PositronEmitting Pharmaceuticals for Imaging of Tumor Hypoxia

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Mt Sinai Medical CenterNew York, NY, March 25,
2005
Optical in vivo ImagingPrinciples and
Applications
Pat Zanzonico, PhDMemorial Sloan-Kettering
Cancer CenterNew York, NY ZanzoniP_at_MSKCC.ORG
Disclosure Ivis (Xenogen) devices used
exclusively at MSKCC
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MSKCC Small-Animal Imaging Core

• microPET - R4 - Focus 120 (SIG-funded)
• microCT - microCAT II
• SPECT-CT - X-SPECT X-O (SIG-funded)
• Optical - Ivis (Bioluminesce Fluorescence)
• MRI/MRS - 4.7-T Unit Bruker console - 7-T Unit
  

All small-animal imaging modalities will be
incorporated into a clean Vivarium in a new
Research Tower (2006)
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Optical in vivo Imaging Luminescence and
Fluorescence
Fluorescence
Luminescence
2DProjection ImagesNO depth (3D)information
Light originates in labeled cell
Light originatesexogeneously
?em - ?ex Stokes shift
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Optical in vivo Imaging Applications

• Non-invasive detection, localization, and
  quantitation of cells
• Longitudinal studies of natural history of
  disease? Disease progression and
  dissemination? Response to therapy
• Stem cell / Viral trafficking

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Optical in vivo Imaging The Pros

• Long-term labeling of cells? Cells and their
  progeny stably transduced
• Light ? of Cells ? Quantitation
• Low BGs ? High SNRs
• Non-invasive? Does not require animal to be
  sacrificed? Longitudinal imaging studies? Each
  animal serves as its own control
• No radioactivity / radiation
• Simple instrumentation and analysis software?
  Operated by end-user
• Inexpensive - lt ¼ cost of microPET etc

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Optical in vivo Imaging The Cons

• Requires stable genetic transduction ? Loss of
  signal may reflect silencing of reporter
  rather than regression of cells
• Coarse spatial resolution (1 cm)
• Signal is only semi-quantitative? No correction
  for attenuation of light? Decreasing signal with
  increasing depth
• 2D projection images provide no 3D(depth)
  information

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Optical in vivo Imaging Paradigm

• Visible light is emitted by exogeneous labeled
  (ie genetically transduced) cells within animal
• Most light traveling through tissue is absorbed
  but some is scattered - many times - and creates
  a "fuzzy" signal at animals surface
• IVIS Imaging System views the diffuse surface
  image with a charged-couple detector (CCD) - a
  digital camera - and produces a 2D projection
  image of light distribution

Rule of thumb Area (fuzziness) of
surfacesignal ? Depth of signal
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Optical in vivo ImagingThe Reporter
Gene-Reporter Substrate System
OPTIONAL cell-surface markerfor
FACS/Fluorescence microscopy imaging
Light
GFP
nucleus
NUCLEUS
ATP O2
AdministeredLuciferin
Luciferin
Oxy-luciferin
FLuc
Diffusible
ReporterGene Product
ReporterGene(s)
Vector Retrovirus w/FLuc GFP genes

• Luciferases other than Firefly Luciferase are
  used
• Vectors other than Retroviruses are used

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Optical in vivo Imaging Quantitation (ie
Semi-Quantitation) Light emitted at surface
increases withnumber of living - luminescent or
fluorescent - cells in tumor ? Measure of
tumor size (growth) at a fixed location
Other than tumor cells, labeled cells may be
stem cells, bacteria etc
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The Electromagnetic (EM)- and Visible Light -
Spectra
Increasing Tissue Penetrability
700 nm
400 nm
Infrared
Ultraviolet
Increasing Energy and FrequencyDecreasing
Wavelength
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Solid Angle andQuantitation of Light
Area A
Inverse Square Law F1 r12 F2 r22 where Fi
the photon flux at distance ri
Field of View (FOV)Area A
?
Sphere Radius r
Detector
Solid Angle ?steradian(sr)
Point P
Distance r
For a given light source, the detected photon
flux F (photons/sec) normalized to the FOV A
and solid angle ? - ie is independent of the
source-to-detector distance r and f stop, ie
photon/sec/cm2/sr is constant
Corrects for inverse-square law effect
Corrects for effect of different lens apertures
(f stops)
Radiance
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Charge-Coupled Detector Digital Camera
Charge produced?Light absorbed
Dx
Dy
n x n Binning
Small (Hi-Res) 1x1 Medium 2x2
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Charge-Coupled Detector Dark Current
Even at room T, some e may be sufficiently
energized to jump from the valence to conduction
band in the absence of light, producing a
background, or dark, current
Operating T ? -120 ?C
Thermal energy
CooledCCD
Refrigerant Line
RefrigerantControl Unit
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The Ivis 100 Optical Imaging System
CCDChip
Cooled CCD
Lights
HeatedAnimal Stage
Data Acquisition and Analysis PC
Light-tight Enclosure
RefrigerantControl Unit
OPTIONALFluorescence Light Source
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The Ivis 200 Optical Imaging System
New Features

• Integrated design
• New lens
• Improved light collection
• zooms closer
• Built-in fluorescence
• Computerized alignment
• Surface topography
• Quasi-tomographic (3D) imaging capability

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Galvanometer
Ivis 200 Optical Imaging System

• Mirror pair with magnetic actuators allows
  computer controlled steering of laser beam
  (Green l 532 nm)

Alignment
Surface Topography
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Ivis Optical Imaging System Basic Protocol

• Inject / infuse genetically transduced (eg
  Luciferase- or GFP-expressing) cells - 1x106
  cell iv, ip, or subQ
• Days to weeks later, for luminescent imaging
  inject Luciferin - 300 mg in 200 ml PBS iv
  or ip 10-20 min prior to imaging
• Initialize system - T ? -120 ?C
• Anesthetize animals for imaging - 1.5
  Isofluorane _at_ 1 l/min
• Select stage position - A for 5 mice ? D (or E)
  for 1 mouse
• Image anesthetized animal(s) - Photograph ? 2-4
  min for luminescent or fluorescent imaging with
  Medium binning and f stop 16
• Noisy images - Increase exposure time (Decrease
  f stop)Saturated Images - Decrease exposure
  time
• Viewing/analyzing images - Absolute units
  photons/sec/cm2/sr

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The Ivis Optical Imaging SystemLiving Image
SoftwareControl Panel Window
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The Ivis Optical Imaging System
Overlay of Sequential Photographand Luminescent
or Fluorescent Image
1) Photograph
2) Luminescent or Fluorescent Image
1 sec
2 min

3) Overlayed Images
Shorter w/ Smaller f stop (larger
aperture) Coarser binning
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Ivis Fields of View (FOVs)
A 25 cm
B 19.5 cm
C 13 cm
D 6.5 cm
E 3.9 cm
FOVcm
Resolutionmm
200 System only
A 3.9 B 6.5 C 13 D 19.5 E
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60 100 130 190 330

NOT forFluoro
System is calibrated and therefore
quantitative ONLY at the preset stage positions
A, B, C, D, and E
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Attenuation of Light Beers Law aka Beer-Lambert
Law
Attenuation Scatter Absorption
dI ? I dx dI m I dx dI / I m dx (dI / I)
/ x m I(x)?I(0)dI / I x?0 mx dx
I
I-dI
I(0)
I(x)
I(x) I(0) e-mx
dx
where I Intensity in photons/cm2/sec x thi
ckness of absorber in mm m linear
attenuation coefficient in mm-1
x
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Attenuation of Light Beers Law aka Beer-Lambert
Law Transmitted Intensity vs Depth in Tissue
Linear Plot
Semi-Log Plot

• Optical in vivo imaging is only SEMI-QUANTITATIVE
• The image signal depends on the DEPTH of the
  cells
• NO correction is currently available for this
  depth-dependence

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Tissue is not transparent to visible
light Transmission depends on wavelenght (l)
Luciferase Emission Spectra
Detectable Cells in Tissue
SensitivityMinimum ofdetectable cells???
Exogenously introduced PC3M cells
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Tissue is not transparent to visible
light Transmission depends on tissue composition
Less transmission through bone (skull) than soft
tissue
FLuc Bioluminescence
Partially differentitated human neuronal stem
cells
175,000 FLuc cells
60,000 FLuc cells
Courtesy of Michelle Bradbury, MSKCC
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Comparative "Depth" SensitivityOptical vs
Radionuclide Imaging
Detection of Hypoxia in R3327-AT Prostate Tumor
Xenografts in Nude Mice
Expression of hypoxia-inducedhsv tk by I124-FIAU
microPET imaging
Expression of hypoxia-inducedGFP by
Ivisfluorescent imaging
Genetically transduced cellsGFP and hsv tk
genesunder control of hypoxia responsive
element
8 hr pi of 150 mCi
Courtesy of Gloria Li, MSKCC
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Fluorescence
Ivis 100 Optical Imaging System

• 6-position computer-controlled Excitation and
  Emission filter wheels

GFP RFP Cy5.5 ICG
150-W Tungsten/Halogen lampw/ computer-controlled
intensity
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Fluorescence
Ivis 200 Optical Imaging System

• 24-position computer-controlled emission filter
  wheels
• 6-position computer-controlled excitation filter
  wheel

150-W Tungsten/Halogen lamp with
computer-controlled intensity
Low autofluorescence optics and fiber optics
with out-of-band blocking filters
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Bioluminescence vs FluorescenceOptical in vivo
Imaging
Bioluminescent Image
Fluorescent Image
Bioluminescence (107 cells) Background flux
4.3?103 p/s Signal flux 1.2?107
p/s Signal/background 2700
DsRed transient transfection Background flux
5.1?109 p/s Signal flux 1.1?1010
p/s Signal/background 2.2
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MultispectralFluorescence in vivo Imaging
Maestro, Cambridge Research and Instrumentation
(CRI)
Proprietary filter- and modeling- based
technology for reducing autofluorescence
Standard fluorescent image
Maestro
Comparison of 630 20 nm bandpass image vs
Unmixed quantum dot antibody image Mouse on
right Negative control
Courtesy of CRI and Shuming Nie, Emory University
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MultispectralFluorescence in vivo Imaging
Maestro, Cambridge Research and Instrumentation
(CRI)
GFP
food autofl.
mPlum
RFP
Standard fluorescent image
GFP
mPlum
RFP
Normalized Spectra
Unmixed composite - Maestro
Courtesy of CRI and Roger Tsien, UCSD
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The Ivis Optical Imaging SystemQuality Control
(QC) - Daily
1) Constancy and Uniformity
10 LSC vials filled with equal amounts of 3H-H2O
and of scintillation cocktail plus 1 vial filled
with scintillation cocktail only (BG)
Uniformity SD of Radiance
Service
Constancy Mean Radiance
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The Ivis Optical Imaging SystemQuality Control
(QC) - Daily
2) Linearity
5 LSC vials filled with serial dilutions of
3H-H2O and scintillation cocktail plus 1 vial
filled with scintillation cocktail only (BG)
Service
Todays Data andStraight-line FitRadiancevs
3H Activity
Slope ofRadiance vs 3H ActivityStraight-line Fit
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Disease Detection and Progression Bioluminescent,
microPET, and microCT ImagesMouse 21 d post-iv
injection of Raji lymphoma cells
FLuc Bioluminescence
FusedmicroPET microCT
18F-FDGmicroPET
HE
Sagittal
Dorsal
Ventral
Transverse
Courtesy of Dr. Elmer Santos, MSKCC
Brentjens et al. Nat Med 9 279, 2003
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Disease Detection and Progression Serial
Bioluminescent Images of Myeloma-Inoculated Mice
SCID mice inoculated iv w/ MM.1/eGFP-FLuc Cells
(10x106)
Day 23
Day 37
Day 43
Dorsal
Ventral
Courtesy of Kaida Wu and Malcolm AS Moore, MSKCC
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Monitoring of Therapeutic Response Serial
Bioluminescent, MRI, and microCT Images Mouse
PSMA FLuc-transduced RM1 prostate cancer cells
ivtreated w/ PSMA-specific (PZ1) or non-specific
(19Z1) T cells
FLucBioluminescence
microMRI
microCT
ControlNon-specific (19Z1) T cells
Day 7
3
x106
2
Day 13
1
TxPSMA-specific (PZ1) T cells
Therapeutic Effect orSilencing of Reporter?
Day 13
Courtesy ofElmer Santos, MSKCC
Dorsal
Ventral
Transverse
Transverse
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Trafficking of Anti-tumor Virus to Metastatic
Disease ES-2-Tumor Inoculated SCID Mice
Treated with FLuc-Transduced Sindbis Virus
ES-2 ovarian ca cell inoculation ip 2x106 cells
Sindbis/FLucvirus ip
High-affinity binding to laminin receptors
(LAMR) upregulated in many human tumors,
including ES-2 ovarian ca
5 d
Tseng and Meruelo, Nat Biotech 2270, 2004 Tseng
and Meruelo, Cancer Res 64 6684, 2004
Courtesy of Jen-Chieh Tseng and Daniel Meruelo,
NYU
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Trafficking of Anti-tumor Virus to Metastatic
Disease ES-2-Tumor Inoculated SCID Mice
Treated with FLuc-Transduced Sindbis Virus
3 Daily ip Treatments
Sindbis/Fluc
Mean counts/sec/cm2/sr
Sindbis/HSV tk
microPET image 2 hr post-iv injectionof
partiallymetabolized (trapped)thyrmidine
analog F18-FEAU
Courtesy of Jen-Chieh Tseng and Daniel Meruelo,
NYU
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• Trafficking of Hematopoietic Stem Cells
• C57BL/6 mouse bone marrow cells retrovirally
  transduced to express the HSV TK-EGFP-FLuc
  reporter gene
• Serial bioluminescence imaging following systemic
  administration to lethally irradiated recipients

Day Post-Administration
1
2
3
4
14
21
28
Courtesy of Ron Blasberg, MSKCC
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Optical in vivo Imaging 2D Projection Images No
depth (3D) information

• Image multiple projections - Dorsal and
  Ventral - Right and Left Lateral

Cells aremore dorsal
Dorsal
Ventral

• Tomographic (3D) imaging

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Optical in vivo Imaging Tomographic (3D) Imaging
- Work in progress
Measuredlight signal
5.4 mm
Multiple projections of body contoursSurface
Topography (Ivis 200)
Top(Ventral)View
LateralPerspectiveView
Source
Reconstruction - based on measured light signal
at multiple angular views, surface topography,
modeling of light attenuation - of 3D source
distribution in vivo
Courtesy ofXenogen Corp
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Summary and ConclusionOptical in vivo Imaging

• Turn-key, easy-to-use systems - Marketed by
  Xenogen (and others)
• Luminescent and Fluorescent systems
• Semi-quantitative - No depth (3D) information or
  correction for light attenuation
• Routinely used in many animal models Tumor,
  pathogen, stem-cell, and virus location, growth
  and dissemination, and/or therapy-induced
  regression
• Tomographic (3D) optical imaging systems - Under
  development
• The most widely used and cost-effective (?)
  small-animal imaging modality

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Thank You!