Development of a new positron emission tomography tracer for targeting tumor angiogenesis: synthesis, small animal imaging, and radiation dosimetry.

Angiogenesis plays a key role in cancer progression and correlates with disease aggressiveness and poor clinical outcomes. Affinity ligands discovered by screening phage display random peptide libraries can be engineered to molecularly target tumor blood vessels for noninvasive imaging and early detection of tumor aggressiveness. In this study, we tested the ability of a phage-display-selected peptide sequence recognizing specifically bone marrow- derived pro-angiogenic tumor-homing cells, the QFP-peptide, radiolabeled with 64Cu radioisotope to selectively image tumor vasculature in vivo by positron emission tomography (PET). To prepare the targeted PET tracer we modified QFP-phage with the DOTA chelator and radiolabeled the purified QFP-phage-DOTA intermediate with 64Cu to obtain QFP-targeted radioconjugate with high radiopharmaceutical yield and specific activity. We evaluated the new PET tracer in vivo in a subcutaneous (s.c.) Lewis lung carcinoma (LLC) mouse model and conducted tissue distribution, small animal PET/CT imaging study, autoradiography, histology, fluorescence imaging, and dosimetry assessments. The results from this study show that, in the context of the s.c. LLC immunocompetent mouse model, the QFP-tracer can target tumor blood vessels selectively. However, further optimization of the biodistribution and dosimetry profile of the tracer is necessary to ensure efficient radiopharmaceutical applications enabled by the biological specificity of the QFP-peptide.

Recombinant human interleukin-11 treatment enhances collateral vessel growth after femoral artery ligation.

OBJECTIVE: To investigate the role of recombinant human interleukin-11 (rhIL-11) on in vivo mobilization of CD34(+)/vascular endothelial growth factor receptor (VEGFR) 2(+) mononuclear cells and collateral vessel remodeling in a mouse model of hindlimb ischemia. METHODS AND RESULTS: We observed that treatment of Sv129 mice with continuous infusion of 200-µg/kg rhIL-11 per day led to in vivo mobilization of CD34(+)/VEGFR2(+) cells that peaked at 72 hours. Sv129 mice pretreated with rhIL-11 for 72 hours before femoral artery ligation showed a 3-fold increase in plantar vessel perfusion, leading to faster blood flow recovery; and a 20-fold increase in circulating CD34(+)/VEGFR2(+) cells after 8 days of rhIL-11 treatment. Histologically, experimental mice had a 3-fold increase in collateral vessel luminal diameter after 21 days of rhIL-11 treatment and a 4.4-fold influx of perivascular CD34(+)/VEGFR2(+) cells after 8 days of therapy. Functionally, rhIL-11-treated mice showed better hindlimb appearance and use scores when compared with syngeneic mice treated with PBS under the same experimental conditions. CONCLUSIONS: These novel findings show that rhIL-11 promotes in vivo mobilization of CD34(+)/VEGFR2(+) mononuclear cells, enhances collateral vessel growth, and increases recovery of perfusion after femoral artery ligation. Thus, rhIL-11 has a promising role for development as an adjunctive treatment of patients with peripheral vascular disease.

Efficient In Vivo Selection of a Novel Tumor-Associated Peptide from a Phage Display Library.

We developed a screening procedure to identify ligands from a phage display random peptide library that are selective for circulating bone marrow derived cells homing to angiogenic tumors. Panning the library on blood outgrowth endothelial cell suspension in vitro followed by in vivo selection based on homing of bone marrow-bound phage to angiogenic tumors, yielded the peptide QFPPKLTNNSML. Upon intravenous injection phage displaying this peptide homed to Lewis lung carcinoma (LLC) tumors in vivo whereas control phage did not localize to tumor tissue. Phage carrying the QFPPKLTNNSML peptide labeled with 64Cu radionuclide when administered intravenously into a tumor bearing mouse was detected noninvasively with positron emission tomography (PET) around the tumor. These proof-of-principle experiments demonstrate the ability of the QFPPKLTNNSML peptide to deliver payload (radiolabeled phage conjugates) in vivo to sites of ongoing angiogenesis and point to its potential clinical utility in a variety of physiologic and pathologic processes where neovascular growth is a critical component.

Selective endothelial cell attachment to peptide-modified terpolymers.

In a previous report we screened a combinatorial peptide library to identify novel ligands that bind with high affinity and specificity to human blood outgrowth endothelial cells (HBOEC). In this study we demonstrate the use of the phage display-selected-HBOEC-specific peptides as a tool to direct and modulate endothelial cell (EC) behavior with a focus on designing functional biomaterials intended for use in cardiovascular applications. First, we ensured that our peptide ligands did not interfere with EC function as tested by proliferation, migration, tube formation, and response to vascular endothelial growth factor. Second, peptides that supported EC function were incorporated into methacrylic terpolymers via chain transfer free radical polymerization. The HBOEC-specific peptide, TPSLEQRTVYAK, when covalently coupled to a terpolymer matrix, retained binding affinity towards HBOEC in a serum-free medium. Under the same binding conditions, the attachment of human umbilical vein endothelial cells (HUVEC) was limited, thus establishing HBOEC specificity. To our knowledge, this is the first report demonstrating specificity in binding to peptide-modified biomaterials of mature EC, i.e., HUVEC, and EC of progenitor origin such as HBOEC. The findings from this work could facilitate the development of autologous cell therapies with which to treat cardiovascular disease.

Data on biodistribution and radiation absorbed dose profile of a novel (64)Cu-labeled high affinity cell-specific peptide for positron emission tomography imaging of tumor vasculature.

New peptide-based diagnostic and therapeutic approaches hold promise for highly selective targeting of cancer leading to more precise and effective diagnostic and therapeutic modalities. An important feature of these approaches is to reach the tumor tissue while limiting or minimizing the dose to normal organs. In this context, efforts to design and engineer materials with optimal in vivo targeting and clearance properties are important. This Data In Brief article reports on biodistribution and radiation absorbed dose profile of a novel high affinity radiopeptide specific for bone marrow-derived tumor vasculature. Background information on the design, preparation, and in vivo characterization of this peptide-based targeted radiodiagnostic is described in the article "Synthesis and comparative evaluation of novel 64Cu-labeled high affinity cell-specific peptides for positron emission tomography of tumor vasculature" (Merrill et al., 2016) [1]. Here we report biodistribution measurements in mice and calculate the radiation absorbed doses to normal organs using a modified Medical Internal Radiation Dosimetry (MIRD) methodology that accounts for physical and geometric factors and cross-organ beta doses.

Synthesis and comparative evaluation of novel (64)Cu-labeled high affinity cell-specific peptides for positron emission tomography imaging of tumor vasculature.

Tumor angiogenesis, the formation of new tumor blood supply, has been recognized as a hallmark of cancer and represents an important target for clinical management of various angiogenesis-dependent solid tumors. Previously, by screening a bacteriophage peptide library we have discovered the FHT-peptide sequence that binds specifically to bone marrow-derived tumor vasculature with high affinity. Here in an effort to determine the potential of the FHT-peptide for in vivo positron emission tomography (PET) imaging of aggressive tumor vasculature we studied four FHT-derivatives: NOTA-FHT, NOTA-(FHT)2, NOTA-PEG-FHT, and NOTA-PEG-(FHT)2. These peptide analogs were synthesized, labeled with the PET radionuclide (64)Cu, and characterized side-by-side with small animal PET and computed tomography imaging (microPET/CT) at 1 h, 4 h, and 24 h post injection in a subcutaneous Lewis lung carcinoma (LLC) tumor model. Because of its excellent in vivo kinetic properties and high tumor-to-background ratio, the (64)Cu-NOTA-FHT radiopeptide was selected for more detailed evaluation. Blocking studies with excess of unlabeled peptide showed specific and peptide mediated (64)Cu-NOTA-FHT tumor uptake. Biodistribution experiments in the same tumor model confirmed microPET/CT imaging results. Human radiation absorbed dose extrapolated from rodent biodistribution of (64)Cu-NOTA-FHT revealed favorable dosimetry profile. The findings from this investigation warrant further development of (64)Cu-NOTA-FHT as a potential targeted diagnostic radiopharmaceutical for PET imaging of aggressive tumor vasculature.

Oxidative and hydrolytic stability of a novel acrylic terpolymer for biomedical applications.

Oxidative and hydrolytic biostability assessment was carried out on a novel acrylic material made of hexamethyl methacrylate (HMA), methyl methacrylate (MMA), and methacrylic acid (MAA). To simulate the in vivo microenvironment, solutions of H2O2/CoCl2 and buffered solutions of cholesterol esterase (CE) and phospholipase A2 (PLA) were used. As controls, film specimens were incubated in deionized water. Samples were incubated in these solutions at 37 degrees C for 10 weeks before physical and mechanical properties were evaluated by size exclusion chromatography (SEC), 1H- nuclear magnetic resonance (1H-NMR), acid-base titration, and Instron tensile testing. The results from this study indicate excellent biostability of HMA-MMA-MAA terpolymers and thus their potential for use in biomedical devices for long-term implantation.

Selection and initial characterization of novel peptide ligands that bind specifically to human blood outgrowth endothelial cells.

Using phage display technology, we have isolated 12-mer peptide ligands that bind to human blood outgrowth endothelial cells (HBOEC). To avoid non-specific binding we apply negative-positive selection approach by pre-incubating the library with non-HBOEC. The selected phage clones bind to their target cell population with high recovery. Moreover, the isolated clones display outstanding cell specificity as no significant binding is observed on a panel of other cell types. We anticipate the findings from this work to be exploited in the development of future cell-based therapeutic revascularization approaches to ischemic disease and endothelial injury or in combination with biomedical devices.