Non Invasive Whole Body Molecular Tumor imaging

Document Sample
Non Invasive Whole Body Molecular Tumor imaging Powered By Docstoc
					Developments in Non-Invasive Whole-
   Body Molecular Tumor imaging

           Lu Yin (Lucy)

• Molecular imaging

• Whole body photonic tumor imaging.

• Radiolabelled peptides in molecular imaging

• Cell penetrating peptide in whole body tumor
                 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 24;302(5645):605-8.

                              Imaging probes

•    Light- or near-infrared (NIR) emitting molecules
     Radioisotopes labeled molecules
      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-
    [18F]fluoroethyl)spiperone([18F]FESP)}used to monitor the dopamine
    receptors of the striatum

    Indirect binding ----- monitor the activity of their targets e.g. hexokinase
     substrate 2-deoxy-2-[18F]fluoro-Ddeoxyglucose (FDG), which monitors
     glucose metabolism

  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 (<1mm)
               Obscured signal due to tissue heterogeneity
               limited spatial resolution
• Tomography imaging
  Fluorescence molecular tomography (FMT)
  Bioluminescence tomography
  Photoacoustic tomography
        Spectral imaging applied to in vivo fluorescence


(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.
Planar imaging VS Tomography imaging

             Planar imaing
   Performance characteristics of Planar and
            Tomography imaging

A. FMT gives better resolution with depth
B. FMT gives better signal with varying
C. FMT is able to image fluorochromes in
   highly diffusive medium that simulates
   tissue optical heterogeneity.
               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.
In vivo fluorescence imaging
        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
Radiolabeled peptide in oncology

             Weiner RE, Thakur ML. BioDrugs. 2005;19(3):145-63.
     Radionuclide imaging of small-cell lung cancer (SCLC)
         using 99mTc-labeled neurotensin peptide 8–13

•Neurotensin (NT) is a 13 amino acid
peptide originally isolated from calf
                                                                neuromedin N
It has dual function of neurotransmitter
or neuromodulator in the nervous                                neurotensin
system and of local hormone in the
•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) 505–512
 Synthesis of (Na-His)Ac-NT(8–13) derivative

  NT(8–13), the C-terminal hexapeptide fragment (Arg8–Arg9–
  Pro10–Tyr11–Ile12–Leu13) of NT, contains the amino acids
  essential for binding NT receptors

Redio-labeled with [99mTc(H2O)3(CO)3]+
Competitive Binding Assays and Affinity
Internalization study
Biodistribution of 99mTc-(Nα-His)Ac-NT(8–13)
In vivo Imaging
              Cell-penetrating peptides (CPPs)
     • The first CPP came from the discovery that the
       third helix of Antennapedia homeodomain,
       pAntp(43–58), 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,                   ople/billeter/antphd.jpg
       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.

Sílvia Pujals et al. Biochimica et Biophysica Acta 1758 (2006) 264–279
                    Internalization mechanisms


                                                     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
  .     Endocytosis                                  mechanism, but a re-evaluation of the
                                                     internalisation mechanism yielded a new
Sílvia Pujals et al. Biochimica et Biophysica Acta   CPP concept.
1758 (2006) 264–279
                            Principal classes of CPPs

Sílvia Pujals et al. Biochimica et Biophysica Acta 1758 (2006) 264–279
          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.
                   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
                                MMP-2 and MMP-9, have been
                                widely studied in breast and prostatic
                                tumors, the growth of which is often
    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.
Until Cleaved Off, Polyanionic Sequences
 Inhibit Association of CPPs with Cells
      MMP-2 Cleavable ACPPs Concentrate in
       Human Tumors Xenografted into Mice

 (fluorescence intensity of tumor -
 of normal contralateral region -

                   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.)
  Quantitation of Tissue Distribution by Cy5
      moles of recovered peptide/weight of tissue sample
         moles injected into animal/total body weight
       ACPPs Light Up Human Squamous Cell

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.
• Contrast, (tumor tissue fluorescence - autofluorescence)/(normal
  tissue fluorescence - autofluorescence),was almost eight in this
• 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
• 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.

• 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 8–13 could image the small-
  cell lung cancer (SCLC) by targeting overexpressed neutotesin

• 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.
Thank you

mikesanye mikesanye