title: Utilizing the Multiradionuclide Resolving Power of SPECT and Dual Radiolabeled Single Molecules to Assess Treatment Response of Tumors.
abstract: OBJECTIVE
  Single photon emission computed tomography (SPECT) radionuclide pairs having distinct decay rates and different energy maxima enable simultaneous detection of dual gamma signals and real-time assessment of dynamic functional and molecular processes in vivo. Here, we report image acquisition and quantification protocols for a single molecule labeled with two different radionuclides for functional SPECT imaging.
  
  
  METHODS
  LS370 and LS734 were prepared using modular solid phase peptide synthesis. Each agent has a caspase-3 cleavable reporting motif, flanked by a tyrosine residue and a chelator at the opposite end of molecule. Cell uptake and efflux were assessed in human MDA-MB-231 breast cancer cells. Biodistribution studies were conducted in tumor naive and orthotopic 4T1 metastatic breast cancer tumor mice. NanoSPECT dual-imaging validation and attenuation correction parameters were developed using phantom vials containing varying radionuclide concentrations. Proof-of-principle SPECT imaging was performed in MMTV-PyMT transgenic mice.
  
  
  RESULTS
  LS370 and LS734 were singly or dually radiolabeled with (125)I and (111)In or (99m)Tc. Cell assays demonstrated 11-fold higher percent uptake (P < 0.001) of [(125)I]LS734 (3.6 ± 0.5) compared to [(125)I]LS370 (0.3 ± 0.3) at 2 h. Following chemotherapy, cellular retention of [(125)I]LS734 was 3-fold higher (P < 0.05) than untreated cells. Pharmacokinetics at 1 h postinjection demonstrated longer blood retention (%ID/g) for [(125)I]LS734 (3.2 ± 0.9) compared to [(125)I]LS370 (1.6 ± 0.1). In mice bearing bilateral orthotopic 4T1 tumors, the uptake (%ID/g) was 2.4 ± 0.3 for [(125)I]LS734 and 1.2 ± 0.03 for [(125)I]LS370. The iodinated tyrosine peptide residue label was stable under in vitro conditions for up to 24 h; rapid systemic deiodination (high thyroid uptake) was observed in vivo. Phantom studies using standards demonstrated deconvolution of radionuclide signals based on different gamma ray energies. In MMTV-PyMT mice imaged with dual-labeled [(111)In]-[(125)I]LS734, the gamma signals were separable and quantifiable.
  
  
  CONCLUSIONS
  Image processing protocols were developed for quantitative signal separation resulting from a caspase-3 responsive dual-radiolabeled SPECT probe. Crosstalk unmixing was obtained for multiradionuclide NanoSPECT imaging. In vitro and in vivo data demonstrated structure-activity relationships for developing functional agents for ratiometric SPECT imaging.
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA. baogangx@mir.wustl.edu.
authors: Baogang
authors: B
authors: Xu
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA.
authors: Monica
authors: M
authors: Shokeen
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA.
authors: Gail P
authors: GP
authors: Sudlow
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA.
authors: Scott E
authors: SE
authors: Harpstrite
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA.
authors: Kexian
authors: K
authors: Liang
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA.
authors: Philip P
authors: PP
authors: Cheney
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA.
authors: W Barry
authors: WB
authors: Edwards
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA.
authors: Vijay
authors: V
authors: Sharma
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA.
authors: Richard
authors: R
authors: Laforest
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA.
authors: Walter J
authors: WJ
authors: Akers
authors: Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, MO, 63110, USA. achilefus@mir.wustl.edu.
authors: Samuel
authors: S
authors: Achilefu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008807:Mice, Inbred BALB C; D057054:Molecular Imaging / Q000379:methods*; D009369:Neoplasms / Q000000981:diagnostic imaging* / Q000188:drug therapy* / Q000473:pathology; D019275:Radiopharmaceuticals / Q000737:chemistry / Q000493:pharmacokinetics*; D014018:Tissue Distribution; D015899:Tomography, Emission-Computed, Single-Photon / Q000379:methods*
chemical_list: D000970:Antineoplastic Agents; D019275:Radiopharmaceuticals; D053148:Caspase 3
keywords: Apoptosis; Cancer; Caspase; Cleavable peptide; Programmed cell death; Radionuclide; SPECT