Peptide discovery and characterization
In vivo phage display technology was used to identify BC-specific peptides in our previous study (17). After three rounds of biopanning, phage clone P7 was selected based on its high affinity for RT112 (a BC cell line). P7 was isolated, sequenced, and translated into the corresponding peptide, CSDRIMRGC (PLSWT7), and labeled with IRDye800CW (PLSWT7-DMI) or fluorescein isothiocyanate (PLSWT7-FITC). The chemical structure is shown in Fig. 1A. Mass spectrometry was used to determine the mass/charge ratio (m/z) of PLSWT7-DMI, and its monoisotopic mass based on this was determined to be 2514.8 Da (Fig. 1B). The fluorescence spectra for PLSWT7-DMI and IRDye800CW in phosphate-buffered saline (PBS) at an excitation wavelength of 774 nm revealed an emission peak at 789 nm (Fig. 1C). Thus, no shift was observed after IRDye800CW labeling. The apparent dissociation constant (Kd) and association rate constant (K) of PLSWT7-FITC were 42 nM (Fig. 1D) and 0.14 min-1 (Fig. 1E), respectively. The results were determined using the least-squares fit of the data. To evaluate the specificity of the peptide in vitro, RT112, EJ, and SV-HUC-1 (a normal urothelium cell line) were incubated with the probes, and higher fluorescence intensity was observed in tumor cells incubated with PLSWT7-DMI, indicating efficient binding to RT112 and EJ cells. However, no fluorescence was observed following incubation with cPLSWT7-DMI (scrambled peptide CQRSPIHDC, labeled with IRDye800CW) or PBS. Additionally, pretreatment with PLSWT7 prevented PLSWT7-DMI from binding (Fig. 1F), and minimal fluorescent signals were observed in SV-HUC-1 cells incubated with PLSWT7-DMI, suggesting that the binding of PLSWT7-DMI occurred via the targeting of specific sites on tumor cells. The binding of CD44v6 to RT112 cells also occurred in a dose-dependent manner. The level of PLSWT7-DMI binding was higher in non-transfected cells than in silenced cells (Fig. 1G). These peptides bind to RT112 mouse bladder tumor cells, which express murine CD44v6 of RT112 cells, and an anti- CD44v6 blocking antibody inhibited the cellular binding of CD44v6. These findings indicate that the binding of CD44v6 to CD44v6-overexpressing cells is specifically mediated by CD44v6. In addition, CD44v6 preferentially binds to plates coated with either human or mouse recombinant CD44v6 compared to control plates coated with albumin (Fig. 1H).
For pharmacological/toxicological analysis of PLSWT7-DMI, rats were administered four different doses of PLSWT7-DMI by oral gavage (n = 12 animals per dose). After 15 days, the rats showed no peptide-related acute adverse effects according to clinical signs, chemistries, or necropsy (Table S1).
Ex vivo endoscopic NIR imaging
Specific binding of PLSWT7-DMI to NMIBC was confirmed ex vivo using fresh human bladder specimens (n = 8) from patients who had undergone radical cystectomy. Multifocal tumors and CIS were present in the mucosa. After incubation with PLSWT7-DMI, tumors were clearly visible, but the normal mucosa was not, indicating specific targeting of the probe (Fig. 2A). Quantitative analysis showed that the fluorescence density was significantly higher in urothelial carcinoma and CIS tissue than in the normal mucosa after incubation with PLSWT7-DMI, with no difference observed with cPLSWT7-DMI incubation (Fig. 2B). These results suggest that PLSWT7-DMI binds specifically to human BC tissue, and thus can be translated into clinical applications.
Endoscopic NIR imaging with PLSWT7-DMI in NMIBC patients
To determine whether PLSWT7-DMI specifically binds to NMIBC in vivo, we enrolled 22 NMIBC patients and compared the absolute fluorescence of tumor tissue with that of the adjacent normal tissue following intravesical probe administration. The bladders were washed and emptied, and intravesically instilled with a control probe (cPLSWT7-DMI), and images were obtained using an in-house developed endoscopy system. After washing to remove the control probe, bladders were re-instilled with PLSWT7-DMI and imaged again. With the control probe, no contrast was detected between the normal and apparent tumor mucosa (Fig. S1). After instillation with PLSWT7-DMI, urothelial carcinomas at stage Ta and T1 were clearly stained (Fig. 3A), but the normal mucosa was not (Fig. 3B). Quantitative analysis revealed that the mean fluorescence density was significantly higher in the tumor tissue than in the normal mucosa after incubation with PLSWT7-DMI, while no difference was observed with the control probe cPLSWT7-DMI (Fig. 3C), indicating that PLSWT7-DMI specifically binds to tumors. Moreover, the TBR of PLSWT7-DMI was 4.58-fold higher than that of cPLSWT7-DMI (Fig. 3D). After treatment with PLSWT7-DMI, the signal ratio between the lesion and margin in stage Ta and T1 tumors was higher than that in the normal tissue (Fig. 3E). Furthermore, T1 urothelial carcinomas showed higher TBRs than the patient of Ta stage (Fig. 3F).
The illuminated lesions were biopsied using NIR imaging. Pathological examination confirmed that the probe-bound lesions corresponded to NMIBC tissue, while the lesions without probe binding were identified as normal mucosa.
Detection accuracy of PLSWT7-DMI fluorescence endoscopy for NMIBC
A total of 108 lesions (68 cancerous lesions and 40 benign lesions) were analyzed to compare PLSWT7-DMI fluorescence endoscopy and histopathological diagnosis (Table S2). For urothelial neoplasms, 20.6% (n = 14) were found only by NIR imaging, and 7.4% (n = 5) were found only by WL imaging. Small satellite tumors and CIS overlooked under WL imaging were identified using probe-based NIR imaging (Fig. 4A). Under WL imaging, 54 NMIBC lesions were detected, while nine additional lesions were detected by NIR imaging (Table S2), including six small foci and three CIS that were overlooked by WL endoscopy. These results indicate that PLSWT7-DMI-based NIR fluorescence endoscopy may reduce the number of overlooked NMIBC lesions, thus facilitating complete resection and reducing the recurrence rate of BC.
Suspicious benign lesions that were difficult to discriminate under WL, such as inflammation and ulcers, could be easily differentiated by PLSWT7-DMI-based NIR fluorescence endoscopy (Fig. 4B). The mean fluorescent density did not differ among normal mucosa, ulcers, and inflammation (Fig. 4C), indicating negligible probe binding to benign lesions. Ten out of thirteen inflammatory lesions and three out of three ulcers were invisible under NIR (Table S2), suggesting that mistaken resection of benign lesions would be minimized if our technique was used during tumor resection.
Overall, PLSWT7-DMI-based NIR fluorescence endoscopy resulted in a more thorough detection of urothelial neoplasms at different tumor stages than WL endoscopy (Fig. 4D). The sensitivity of probe-based NIR imaging was 91.2% (62/68), specificity was 90% (36/40), and the false positive rate was 10% (4/40), as shown in Table 2.
Distribution of PLSWT7-DMI in bladder tissue
We used immunofluorescence to further examine the distribution of PLSWT7-DMI in the tumor and normal tissue. Frozen sections of biopsies from the detected lesions and the adjacent normal mucosa were obtained during the present study. In representative images of NMIBC and normal mucosa, we found that the PLSWT7-DMI signal was higher in NMIBC tissue than in normal tissue and overlapped with CK20 expression (Fig. S2), indicating that PLSWT7-DMI was localized in NMIBC tissue but not in the normal urothelium. The tumor-selective distribution of PLSWT7-DMI may serve to illuminate tumor lesions during intraoperative imaging.
Safety and adverse effects
PLSWT7-DMI was well-tolerated by the study subjects, and no adverse pharmacological activity or allergic reactions occurred during the study. Three patients experienced surgical fevers within the first postoperative day (36.8–38.5°C), which subsequently subsided. No damage to liver or renal function was observed. None of the patients complained of nausea, vomiting, or gross hematuria within one week after surgery. No symptoms of urinary tract infection were reported (Table S3).