carbocyanines and Urinary-Bladder-Neoplasms

Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore,

Topics: Animals; Carbocyanines; Carcinoma, Transitional Cell; Cell Line, Tumor; Contrast Media; Dogs; Elastin; Epidermal Growth Factor; ErbB Receptors; Humans; Mice, Nude; Peptides; Recombinant Fusion Proteins; Urinary Bladder Neoplasms

Bladder cancer (BC) is a prevalent disease with high morbidity and mortality; however, in vivo optical imaging of BC remains challenging because of the lack of cancer-specific optical agents with high renal clearance. Herein, a macromolecular reporter (CyP1) was synthesized for real-time near-infrared fluorescence (NIRF) imaging and urinalysis of BC in living mice. Because of the high renal clearance (ca. 94 % of the injection dosage at 24 h post-injection) and its cancer biomarker (APN=aminopeptidase N) specificity, CyP1 can be efficiently transported to the bladder and specially turn on its NIRF signal to report the detection of BC in living mice. Moreover, CyP1 can be used for optical urinalysis, permitting the ex vivo tracking of tumor progression for therapeutic evaluation and easy translation of CyP2 as an in vitro diagnostic assay. This study not only provides new opportunities for non-invasive diagnosis of BC, but also reveals useful guidelines for the development of molecular reporters for the detection of bladder diseases.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Aminopeptidases; Animals; Biological Transport; Biomarkers, Tumor; Carbocyanines; Fluorescent Dyes; Humans; Macromolecular Substances; Mice; Neoplasms, Experimental; Optical Imaging; Spectroscopy, Near-Infrared; Urinary Bladder Neoplasms

Achieving the activation of drugs within cellular systems may provide targeted therapies. Here we construct a tumour-selective cascade activatable self-detained system (TCASS) and incorporate imaging probes and therapeutics. We show in different mouse models that the TCASS system accumulates in solid tumours. The molecules show enhanced accumulation in tumour regions via the effect of recognition induced self-assembly. Analysis of the molecular penetration in tumour tissue shows that in vivo self-assembly increases the penetration capability compared to typical soft or hard nanomaterials. Importantly, the in vivo self-assembled molecules exhibit a comparable clearance pathway to that of small molecules, which are excreted from organs of the reticuloendothelial system (liver and kidney), while are relatively slowly eliminated from tumour tissues. Finally, this system, combined with the NIR probe, shows high specificity and sensitivity for detecting bladder cancer in isolated intact patient bladders.

Topics: Amino Acid Motifs; Animals; Antibiotics, Antineoplastic; Biological Availability; Carbocyanines; Carcinoma, Transitional Cell; Cell Line, Tumor; Coloring Agents; Doxorubicin; Drug Delivery Systems; HEK293 Cells; Humans; Kidney; Liver; Mice; Neoplasm Transplantation; Protein Engineering; Sensitivity and Specificity; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

To develop bladder cancer-specific ligands using a combinatorial chemistry approach.. We performed a high-throughput one-bead one-compound combinatorial chemistry approach to identify ligands that bound to bladder transitional cell carcinoma cells. The whole-cell binding assay allowed successful identification of a few peptides that bound selectively to bladder cancer cells. Single cell suspensions derived from clinical bladder cancer specimens and cell lines were used to determine the binding specificity. Studies with mouse xenografts were performed to determine the in vivo binding and targeting efficiency, specificity, and biodistribution of one of the ligands.. One cyclic peptide named PLZ4 (amino acid sequence: cQDGRMGFc) was identified that could selectively bind to bladder cancer cell lines and all of the 5 primary bladder cancer cells from human patients, but not to normal urothelial cells, cell mixtures from normal bladder specimens, fibroblasts, and blood cells. Comparison of PLZ4 binding to cell lines of different cancer origins showed that it was bladder cancer-specific (P < 0.05). PLZ4 could bind to tumor cells treated with urine at pH 6.0, but not to noncancerous cells collected from the urine of 4 patients actively being treated with intravesical Bacillus Calmette-Guerin therapy. In vivo and ex vivo imaging studies showed that PLZ4 linked to Cy5.5 fluorescent dye administered via tail vein injection was specifically taken up in mouse xenografts developed from excised fresh human bladder cancer specimens. Several ligands contain the same DGR motif, but only PLZ4 was bladder cancer-specific. We performed alanine walk and rainbow bead coding experiments, and found that the C-terminal GF residues were also important for cell binding and modulated the binding specificity.. PLZ4 has the potential to be used for targeted therapy and imaging detection during diagnosis and follow-up/surveillance of noninvasive and advanced bladder cancer.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Carbocyanines; Carcinoma, Transitional Cell; Cell Line, Tumor; Combinatorial Chemistry Techniques; Female; Humans; Jurkat Cells; Ligands; Mice; Mice, Nude; Microscopy, Fluorescence; Neoplasms, Experimental; Peptide Library; Peptides; Peptides, Cyclic; Protein Binding; Tissue Distribution; Transplantation, Heterologous; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Here we evaluate an improved orthotopic rat bladder tumor model, to be used for the evaluation of the therapeutic potential of novel cancer therapeutics. Before instilling AY-27 tumor cells into chemically denudated rat bladders, AY-27 cells were labeled with the fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). We found that the presence of Dil did not alter the in vitro AY-27 cell proliferation and that the Dil label was strongly associated with the cells. We further provide evidence that the use of fluorescently labeled AY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inoculation process. Using this technique it was possible to track down the tumor cells after inoculation into the bladder, which makes it straightforward to distinguish tumor cells from remaining or regenerated normal urothelium over a period of 5 d. The results also demonstrated that malignant AY-27 tissue exists as an intact non-muscle invasive bladder tumor only for 1-3 d after cell implantation. Accordingly the AY-27 bladder tumor model was used to evaluate the antitumoral effect of a single intravesical MM-C instillation. All rats instilled with 1 mM MM-C survived the final endpoint of 30 d after intravesical MM-C. Moreover, 10 and 30 d after treatment the urothelium of the MM-C-treated animals was completely restored. Remarkably, after MM-C treatment distinct patchy fluorescent dots were found into the submucosa and the regenerated urothelium, suggesting that dye retention is secondary to the digestion of Dil-loaded AY-27 cells and cellular debris by macrophages and related immune cells.

Topics: Animals; Antibiotics, Antineoplastic; Carbocyanines; Cell Line, Tumor; Disease Models, Animal; Female; Fluorescence; Fluorescent Dyes; Mitomycin; Rats; Rats, Inbred F344; Staining and Labeling; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

To investigate the feasibility of molecular beacons (MBs) for screening urine samples from patients with suspected bladder cancer. Our previous study showed that MBs could detect bladder cancer cells and cells shed in the urine from patients with bladder cancer.. Samples from 35 patients with bladder cancer and 35 healthy adults were initially evaluated. Cyanine 3-labeled MBs linked to a survivin mRNA probe were used to detect exfoliative cells in urine. Exfoliative cytology, enzyme-linked immunosorbent assay, and Western blotting of the tissues were used to confirm the MB results. We then evaluated the urine samples from 187 patients with suspected bladder cancer. All 187 patients also underwent cystoscopy.. In the initial cohort evaluated, MBs detected cancerous cells in 28 (80%) of the 35 patients with confirmed bladder cancer. Survivin protein was detected by Western blotting in 25 (71.4%) of the 35 patients. The sensitivity and specificity of enzyme-linked immunosorbent assay was 54.3% (20 of 35) and 68.6% (24 of 35), respectively. In a large group of patients with suspected bladder cancer, the sensitivity of MBs was 77.3% (85 of 110) and the specificity was 76.6% (59 of 77) compared with the cystoscopy data. Differences in the protein levels between the tumor grades and stages were not significant.. Our results have demonstrated that it is feasible to detect survivin mRNA in the exfoliated cells in urine using MBs. With further development, MBs could be used in a noninvasive clinical diagnostic procedure for the early detection of bladder cancer and postoperative follow-up.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carbocyanines; Carcinoma, Transitional Cell; Feasibility Studies; Female; Humans; Inhibitor of Apoptosis Proteins; Male; Microtubule-Associated Proteins; Middle Aged; Molecular Diagnostic Techniques; Sensitivity and Specificity; Survivin; Urinary Bladder Neoplasms; Urine; Young Adult

A novel near-infrared (NIR) cyanine 1-(epsilon-succinimidyl-hexanoate)-1'-methyl-3,3,3',3'-tetramethyl-indocarbocyanine-5,5'-disulfonate potassium (MeCy5-OSu) has been developed in our laboratory. Simultaneous determination of MeCy5-OSu-derivatized polyamines spermine (Spm), spermidine (Spd), cadaverine (Cad), and putrescine (Put) based on the separation by CE combined with diode LIF detection has been accomplished. The highest derivatization efficiency was achieved in 0.2 mol/L borate buffer (pH 8.8) for 20 min at 25 degrees C. Polyamine derivatives were separated within 14 min in the phosphate running buffer (pH 3) containing 50 mmol/L phosphoric acid, 40 mmol/L SDS, and 35% methanol v/v. Linearity of response was obtained in the range of 10-200 nmol/L. The detection limits (S/N = 3) for Spm, Spd, Cad, and Put were 0.8, 1, 3, and 2 nmol/L, respectively. The proposed method has been successfully applied to the analysis of polyamines in erythrocytes of two healthy persons and one cancer patient. Average recoveries for erythrocyte samples were 93.6-106% and coefficients of variation ranged from 1.8 to 5.4%. The analysis of polyamines in erythrocytes can be used for studying the relationship between their changes and the carcinogenesis process involved in erythrocytes.

Topics: Carbocyanines; Electrophoresis, Capillary; Erythrocytes; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Polyamines; Spectroscopy, Near-Infrared; Urinary Bladder Neoplasms

In the context of investigation of a prognostic marker applicable to bladder tumours, the authors propose the cytofluorometric study of the membrane potential of malignant urothelial cells using a molecular probe, 3,3' dihexyloxacarbocyanine. This preliminary study demonstrated a significant increase in the membrane potential of malignant urothelial cells in comparison with normal cells, which leads the authors to propose this cellular parameter as a new tool in the prognostic evaluation of bladder tumours.

Topics: Biomarkers, Tumor; Carbocyanines; Dimethyl Sulfoxide; Epithelium; Flow Cytometry; Fluorescent Dyes; Humans; Membrane Potentials; Molecular Probes; Prognosis; Urinary Bladder Neoplasms

We investigated mechanisms of mitochondrial phototoxicity caused by the cationic cyanine dye N,N'-bis(2-ethyl-1,3-dioxylene)kryptocyanine (EDKC), examining the role of the mitochondrial membrane potential on the dye uptake by carcinoma cells in vitro, and both the dark and photosensitizing effects of the dye on the function of isolated mouse liver mitochondria. When human bladder carcinoma cells (EJ) were pretreated with 2,4-dinitrophenol or nigericin, cellular uptake of EDKC decreased or increased, respectively, consistent with dye uptake that is dependent on membrane potentials. In isolated liver mitochondria, during NADH linked substrate oxidation (using glutamate plus malate or beta-hydroxybutyrate as substrates), low concentrations of the dye (0.25-0.5 microM) sensitized mitochondria to illumination with long wavelength light and inhibited both basal and ADP-stimulated respiration. Similar effects were observed during succinate oxidation, but only at higher concentrations of EDKC (greater than 5 microM) and at 10-fold greater light doses. NADH coenzyme Q reductase (Complex I) activity was inhibited by dye with or without light to an extent comparable to the inhibition of glutamate plus malate oxidation. Activity of cytochrome c oxidase, the terminal enzyme in the electron transport chain, was photosensitized with high dye doses (greater than 5 microM) and light, but the extent of inhibition was much less than the inhibition of respiration with succinate as substrate. ATP synthetase (F0F1 ATPase) activity was minimally affected by 4.0 microM EDKC with or without 24 J/cm2 light. We conclude that at low concentrations of dye, respiratory Complex I is a primary target for EDKC dark and light-induced toxicities. If Complex I is bypassed by using succinate as a respiratory substrate, the mitochondria can tolerate much higher dye concentrations and light doses.

Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Carbocyanines; Coloring Agents; Dinitrophenols; Electron Transport; Humans; Light; Membrane Potentials; Mitochondria; Mitochondria, Liver; NAD(P)H Dehydrogenase (Quinone); Nigericin; Oxygen Consumption; Quinolines; Quinone Reductases; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Urinary Bladder Neoplasms