Molecular Imaging: Monitoring Gene Expression and Protein Function In Vivo David PiwnicaWorms, M'D',

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Molecular ImagingMonitoring Gene Expression and
Protein Function In Vivo David Piwnica-Worms,
M.D., Ph.D.Director, Molecular Imaging
CenterMallinckrodt Institute of Radiology and
Department of Molecular Biology
Pharmacology,Washington University Medical
School,St. Louis, MO.
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Molecular Medicine Here and Now
Cluster Analysis of Gene Expression in Clinical
Breast Cancer
Differential Display of Expressed Genes in Drug
Resistant Breast Cancer Cells
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New Linkages in Imaging
Molecular Biology
Radiology
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What is Molecular Imaging?How is it Different
from Conventional Radiological Imaging?
Molecular Imaging Fundamental source of imaging
signal is derived from a specific biomolecule
(receptor, enzyme, transporter, ligand)
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Molecular Targets in Oncology
Gene Expression in Cancer Abnormal Regulation of
Genes Overexpression Up-regulation Down-regu
lation Abnormal Signal Transduction Abnormal
Cell Surface Receptors Drug Resistance Gene
Therapy/Reporter Genes
Cell 100, 5770, 2000
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Molecular Targets in Oncology

• Signal Transduction
• PI 3-Kinase
• Cell Cycle
• Cyclins
• Checkpoint Control Pathways (p53, G2-M)
• Cyclin-dependent Kinases (Cdk)
• Cdk Inhibitors (p16, p21)
• Multidrug Resistance
• MDR1 P-glycoprotein
• MRP
• LRP
• Topoisomerase

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Molecular Targets in Oncology

• Angiogenesis
• p53
• Retinoblastoma tumor suppressor protein (RB)
• Apoptosis
• Bcl-2
• Bcl-x
• Extracellular matrix
• Immune response
• Telomerase

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Challenges of Molecular Imaging

• Examine molecular, biochemical and cellular
  events in vivo
• Low capacity systems require strategies to
  enhance signal-to-noise
• Highly specific and targetable imaging agents
  are usually required

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General Strategies for Molecular Imaging

• Signal generation by transport, binding, or
  sequestration of a labeled reagent
• Signal amplification by an enzymatic activity
  acting on a reagent
• Reporter gene acting on a reporter probe

One main focus of molecular imaging is on
discovery and validation of image enhancing
agents that interact with their cellular and
molecular environment
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How Does Molecular Imaging Relate to
Oncology? Contrast Agents, Radiopharmaceuticals
and Target Selection in Medical Imaging

• Guiding Therapeutic Choices
• Monitoring Drug Treatment
• Prognosis
• Early Response Indicators
• Monitoring Gene Therapy
• Basic/Translational Research
• Basic investigations of disease mechanisms in
  vivo
• Transgenic mouse models of disease

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• Molecular Imaging Today
• Nuclear Medicine Positron Emission Tomography
  (PET), Single Photon
• Emission Computed Tomography (SPECT), Planar
  Scintigraphy
• Imaging receptors, transporters and enzymes in
  vivo with radiopharmaceuticals
• 18FFDG PET
• Glut 1 transporter and hexokinase activity
• 111InOctreoscan SPECT
• Somatostatin receptor type 2

18FFDG PET Lung Ca
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MicroPET
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Mouse 18FNaF Bone Scan
J. Lewis
MicroPET Washington University Molecular Imaging
Center
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Molecular Imaging Tomorrow Activated MR Contrast
Agents Cleavage of Galactopyranose Linked
Gadolinium-DOTA (EgadMe) by b-Galactosidase

No relaxivity
High T1 relaxivity
b-Galactosidase mRNA(-) mRNA()
Meade, et al, Nature Biotech 18321, 2000
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Molecular Imaging Tomorrow Imaging
Overexpressed Cancer Genes Protease-Mediated
Activation of an Imaging Probe Cleavage of
Auto-Quenched Near Infrared Fluorescence (NIRF)
OFF
NIRF Probe (Cy5.5)
Autoquenching
u
Cleavable linkers
(
)
Polymer Backbone
Tung, et al. Cancer Research 604953, 2000
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Molecular Imaging Tomorrow Near Infrared
Fluorescence Optical Imaging of Protease Activity
In Vivo in a Xenograft Tumor Model
cathepsin D(-)
cathepsin D ()
Visible light NIRF signal Threshold
false (700 nm) color map
Tung, et al. Cancer Research 604953, 2000
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Case Study Molecular Imaging of Cancer Drug
Resistance In Vivo Multidrug Resistance (MDR1)
P-glycoprotein
-Efflux transporter -Normally expressed in liver,
kidney, intestine, choroid plexus and
capillaries of BBB -Pumps xenobiotics and cancer
chemotherapeutic drugs out of MDR cancer
cells -MDR cancers breast, lung, lymphoma,
colon, renal, Rb -MDR drugs include taxol,
doxorubicin, vinblastine, vincristine, VP16,
Gleevec (lipophilic cations) -MDR modulators
(inhibitors) in clinical trials
Immunotech
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• MODEL OF 99mTc-SESTAMIBI ACCUMULATION
• IN TUMOR CELLS
• MEMBRANE POTENTIAL-DRIVEN PASSIVE INFLUX
• MDR1 P-GLYCOPROTEIN-MEDIATED EFFLUX TRANSPORT

cell membrane
Tc-Complex
Tc-Complex
mito
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Electron Probe X-ray Microanalysis (EPXMA) of a
Cultured Heart Cell Incubated in
Technetium-99-Sestamibi
Electron Density Map Technetium Map
Mitochondrial rich tissue No P-glycoprotein
expression
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Baculoviral Expression of Recombinant Human MDR1
in Host Sf9 Cells Effect on 99mTc-Sestamibi
Accumulation
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Expression of Human MDR1 in Drug-Sensitive MCF-7
Breast Cancer Cells Effect on Doxorubicin
Cytotoxicity and 99mTc-Sestamibi Accumulation
MCF-7/MDR1
Western (mAb C219)
Immunofluorescence (mAb C219)
IRES Plasmid
99mTc-Sestamibi Transport
Cytotoxicity Assay
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Imaging Multidrug Resistance (MDR1)
P-glycoprotein (Pgp) Transport Activity in KB
Tumor Xenografts In Vivo with 99mTc-Sestamibi
99mTc-Sestamibi
201Tl
Pgp-
Pgp-
Pgp
Pgp
Cancer Research
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Tc-99m-Sestamibi Tracer Efflux from Rat Mammary
Tumors In Vivo
Ballinger, et al.
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Molecular Imaging of MDR1 P-glycoprotein Activity
In Breast Cancer 99mTc-Sestamibi
Mammoscintigraphy
1 hr post-injection
1 Tumor
4 hr post-injection
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99mTc-Sestamibi Clearance From Breast Cancers
In Vivo
Low Pgp
High Pgp
Del Vecchio, et al, Eur J Nucl Med 24150, 1997
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Correlation of Tc-Sestamibi Efflux Rate Constants
and MDR1 P-glycoprotein Expression in Breast
Cancer
Del Vecchio, et al, Eur J Nucl Med 24150, 1997
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Correlation of 99mTc-Sestamibi Tumor Efflux Rates
with Expression of MDR1, but not MRP1 or LRP in
Lung Cancer
Zhou, et al. J Nucl Med 421476, 2001
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Effect of the MDR modulator LY 335979 on
Tc-99m-Sestamibi Accumulation into
Drug-Sensitive KB3-1 and MDR KB8-5 Cells
KB 3-1 KB 8-5 KB 8-5-11
Pgp
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Effect of the MDR Modulator PSC 833 on
Transport and Pharmacokinetics of
Tc-99m-Sestamibi In Vivo
Whole Body Posterior View 2 hrs Post-Injection of
Tracer
Pre-PSC 833
Post-PSC 833
Heart
Liver
Kidneys
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Modulator Challenge Test
Adrenocortical Cancer Pulmonary
Metastases Effect of XR-9576 on 99mTc-Sestamibi
Retention 3 Hrs p.i.
Nature Reviews Cancer 2 48-58 (2002)
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Effect of XR9576 on 99mTc-Sestamibi Retention
Fojo, et al., ASCO Meeting (2002)
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Vectors and Vector Delivery
Gene Therapy and Drug Delivery Viral Vectors
(adenovirus, lentivirus) Non-Viral Vectors PEG
co-polymers Naked DNA, RNAi Liposomes Membrane
permeation peptides Tat basic domain Cationic
detergents
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Molecular Imaging Technetium-99mTat Peptide
Conjugates
Bioconjugate Chem 11 762-771, 2000
99mTc-Tat Peptide Complex
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Enzymatic Cleavage in Molecular ImagingGeneral
Structure of a Cell Membrane Permeant Peptide
Imaging Conjugate
Cleavage
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Enzymatically Activated Trapping of an Imaging
Probe 99mTc-Tat Peptide Fragments and
Caspase-3 Protease
Tat peptide Caspase motif
Tc-99m-Chelate
Normal Cell
Committed Apoptotic Cell
(Active Caspase)
(Non Active Pro-Caspase)




Pro-Caspase-3
Caspase-3


Cleavage

Washout
Washout
Enzymatically Amplified Signal Caspase-3
Specific Retention of Tc-99m-Chelate




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Proposed Structure for a Cleavable 99mTc-Tat
Peptide Complex
Tat peptide
Caspase-3 Substrate Sequence
Chelate
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Molecular Imaging

• Monitoring Gene Expression In Vivo
• - Imaging Reporter Genes
• Constitutive exogenous promoter to drive an
  imaging reporter gene for monitoring cells or
  tissues (CMV promoter reporter gene)
• Study regulation of an endogenous promoter with a
  reporter gene (MDR1 promoter reporter gene)
• Engineer imaging transgenic mouse models

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Recombinant Lentivirus Carrying a Firefly
Luciferase Report Gene In Vivo Bioluminescence
Imaging of Luciferase Reporter Stably Transduced
into Hela Cell Tumor Xenografts
Lentivirus with CMV-FLuc Reporter Gene
Transduced Hela Cells
WU Molecular Imaging Center
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Noninvasive Bioluminescence Imaging of Herpes
Simplex Virus 1 (HSV-1) Infection and Therapy in
Living CD-1 Mice
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Bioluminescence Imaging of MDR1 Pgp Transport
Activity InVivo Effect of the MDR Modulator
GF918 on Coelenterazine Accumulation into
Drug-Sensitive KB3-1 and MDR KB8-5-11 Tumor
Xenografts Stably Transfected with R Luc
KB3-1
KB3-1
KB8-5-11
KB8-5-11
(-) GF918
() GF918
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The p53 Pathway
Multiple ways to affect p53 -Loss of sensory
tumor suppressors -Overexpression of cellular
oncogenes or viral oncogenes -Mutation of
p53 itself (most common) but not obligatory
All result in an inability to correctly halt the
cell cycle or induce apoptosis in response to
tumorigenic changes
Modified from J. Weber, Washington Univ.
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Protein Interaction Systems

• Two-hybrid
• Split-ubiquitin
• Protein fragment complementation
• Ras-recruitment
• Resonance energy transfer

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PET Imaging of Reporter Gene Expression
Reporter Gene Construct
Promoter
HSV1-tk Gene
Gene Transfection (Plasmid Vector or Virus)
Endogenous Gene Promoter Elements
Exogenous Reporter Gene
Nucleosides
Reporter Substrate
Reporter Gene HSV1-tk
TK
DRK
18F-FHBG
DPK
Nuc Transporter
18F-FHBG-(PO4)
HSV1-tk mRNA
Blasberg, et al.
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Ribbon Plot of Wild Type HSV1-TK
Gene Ther 61415, 1999
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Expression of Mutant HSV1-tk Reporter Proteins
in Transiently Transfected HeLa Cells
WT tk-GFP sr39tk-GFP mNLS-sr39tk-GFP
Fluorescence Microscopy
Cellular Retention of 8-3H-penciclovir
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Analysis of Protein-Protein Interactions with
Two-Hybrid Technology
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Analysis of Protein-Protein Interactions In Vivo
Inducible Two-Hybrid Imaging System
Inducible Hybrid Proteins
Tet Promoter
Fusion Reporter
mNLS-sr39tk
GFP
Gal4
Gal4
Gal4
Gal4
Gal4
Gal4 Promoter
PNAS 996961, 2002
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MicroPET Imaging of Protein-Protein Interactions
in Living Mice p53/Coat Protein (Negative
Interaction) Versus p53/TAg (Positive Interaction)
Gal4
PNAS 996961, 2002
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MicroPET Imaging of Protein-Protein Interactions
in Living Mice Proportional Reporter Response
with Increased Induction of Protein
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Molecular Imaging
David Piwnica-Worms, M.D., Ph.D. Molecular
Imaging Center
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Acknowledgments

• Molecular Imaging Group, Washington University
• Vijay Sharma
• Gary Luker
• Kathy Luker
• Stefania Violini
• Andrea Pichler
• Julie Prior
• Chris Pica
• Joe Ocheskey
• Wei Li

• Washington University, Medical Oncology
• Nancy Bartlett
• Paula Fracasso
• Joanne Mortimer
• Phil Miller

• Notre Dame
• Alicia Beatty

• Tyco-Mallinckrodt
• Mary Dyszlewski
• Dan Burleigh
• Elizabeth Webb

• Helen Piwnica-Worms
• Jeff Milbrandt
• Farrokh Dehdashti
• Mark Minton
• Barry Siegel
• Michael Welch

FUNDING NCI, DOE