypoxic plates: blocked/hindered migration -may be migration inducing factor

14. MBowers/Aboody Lab 2005 Future Experiments Changes for future experiments: -culture stem cells in 1%O 2 for less than 24 hours -run many tests to find good time span -real time-PCR on SC cultured for different amounts time -use U251 and Brain cell line 5904, not SKNAS -SKNAS: lower response to F3.C1 SC Repeat Boyden Chamber -making changes -compare migration results

15. MBowers/Aboody Lab 2005 Next Step Extract stem cell RNA and perform real time-PCR - Use mRNA expression to determine regulation of candidate ‘homing’ genes during migration

16. MBowers/Aboody Lab 2005 Use female immune compromised nude mice -inject mice with: -‘normal’ human brain tumor or - HIF siRNA brain tumor: HIF expression blocked -decrease cytokine expression - both groups will receive injection of stem cells In Vivo Model

17. MBowers/Aboody Lab 2005 harvest mice, remove tumor tissue for RNA extraction and real time-PCR -determine gene expression of tumor tissue and NSC -expect: gene expression to be different in normal and HIF siRNA tumors -use immnuohistochemical staining compare distribution stem cells in brains -expect: more stem cells in normal tumor than HIF siRNA tumor Data Analysis

18. MBowers/Aboody Lab 2005 Thank You Dr. Karen Aboody Donghong Zhao, PhD The Aboody Lab The Barish Lab The Glackin/Flannigan Labs Queenie Du and the Summer Student Program