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Developments in Non-Invasive Whole-Body Molecular Tumor imaging

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Developments in Non-Invasive Whole-Body Molecular Tumor imaging Lu Yin (Lucy) Outline Molecular imaging Whole body photonic tumor imaging. Radiolabelled peptides in ... – PowerPoint PPT presentation

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Title: Developments in Non-Invasive Whole-Body Molecular Tumor imaging


1
Developments in Non-Invasive Whole-Body Molecular
Tumor imaging
  • Lu Yin (Lucy)

2
Outline
  • Molecular imaging
  • Whole body photonic tumor imaging.
  • Radiolabelled peptides in molecular imaging
  • Cell penetrating peptide in whole body tumor
    imaging

3
Molecular imaging
  • Molecular imaging, which I define here as the
    noninvasive, quantitative, and repetitive
    imaging of targeted macromolecules and biological
    processes in living organisms, requires two
    basic elements
  • (i) molecular probes whose concentration
    and/or spectral properties are altered by the
    specific biological process under investigation
    and
  • (ii) a means by which to monitor these probes.
  • ---------Harvey R. Herschman
  • Science. 2003 Oct 24302(5645)605-8.

www.medical.philips.com/.../mi_home.jpg
4
Imaging probes
  • Light- or near-infrared (NIR) emitting molecules
  • Radioisotopes labeled molecules
  • Detections
  • Photonic imaging
  • Positron emission tomography (PET)
  • Single photon emission computed
    tomography (SPECT)
  • Magnetic resonance imaging (MRI)
  • X-ray computed tomography (CT)
  • Direct binding ----- monitor receptor e.g.
    (3-(2-18Ffluoroethyl)spiperone(18FFESP)used
    to monitor the dopamine receptors of the striatum
  • Indirect binding ----- monitor the activity
    of their targets e.g. hexokinase substrate
    2-deoxy-2-18Ffluoro-Ddeoxyglucose (FDG), which
    monitors glucose metabolism
  • .

5
Whole-body photonic imaging of small animal
  • Small animal imaging is an important translation
    tool between in vitro research and clinical
    application.
  • Whole-body photonic imaging
  • Planar imaging (muti-photon or confocal
    microscopy)
  • The simplest technique for detecting optical
    reporter molecules in vivo, uses photographic
    principles to capture light emitted from the
    animals---- fluorescence reflectance imaging
    (FRI).
  • Limitations Signal projection view
  • Limited penetration depth
    (lt1mm)
  • Obscured signal due to
    tissue heterogeneity
  • limited spatial resolution
  • Tomography imaging
  • Fluorescence molecular tomography (FMT)
  • Bioluminescence tomography
  • Photoacoustic tomography

6
 Spectral imaging applied to in vivo fluorescence
detection
(a) Standard color image obtained from a green
fluorescence protein (GFP)-expressing mouse
implanted with a red fluorescent protein
(RFP)-expressing tumor. (b) Spectral imaging and
processing improves visualization of the RFP
signal, which can be separated from the mouse
intrinsic auto-fluorescence and GFP fluorescence.
7
Planar imaging VS Tomography imaging
Planar imaing
8
Performance characteristics of Planar and
Tomography imaging
  • FMT gives better resolution with depth
  • FMT gives better signal with varying background
  • FMT is able to image fluorochromes in highly
    diffusive medium that simulates tissue optical
    heterogeneity.

9
Improving spatial sampling
Image reconstruction is based on mathematical
models that describe the composite photon
propagation in tissue and in air. The animal
surface was captured using photo-grammetry, that
is, the mathematical combination of photographs
obtained under different angles to deduce the
physical dimension of the animal.
10
 In vivo fluorescence imaging
11
 Visualization of brain structure and function
using photoacoustic tomography (PAT)
PAT also referred to as optoacoustic or
thermoacoustic tomography, attains the advantage
of combining ultrasonic-scale spatial resolution
with high sensitivity to tissue light absorption
and can yield information on physiology or on
exogenously administered light absorbers.
(a,b) Functional maps of brain activities
corresponding to the left-side (a) and right-side
(b) whisker stimulations, respectively, acquired
with the skin and skull intact.
12
Radiolabeled peptide in oncology
Weiner RE, Thakur ML. BioDrugs.
200519(3)145-63.
13
Radionuclide imaging of small-cell lung cancer
(SCLC) using 99mTc-labeled neurotensin peptide
813
  • Neurotensin (NT) is a 13 amino acid peptide
    originally isolated from calf hypothalamus.
  • It has dual function of neurotransmitter or
    neuromodulator in the nervous system and of local
    hormone in the periphery.

neuromedin N
neurotensin
  • Three subtypes of neurotensin receptors two of
    them belong to the family of G protein-coupled
    receptors, whereas the third one is an entirely
    new type of neuropeptide receptor and is
    identical to
  • gp95/sortilin, a 100 kDa-protein with a single
    transmembrane domain.

Neurotensin (NT) receptors are overexpressed in a
variety of tumors e.g. prostatic tumors and
pancreatic tumor.
Jean-Pierre Vincent, et al. TiPS July 1999
(Vol. 20) Kaijun Zhang, et al. Nuclear Medicine
and Biology 33 (2006) 505512
14
Synthesis of (Na-His)Ac-NT(813) derivative
NT(813), the C-terminal hexapeptide fragment
(Arg8Arg9Pro10Tyr11Ile12Leu13) of NT,
contains the amino acids essential for binding NT
receptors
Redio-labeled with 99mTc(H2O)3(CO)3
15
(No Transcript)
16
Competitive Binding Assays and Affinity Studies
17
Internalization study
18
Biodistribution of 99mTc-(Na-His)Ac-NT(813)
19
In vivo Imaging
20
Cell-penetrating peptides (CPPs)
  • The first CPP came from the discovery that the
    third helix of Antennapedia homeodomain,
    pAntp(4358), can cross biological membranes.
  • Different CPPs have been described to efficiently
    deliver various types of cargo to the inside of
    cells,from low molecular weight drugs to
    liposomes, plasmids, antibodies or nanoparticles.
  • CPPs are peptides made of less than 30 amino
    acids that are internalised via energy-dependent
    or independent mechanisms. Positively charged
    amino acids, hydrophobicity and amphipathicity
    are common features shared among many of the
    known CPPs.

http//hugin.ethz.ch/wuthrich/people/billeter/antp
hd.jpg
http//homeobox.biosci.ki.se/homeo/antp1.gif
Sílvia Pujals et al. Biochimica et Biophysica
Acta 1758 (2006) 264279
21
Internalization mechanisms
Self-assembly
The exact internalization macheniss are still not
clear yet.Initially, CPPs were defined as short
cationic peptides able to translocate through the
plasma membrane of eukaryotic cells via a
receptor- and endocytosis-independent mechanism,
but a re-evaluation of the internalisation
mechanism yielded a new CPP concept.
Endocytosis
.
Sílvia Pujals et al. Biochimica et Biophysica
Acta 1758 (2006) 264279
22
Principal classes of CPPs
Sílvia Pujals et al. Biochimica et Biophysica
Acta 1758 (2006) 264279
23
Tumor imaging by means of proteolytic activation
of cell-penetrating peptides
Fig. 1. Schematic diagram of activatable CPPs.
Cellular uptake induced by a cationic peptide is
blocked by a short stretch of acidic residues
attached by a cleavable linker. Once the linker
is cleaved, the acidic inhibitory domain drifts
away, and the cationic CPP is free to carry its
cargo into cells.
24
How to target tumor?
  • MMP-2 (matrix metalloproteinase ) Cleavable
    linker PLGLAG
  • MMPs
  • Matrix metalloproteinases (MMPs) are a
    large family of zinc metallo-endopeptidases that
    degrade varying components of the extracellular
    matrix in both normal and diseased tissue.
  • More Than 22 members, all of which share a
    common catalytic core containing a zinc molecule
    in the active site.
  • Aberrant MMP activity is believed to be
    associated with a number of pathological
    conditions including rheumatoid arthritis, tumor
    invasion, and metastasis.

MMP-2 and MMP-9, have been widely studied in
breast and prostatic tumors, the growth of which
is often hormone-dependent.
25
ACPPs Adopt a Hairpin Conformation Before
Cleavage
Nuclear Overhauser effects observed in
two-dimensional NMR of a simple ACPP,
succinyl-e8-XPLGLAG-r9-Xk, where X denotes
6-aminohexanoyl. Dashed red lines indicate
observed nuclear Overhauser effects, and the
green line highlights the peptide outline for
clarity.
26
Until Cleaved Off, Polyanionic Sequences Inhibit
Association of CPPs with Cells
27
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28
MMP-2 Cleavable ACPPs Concentrate in Human Tumors
Xenografted into Mice
quantitation
(fluorescence intensity of tumor -
autofluorescence)/(fluorescence of normal
contralateral region - autofluorescence)
mean SE ACPP
2.1 0.17 n 6 scrambled isomer
1.3 0.16, n 2 all-D-amino acid 1.5
0.11, n 4
HT-1080 tumor xenografts visualized with
activatable CPPs. HT-1080 tumors were implanted
into the mammary fat pad of nude mice and allowed
to grow until they reached 5-7 mm in diameter.
(A1) A live anesthetized animal imaged 50 min
after injection with 6 nmol of cleavable peptide.
(A2 and A3) Tumor and muscle histology from a
different animal killed 30 min after injection.
(B1-B3) A similar experiment with the scrambled
peptide. (Scale bars, 30 µm.)
29
Quantitation of Tissue Distribution by Cy5
fluorescence

moles
injected into animal/total body weight
moles of recovered peptide/weight of tissue sample
SUV
30
ACPPs Light Up Human Squamous Cell Carcinomas
ACPP staining of surgically resected human
squamous cell carcinoma tissue. Fresh tumor
tissue was sliced in 1-mm slices and incubated in
1 µM cleavable (A) or uncleavable (B) peptide for
15 min, washed, and frozen. Sections (5 µm) were
taken for fluorescence microscopy by using a 10
objective, and tissue type was verified by
hematoxylin/eosin stain. (A) The arrow indicates
a differentiated keratin pearl. As a control,
histologically normal tissue from the same
patient was treated similarly with MMP-2
cleavable peptide (C) or scrambled peptide (D).
(C) The arrow indicates a ring of invading tumor
cells.
31
  • Contrast, (tumor tissue fluorescence -
    autofluorescence)/(normal tissue fluorescence -
    autofluorescence),was almost eight in this
    example.
  • Contrast tended to be greatest where the tumor
    tissue had a high histologic grade of malignancy.
  • lymphocytic granulation tissue was nearly as
    bright as the tumors themselves, possibly because
    of the release of MMPs from lymphocytes.
  • Normal tissue immediately adjacent to tumor
    tissue was noticeably brighter than more remote
    normal tissue, possibly because of the presence
    of immune cells or to diffusion of the soluble
    proteases.

32
Conclusion
  • For whole-body photonic imaging, tomography
    imaging gives better results compared with planar
    imaging. PAT will be the possible future
    direction of photonic imaging.
  • Tumor imaging largely rely on probes that can
    target the tumor
  • The 99mTc-labeled neurotensin peptide 813 could
    image the small-cell lung cancer (SCLC) by
    targeting overexpressed neutotesin recpetor.
  • MMP-2 proteolytic activation of cell-penetrating
    peptides can target the MMP-2 positive tumor
    without target specific receptor. It is a novel
    method that can be applied to tumor imaging.

33
Thank you
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