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Feasibility of HIF stabilization and activity in tumor cells

Feasibility of HIF stabilization and activity in tumor cells. Florian Stuker, Katerina Dikaiou, Steffi Lehmann, Markus Rudin. WP6/7: Applications to murine tumor models: tool development and therapy assessment I. Application: assay development (hypoxia cascade)

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Feasibility of HIF stabilization and activity in tumor cells

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  1. Feasibility of HIF stabilization and activity in tumor cells Florian Stuker, Katerina Dikaiou, Steffi Lehmann, Markus Rudin

  2. WP6/7: Applications to murine tumor models: tool development and therapy • assessment • I. Application: • assay development (hypoxia cascade) • in vitro characterization of HIF assay: transient and stable cell transfection • imaging hypoxia signaling in murine tumor xenografts • initial experiments with protease imaging • II. Methodology: • e) development of combined MRI-FMT system

  3. mechanistic studies: HIF signaling Hypoxia Hypoxia inducible factor 1a (HIF1a) transcriptions factor: HIF-1a/HIF1b binding to DNA: gene expression glykolytic enzymes VEGF i-NOS and HO-1 EPO anaerobic metabolism angiogenesis erythropoiesis vasodilation blood volume glucose utilization lactate energy metabolism permeability blood volume

  4. Protein degradation Transcriptional activation HIF-1a regulation by pO2 L. E. Huang et al. JBC 2003

  5. reporter gene for HIF 1a HIF1a Promoter Reporter GOI Linker HIF1a mCherry DNA HIF1a Luciferase DNA S. Lehmann, I. Kotevic, D. Vats, R. Keist

  6. regulation of HIF1a-mCherry fusion protein in cells Mouse embryonic fibroblasts normoxic induced (DMOG) 1) inhibition of degradation machinery stabilizes HIF reporter construct 2) stabilized product is found in cellular nucleus (transcription factor) S.Lehmann, SSN (2007)

  7. hypoxia hypoxia inducible factor (HIF1a) transcriptions Factor: HIF-1a/HIF1b binding to DNA: Gene Expression viral promoter mCherry HIF1a ODD SV40 fluc VEGF i-NOS and HO-1 EPO HRE VEGF angiogenesis erythropoiesis vasodilation blood volume permeability blood volume signaling: HIF signaling assays

  8. d6 hypoxia d8 d10 HIF1a d13 signaling: HIF signaling assays C51: HRE-luciferase multimodal hypoxia readouts HRE Lehmann et al. (submitted)

  9. d6 d8 d10 d13 Angiogenesis (VEGF-R) signaling: HIF signaling assays C51: HRE-luciferase multimodal hypoxia readouts volume Lehmann et al. (submitted)

  10. signaling: HIF signaling assays • No (weak) correlation between level of hypoxia and HIF1a • Correlation between HIF1a and HIF-downstream readout (HRE): HRE induction significantly larger than that of HIF1a • Focus on HRE as molecular readout of hypoxia cascade • Development of HRE assay using mCherry as reporter Lehmann et al. (submitted)

  11. measuring angiogenesis structural / physiological parameters permeability sites of angiogenesis degree of vessel maturation tumor blood volume total vascularization TBV and vessel size vessel architecture molecular factors hypoxia, VEGF, strength of HIF1a & HRE location of angiogenic signaling pro-angiogenic factors time dependence of adhesion molecules activated endothelium proteases efficiency of matrix invasion inflammatory cells tissue response (inflammation) angiogenic ‚endpoint‘ morphology & physiology tumor-host interaction bold: established, red: fluorescence based assays

  12. FMT of protease activity in sc tumor model

  13. outlook next period • I. Application • a) assay development: • establishement of fluorescence based assay for HRE • multimodal assay for HIF1a: GPI-avidin system • Establishement of fluorescence assay for tumor proteases • b) first therapy studies in murine subcutaneous tumor models • c) development of orthotopic tumor models • d) characterization of ODD-m-Cherry and HRE-m-Cherry transfected • tumor cells in vitro • e) In vivo studies in C51 tumor model using m-Cherry transfected • tumor xenografts

  14. methodology: MRI-FMT system

  15. aim and objectives The aim Acquire an MRI- and optical-signal simultaneously! The major focus: Setup design without fibers Free beam illumination and point grid scan Detector operation inside the magnet Investigation of the interaction between the modalities

  16. setup overview

  17. sample platform

  18. setup paramters • MRI: • Field strength: 9.4T operating at 400MHz • Coil: rectangular cylindric shaped surface coil 20x24mm • Illumination: • Laser: cw at 671nm • Exposure time: 5-30sec • Power: 0.5-3mW • Detection: • Detector: 32x32 SPAD array • FOV: 8x8mm • Filters: 660nm and 720nm ± 13nm

  19. preliminary results (MRI)

  20. Thank you for the attention

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