Characterization and Evaluation of 64Cu-Labeled A20FMDV2 Conjugates for Imaging the Integrin αvβ6

PURPOSE: The integrin α(v)β(6) is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for (64)Cu radiolabeling of A20FMDV2, an α(v)β(6) targeting peptide. PROCEDURES: Four chelators were conjugated onto PEG(28)-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4′-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl)) bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with (64)Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted. RESULTS: All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37–50 °C for 15 min) with high specific activity (0.58–0.60 Ci/µmol). All radiotracers demonstrated α(v)β(6)-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers (64)Cu-CB-TE1A1P-1 and (64)Cu-BaBaSar-1 showed improved specificity for the α(v)β(6) positive tumor in vivo over (64)Cu-DOTA-1 and (64)Cu-NOTA-1 (+/− tumor uptake ratios—3.82 +/−0.44, 3.82±0.41, 2.58±0.58, and 1.29±0.14, respectively). Of the four radiotracers, (64)Cu-NOTA-1 exhibited the highest liver uptake (10.83±0.1 % ID/g at 4 h). CONCLUSIONS: We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear “best candidate” for the (64)Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators

Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation

Oral squamous cell carcinoma (OSCC) is the 6th most common cancer worldwide, with approximately 400,000 new cases each year. Current treatment regimens are, to a certain degree, inadequate, with a 5-year mortality rate of around 50%, and novel therapeutic targets are urgently required. Using gene microarrays, we identified the EPS8 gene as being over-expressed in OSCC cell lines relative to normal oral keratinocytes, and confirmed these findings using RT-PCR and Western blotting. In human tissues, we found that Eps8 was upregulated in OSCC (32% of primary tumors) compared with normal oral mucosa, and that expression correlated significantly with lymph node metastasis (p=0.032), suggesting a disease-promoting effect. Using OSCC cell lines we assessed the functional role of Eps8 in tumor cells: Although suppression of Eps8 produced no effect on cell proliferation, both cell spreading and migration were markedly inhibited. The latter cell functions may be modulated through the small GTP-ase, Rac1, and we used pull-down assays to investigate the role of Eps8 in Rac1 signaling. We found that ?v?6- and ?5?1-integrin-dependent activation of Rac1 was mediated through Eps8. Knockdown of either Eps8 or Rac1, inhibited integrin-dependent cell migration similarly, and transient expression of constitutively-active Rac1 significantly restored migration of cells in which Eps8 had been suppressed. We also showed, using an organotypic model of OSCC, that Eps8 promotes tumor cell invasion. These data suggest that Eps8 and Rac1 are part of an integrated signaling pathway modulating integrin-dependent tumour cell motility, and identify Eps8 as a possible therapeutic target

Telomere maintenance as a target for anticancer drug discovery

Maintenance of telomeres - specialized complexes that protect the ends of chromosomes is undertaken by the enzyme complex telomerase, which is a key factor that is activated in more than 80% of cancer cells that have been examined so far, but is absent in most normal cells. So, targeting telomere-maintenance mechanisms could potentially halt tumour growth across a broad spectrum of tumour types, with little cytotoxic effect outside tumours. Here, we describe the current understanding of telomere biology, and the application of this knowledge to the development of anticancer drugs