carbocyanines and Carcinoma--Transitional-Cell

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

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

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