BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3434

Peptides:

Sequences	Affinity Values
CSDRIMRGC	[13.20]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (in vivo)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 30082470

Target Name:

Human bladder cancer cell line RT112

Template Name:

Not determined.

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

Ph.D.-C7C phage display library (CX7C)

Affinity Measurement Method:

ELISA

Affinity Measurement Description:

For ELISA, RT112 and human normal urothelial cell line SV-HUC-1SV-HUC-1 were plated at 1.0e4 cells/well in a 96-well plate and incubated at 37°C in 5% CO2 for 24 hours. Subsequently, cells were washed and fixed with 4% paraformaldehyde for 15 minutes at room temperature. After incubation with 3% H2O2 (100 mL/well) at 37°C for 30 minutes, the plates were washed 3 times with PBS, and the wells were blocked with 200 mL of 5% BSA for 30 minutes at 37°C. The phages (1.0e10 PFU/well) were added to RT112 or SV-HUC-1 cells and incubated at 37°C for 2 hours. The plates were then washed 3 times with TBST before the addition of 100 mL of HRP-anti-M13 mAb (1:5,000) for 1 hour at 37°C. After washing 3 times with 0.05% TBST, 3,30,5,50-tetramethylbenzidine (TMB) was added at room temperature for 30 minutes. The reaction was terminated by the addition of 50 mL of 2 mol/L H2SO4. Subsequently, absorbance was read using an automated ELISA plate reader at 450 nm. Unrelated phages with equal titers were added to the wells in place of selected phage clones to serve as negative controls. Selectivity was determined using the following formula: Selectivity = [RT112(OD450 - OD450negative control)]/[SV-HUC-1(OD450 - OD450negative control)]. The selectivity value was reproduced from Figure 1A and shown.

Brief Description:

Subcutaneous tumor-bearing mice received 3e10 plaque-forming units (PFU) of the phage peptide library via tail-vein injection. After 15 minutes, mice were sacrificed and perfused by injection of 50 mL of PBS through the heart to wash unbound phages. Then, a portion of tumors was harvested and preserved in 4% paraformaldehyde and the rest was manually homogenized and washed with 10 × TBS containing 0.1% Tween-20 (0.1% TBST) to remove nonspecifically bound phages. Cell membrane–bound phages were eluted with 1 mL of elution buffer (0.2 mol/L glycine-HCl, pH 2.2, 1 mg/mL BSA) for 10 minutes on ice and neutralized with 150 mL of 1 mol/L Tris–HCl (pH 9.0). After centrifugation, the supernatant was collected, and the cells in the precipitate were washed once with PBS-BSA and lysed with 0.1% Triton X-100 for 2 hours at room temperature. Thus, the internalized phages in the cell lysate were recovered. A total of 10 mL of eluted phage was titrated on agar plates in the presence of IPTG/X-gel (1 mg/L) and tetracycline (40 mg/mL). The remaining phages were amplified by ER2738 bacteria at 37°C for 4.5 hours and injected into tumor-bearing mice. The biopanning process was repeated for three rounds.

BiopanningDataSet Comments:

A bladder cancer–specific peptide with the sequence of CSDRIMRGC (PLSWT7) was selected by in vivo phage-display technology and labeled with IRDye800CW to synthesize a bladder cancer–specific dual-modality imaging (DMI) probe (PLSWT7-DMI). The feasibility of PLSWT7-DMI–based dual-modality photoacoustic imaging near-infrared (PAI-NIR) imaging was assessed in vitro, in mouse models, and ex vivo human bladders. An air-pouch bladder cancer (APBC) model suitable for probe instillation was established to evaluate the probe-based bladder cancer PAI diagnosis and NIR-imaging–guided resection. Human bladders were used to assess whether the PLSWT7-DMI–based DMI strategy is a translatable approach for bladder cancer detection and resection. The probe exhibited excellent selectivity and specificity both in vitro and in vivo. Postinstillation of the probe, tumors <3mm were detectable by PAI, and NIR-imaging–guided tumor resection decreased the bladder cancer recurrence rate by 90% and increased the survival in the mouse model. Additionally, ex vivo NIR imaging of human bladders indicated that PLSWT7-DMI–based imaging would potentially allow precise resection of bladder cancer in clinical settings. This PLSWT7-DMI–based DMI strategy was a translatable approach for bladder cancer diagnosis and resection and could potentially lower the bladder cancer recurrence rate.

Curator:

Bifang He

Curation Date:

2019-05-28

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