Supplementary Figures S1-S7 from Phage Display-Derived Peptide-Based Dual-Modality Imaging Probe for Bladder Cancer Diagnosis and Resection Postinstillation: A Preclinical Study

Supplementary Figure S1 shows synthesis and characterization of PLSWT7-DMI. (A) Visible absorption spectrum of PLSWT7-DMI. Similar absorption peaks were observed for PLSWT7-DMI and IRDye800CW (~774 nm). (B) Absorbance stability of PLSWT7-DMI. The absorbance dropped to 0.05). (D) Fluorescence intensity of RT112 and EJ cells incubated with various concentrations of PLSWT7-DMI. Cells incubated with 180 pmol/mL PLSWT7-DMI produced the highest fluorescence. Supplementary Figure S2 shows fluorescence microscopy images of SV-HUC-1 cells incubated with PLSWT7-DMI. Minimal fluorescence was observed after incubation. Supplementary Figure S3 shows metabolic profiles of PLSWT7-DMI post-intravenous-injection via the tail vein. (A) IVIS imaging of subcutaneous tumor-bearing mice injected with cPLSWT7-DMI and PLSWT7-DMI. Red circle indicates the tumor region. (B) Fluorescence intensity of the tumor site in (A). PLSWT7-DMI-treated groups exhibited higher fluorescence intensity than that of cPLSWT7-DMI-treated groups. (C) Comparison of TBR profiles of PLSWT7-DMI and cPLSWT7-DMI. TBR of PLSWT7-DMI-treated groups peaked 8 h post-injection and was higher than the TBR of control groups. Values are presented as means {plus minus} SD of 5 animals. Supplementary Figure S4 shows ex vivo imaging of mouse organs at 6-h post-instillation of PLSWT7-DMI. (A) A bright signal was observed in RT112 tumors, whereas signals in organs were minimal. (B) Quantitative analysis of the fluorescence intensity observed in (A). Values are presented as means {plus minus} SD of 5 animals. Supplementary Figure S5 shows orthotopic BC mice imaging. (A) Luciferase imaging showing tumor growth in the orthotopic BC model. (B) NIR imaging of orthotopic BC mice post-instillation of the probes. Bladders of orthotopic BC mice instilled with PBS, cPLSWT7-DMI, PLSWT7-DMI with blockade, or PLSWT7-DMII, followed by imaging using a homemade NIR-imaging system at 30-min post-instillation. Tumors in PLSWT7-DMI groups were clearly visualized; however, the blockade of PLSWT7-DMI binding to the tumor was observed following PLSWT7 pretreatment. The yellow-dashed circle indicates the tumor site. (C) Quantitative analysis of the mean fluorescence density shown in (B). Values are presented as means {plus minus} SD of 5 animals. *p < 0.05 vs. PLSWT7-DMI+blockade groups. Supplementary Figure S6 shows PAI of subcutaneous tumor-bearing mice after intravenous injection via the tail vein. (A) PAI of subcutaneous tumor-bearing mice. Subcutaneous tumor-bearing mice were injected with PBS, cPLSWT7-DMI, PLSWT7-DMI with blockade, or PLSWT7-DMI via tail vein, followed by PAI. PLSWT7-DMI-treated groups exhibited enhanced contrast compared with other groups. The red-dashed circle indicates the tumor site. (B) Quantification of the average PA-signal amplitude of subcutaneous tumor-bearing mice in (A). The PLSWT7-DMI groups exhibited higher signals than those of other groups. Values present means {plus minus} SD of 5 animals. *p < 0.05 vs. PLSWT7-DMI + blockade groups. Supplementary Figure S7 shows ex vivo human bladder imaging post-administration of cPLSWT7-DMI. No optical contrast between cancer and normal tissues was observed post-administration of cPLSWT7-DMI.