uzh | eth | zürich 1 animal imaging center uzh  eth  zürich Feasibility of HIF stabilization and activity in tumor cells Florian Stuker, Katerina Dikaiou, Steffi Lehmann, Markus Rudin

uzh | eth | zürich 2 animal imaging center uzh  eth  zürich WP6/7: Applications to murine tumor models: tool development and therapy assessment I. Application: a)assay development (hypoxia cascade) b)in vitro characterization of HIF assay: transient and stable cell transfection c)imaging hypoxia signaling in murine tumor xenografts d)initial experiments with protease imaging II. Methodology: e) development of combined MRI-FMT system

uzh | eth | zürich 3 animal imaging center uzh  eth  zürich VEGF anaerobic metabolism angiogenesis vasodilation blood volume Hypoxia Hypoxia inducible factor 1  (HIF1  ) transcriptions factor: HIF-1  /HIF1  binding to DNA: gene expression glykolytic enzymes i-NOS and HO-1EPO erythropoiesis glucose utilization lactate energy metabolism permeability blood volume mechanistic studies: HIF signaling

uzh | eth | zürich 4 animal imaging center uzh  eth  zürich Transcriptional activation Protein degradation HIF-1a regulation by pO 2 L. E. Huang et al. JBC 2003

uzh | eth | zürich 5 animal imaging center uzh  eth  zürich reporter gene for HIF 1  DNA HIF1a Promoter mCherry HIF1  GOI Reporter S. Lehmann, I. Kotevic, D. Vats, R. Keist Linker DNA Luciferase HIF1 

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

uzh | eth | zürich 7 animal imaging center uzh  eth  zürich viral promoter HIF1  ODD mCherry permeability blood volume erythropoiesisangiogenesis vasodilation i-NOS and HO-1 VEGF EPO hypoxia hypoxia inducible factor (HIF1  ) transcriptions Factor: HIF-1a/HIF1b binding to DNA: Gene Expression SV40 fluc HRE VEGF signaling: HIF signaling assays

uzh | eth | zürich 8 animal imaging center uzh  eth  zürich d6 d10 d8 d13 signaling: HIF signaling assays Lehmann et al. (submitted) hypoxia HIF1  HRE C51: HRE-luciferase multimodal hypoxia readouts

uzh | eth | zürich 9 animal imaging center uzh  eth  zürich d6 d10 d8 d13 Lehmann et al. (submitted) volume Angiogenesis (VEGF-R) C51: HRE-luciferase multimodal hypoxia readouts signaling: HIF signaling assays

uzh | eth | zürich 10 animal imaging center uzh  eth  zürich Lehmann et al. (submitted) signaling: HIF signaling assays -No (weak) correlation between level of hypoxia and HIF1  -Correlation between HIF1  and HIF-downstream readout (HRE): HRE induction significantly larger than that of HIF1   Focus on HRE as molecular readout of hypoxia cascade  Development of HRE assay using mCherry as reporter

uzh | eth | zürich 11 animal imaging center uzh  eth  zürich measuring angiogenesis structural / physiological parameters permeabilitysites of angiogenesis degree of vessel maturation tumor blood volumetotal vascularization TBV and vessel sizevessel architecture molecular factors hypoxia, VEGF,strength of HIF1  & HRE  location of angiogenic signaling pro-angiogenic factorstime dependence of adhesion moleculesactivated endothelium proteasesefficiency of matrix invasion inflammatory cellstissue response (inflammation) angiogenic ‚endpoint‘ morphology & physiology tumor-host interaction bold: established, red: fluorescence based assays

uzh | eth | zürich 12 animal imaging center uzh  eth  zürich FMT of protease activity in sc tumor model

uzh | eth | zürich 13 animal imaging center uzh  eth  zürich 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

uzh | eth | zürich 14 animal imaging center uzh  eth  zürich methodology: MRI-FMT system

uzh | eth | zürich 15 animal imaging center uzh  eth  zürich 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

uzh | eth | zürich 16 animal imaging center uzh  eth  zürich setup overview

uzh | eth | zürich 17 animal imaging center uzh  eth  zürich sample platform

uzh | eth | zürich 18 animal imaging center uzh  eth  zürich 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

uzh | eth | zürich 19 animal imaging center uzh  eth  zürich preliminary results (MRI)

uzh | eth | zürich 20 animal imaging center uzh  eth  zürich Thank you for the attention