TIP-1 specific antibody blocked the HVGGSSV peptide binding within irradiated tumor.

<p><b>A</b>: Specificity of the TIP-1 antibody as revealed with western blot analysis of whole LLC cell lysate. The endogenous TIP-1 protein (∼14 kD) recognized by the antibody was pointed with arrow. <b>B</b>: Specificity of the TIP-1 antibody as demonstrated in immunofluorescent staining of LLC cells that were transfected with shRNA plasmids. Effect of the TIP-1 targeting shRNA on TIP-1 expression was determined with western blot analysis (upper panel). In the cell staining, the transfected cells were tracked with GFP protein expression from the shRNA plasmids. TIP-1 was stained as red with the TIP-1 antibody. Cell nuclei were stained with DAPI. The LLC cells transfected with the control shRNA that did not affect TIP-1 expression were pointed with arrow head, while the cells transfected with TIP-1 targeting shRNA that abolished TIP-1 expression were pointed with arrows (lower panel). <b>C</b>: ELISA-based <i>in vitro</i> competition assay. Serially diluted antibodies were respectively pre-incubated with the purified GST/TIP-1 proteins (100 ng/well) before the complex was added to the plates coated with the HVGGSSV peptide (50 ng/well). The GST/TIP-1 protein associated to the immobilized HVGGSSV peptide was detected with GST-specific antibody. <b>D</b>: Optical images of LLC tumor-bearing mice that were co-administrated with the Alexa Fluor 750-labeled HVGGSSV peptide and antibodies. LLC tumors in the left hind limbs were irradiated at 5 Gy (pointed with arrows). 200 µg of the TIP-1 antibody, or the control antibody, was injected at 2 hours post the IR treatment, followed by injection of Alexa Fluor 750-labeled HVGGSSV peptide at 4 hours post the IR treatment. Optical images were acquired 24 hours after the peptide injection. The presented data represent three independent experiments.</p

Radiation induced TIP-1 translocation onto the plasma membrane of the cancer cells.

<p><b>A</b>: Flow cytometric profile of TIP-1 expression on the H460 cell surface. TIP-1 on the cell surface was detected with the TIP-1 antibody 24 hours after radiation treatment, a control IgG was included to demonstrate the antibody specificity. <b>B</b>: Fluorescent staining of the TIP-1 expression (green) on the cell surface of the irradiated H460 cells. DAPI was used for counterstaining. <b>C</b>: Time course study. The H460 cells were irradiated at 5 Gy and then fixed at variable time points after irradiation for flow cytometric analysis of the TIP-1 expression on the cell surface. Percentage of the TIP-1 positive cells was presented. <b>D</b>: The dose-dependence study. The H460 cells were irradiated with variable dose of X-ray, the cells were fixed 24 hours post the irradiation for profiling the TIP-1 expression on the cell surface with flow cytometry. Fold change of the TIP-1 positive cells was calculated by comparison to the untreated cells (counted as 1). <b>E</b>: Western blot analysis of TIP-1 expression within the TIP-1 positive or negative cells that were sorted from the irradiated (5 Gy) H460 cells 24 hours after the irradiation. Relative TIP-1 protein level was normalized to that of the actin control (counted as 1) and shown under the image. <b>F</b>: Flow cytometric profile of TIP-1 expression on the cell surface of LLC, H460 or HUVEC cells. The cells were treated with 5 Gy of X-ray, TIP-1 on the cell surface was profiled 24 hours post the radiation treatment. Fold change of the TIP-1 positive cells was calculated by comparison to the untreated cells (counted as 1). * <i>p</i><0.01, <i>n</i> = 3, the Student's <i>t</i>-test, each was compared to the untreated control, respectively.</p

HVGGSSV peptide binds to PDZ domain of the TIP-1 protein.

<p><b>A</b>: Enrichment of phages with selective binding to the HVGGSSV peptide. Phages recovered from the five rounds of screening and those from the original phage library were subjected to selectivity analysis on beads coated with the HVGGSSV peptide or a scrambled peptide control, respectively. Shown are number (PFU) of phages recovered from the beads after the free phages were washed off. 10⁹ PFU of phages were used in this assay. <b>B</b>: Specificity of the TIP-1-expressing phage to the HVGGSSV peptide. <b>C</b>: Diagram of TIP-1 protein shows location of PDZ domain and the critical amino acid (H90) for PDZ ligand binding; <b>D</b>: SDS-PAGE image shows purity of the recombinant TIP-1 proteins and a dysfunctional mutant TIP-1 (H90A). The fusion proteins (∼37 kD) were analyzed along with molecular weight markers. <b>E</b>: Relative association of the purified recombinant proteins to the synthetic peptides was evaluated with ELISA. In these assays, 100 ng of the purified GST/TIP-1 or GST/TIP-1(H90A) proteins were used per wells, all the synthetic peptides were used as 50 ng per well. The mutations in the PDZ binding motif were underlined. Shown are representative data from triplicate experiments. * <i>p</i><0.05, <i>n</i> = 3, the Student's <i>t</i>-test.</p

Optimized Translocator Protein Ligand for Optical Molecular Imaging and Screening

Translocator protein (TSPO) is a validated target for molecular imaging of a variety of human diseases and disorders. Given its involvement in cholesterol metabolism, TSPO expression is commonly elevated in solid tumors, including glioma, colorectal cancer, and breast cancer. TSPO ligands capable of detection by optical imaging are useful molecular tracers for a variety of purposes that range from quantitative biology to drug discovery. Leveraging our prior optimization of the pyrazolopyrimidine TSPO ligand scaffold for cancer imaging, we report herein a new generation of TSPO tracers with superior binding affinity and suitability for optical imaging and screening. In total, seven candidate TSPO tracers were synthesized and vetted in this study; the most promising tracer identified (<b>29</b>, <i>K</i>_d = 0.19 nM) was the result of conjugating a high-affinity TSPO ligand to a fluorophore used routinely in biological sciences (FITC) via a functional carbon linker of optimal length. Computational modeling suggested that an <i>n</i>-alkyl linker of eight carbons in length allows for positioning of the bulky fluorophore distal to the ligand binding domain and toward the solvent interface, minimizing potential ligand–protein interference. Probe <b>29</b> was found to be highly suitable for in vitro imaging of live TSPO-expressing cells and could be deployed as a ligand screening and discovery tool. Competitive inhibition of probe <b>29</b> quantified by fluorescence and ³H-PK11195 quantified by traditional radiometric detection resulted in equivalent affinity data for two previously reported TSPO ligands. This study introduces the utility of TSPO ligand <b>29</b> for in vitro imaging and screening and provides a structural basis for the development of future TSPO imaging ligands bearing bulky signaling moieties

Preclinical TSPO Ligand PET to Visualize Human Glioma Xenotransplants: A Preliminary Study

<div><p>Current positron emission tomography (PET) imaging biomarkers for detection of infiltrating gliomas are limited. Translocator protein (TSPO) is a novel and promising biomarker for glioma PET imaging. To validate TSPO as a potential target for molecular imaging of glioma, TSPO expression was assayed in a tumor microarray containing 37 high-grade (III, IV) gliomas. TSPO staining was detected in all tumor specimens. Subsequently, PET imaging was performed with an aryloxyanilide-based TSPO ligand, [¹⁸F]PBR06, in primary orthotopic xenograft models of WHO grade III and IV gliomas. Selective uptake of [¹⁸F]PBR06 in engrafted tumor was measured. Furthermore, PET imaging with [¹⁸F]PBR06 demonstrated infiltrative glioma growth that was undetectable by traditional magnetic resonance imaging (MRI). Preliminary PET with [¹⁸F]PBR06 demonstrated a preferential tumor-to-normal background ratio in comparison to 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG). These results suggest that TSPO PET imaging with such high-affinity radiotracers may represent a novel strategy to characterize distinct molecular features of glioma growth, as well as better define the extent of glioma infiltration for therapeutic purposes.</p></div

Primary human grade III astrocytoma xenotransplant.

<p>(A) T₂-weighted MRI (coronal) visualizes advanced tumor in the right hemisphere of the brain. (B) Correlative dynamic PET image (coronal) of same advanced tumor in the right hemisphere of the brain, with [¹⁸F]PBR06 uptake primarily confined to the tumor and co-localizing with tumor tissue visualized by MRI. Top arrows indicate tumor and infiltration into left hemisphere. (C) Fused MRI (A) PET (B) image. (<b>D</b>) Time-activity curves of injected [¹⁸F]PBR06 in tumor (<i>green</i>) and contralateral brain (<i>blue</i>). Correlative vimentin immunohistochemistry: (E) Gross; (F) Tumor + White Matter Tract (40X); (G) Tumor (40X). (H) Correlative dynamic PET image, axial view along yellow line in (B); arrows indicate tumor and infiltration into left hemisphere. Correlative TSPO immunohistochemistry: (I) Gross; (J) Tumor + White Matter Tract (40X); (K) Tumor (40X). (L) Dynamic PET image (axial) of control cohort. Correlative CD68 immunohistochemistry: (M) Gross; (N) Tumor + White Matter Tract (40X); (O) Tumor (40X).</p

Side-by-side PET comparison of [¹⁸F]PBR06 and [¹⁸F]FDG in a primary human grade III astrocytoma xenotransplant.

<p>(A) T₂-weighted MRI (<i>coronal</i>) visualizes advanced tumor in the right hemisphere of the brain. (B) [¹⁸F]PBR06 uptake (<i>coronal</i>) is primarily confined to the tumor and co-localizes with tumor tissue visualized by MRI. Arrows indicate tumor and infiltration into left hemisphere. (C) [¹⁸F]FDG uptake (<i>coronal</i>) in normal brain is higher than tumor tissue, resulting in poor imaging contrast. (D) Correlative TSPO immunohistochemistry, Tumor + White Matter Tract (40X).</p

Primary human grade IV glioblastoma xenotransplant.

<p>(A) Dynamic PET image (<i>coronal</i>) of advanced tumor in the right hemisphere of the brain with [¹⁸F]PBR06 uptake confined primarily to the tumor. Arrows indicate tumor and infiltration into left hemisphere. (B) Time-activity curves of injected [¹⁸F]PBR06 in tumor (<i>green</i>) and contralateral brain (<i>blue</i>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141659#pone.0141659.ref030" target="_blank">30</a>]. (C) Correlative TSPO immunohistochemistry, Tumor + White Matter Tract (40X).</p

Human tumor microarrays (TMAs).

<p>Pilocytic astrocytoma TSPO immunohistochemistry (A) and TSPO immunohistochemical scoring (C). Glioblastoma multiforme TSPO immunohistochemistry (B) and TSPO immunohistochemical scoring (D).</p

[¹⁸F]PBR06 selectivity in primary human grade IV glioblastoma xenotransplant.

<p>(A) Dynamic PET image (axial) pre-infusion of cold analog, with [¹⁸F]PBR06 uptake primarily confined to the tumor. (B) Dynamic PET image (axial) post-infusion, showing nearly total displacement of [¹⁸F]PBR06. (C) Time-activity curves of injected [¹⁸F]PBR06 in tumor (<i>green</i>) and contralateral brain (<i>blue</i>). (D) Correlative TSPO immunohistochemistry, Tumor + White Matter Tract (40X).</p