Background:
Bladder cancer (BCa) has a high recurrence rate of 50-70%, particularly for non-muscle-invasive bladder cancer (NMIBC) after surgery and Bacillus Calmette-Guérin (BCG) immunotherapy. Bladder-preserving therapeutic options remain limited to date. Recent intravesical antibody-drug conjugates (ADCs) have shown early clinical promise in bladder cancer, such as EpCAM-targeted oportuzumab monatox, HER2-targeted disitamab vedotin (DV), and nectin-4-targeted enfortumab vedotin. HER2 is expressed in a substantial proportion of reported BCa cases.
Hypothesis/Objective:
Here, we aim to integrate DV with surface-enhanced spatially offset resonance Raman spectroscopic (SESORRS) imaging to track ADCs and visualize lesions in vivo. The theranostic probe (AuNS@DV) consists of a gold nanostar core with potent localized surface plasmon resonance, a resonant near-infrared (NIR) Raman reporter IR792, and DV (anti-HER2-MMAE) for anti-tumor activity. We hypothesize that AuNS@DV exerts therapeutic efficacy against bladder cancer via intravesical administration, guided by non-invasive pelvic SESORRS and microscopic SERRS imaging.
Methods:
First, HER2 expression in BCa was statistically analyzed. AuNSs were synthesized using a previously reported surfactant-free, seedless method. AuNSs, IR792, and DV were subsequently mixed at different ratios, and the resulting product was characterized by Raman spectroscopy, dynamic light scattering, and UV-vis-NIR spectroscopy. Imaging phantoms were used to optimize key parameters of SESORRS imaging in vitro. Next, SESORRS imaging was performed for non-invasive detection in an orthotopic T24 BCa xenograft mouse model, with parallel bioluminescence imaging (BLI) and histopathological sections as references. Finally, the therapeutic efficacy of the probe was assessed in orthotopic T24 xenograft mice by evaluating changes in body weight, gross bladder morphology, and histopathology.
Results:
HER2 was highly expressed in NMIBC tissues and BCa cell lines, confirming its role as a theranostic target for BCa. The anti-HER2 ADC-modified AuNS@DV probe exhibited a uniform hydrodynamic diameter of approximately 100 nm by DLS and maintained stable Raman signal intensity after laser exposure, indicating good photostability. AuNS@DV achieved a sub-picomolar limit of detection, highlighting its high sensitivity for Raman imaging. In phantom studies, the Raman signal reached maximum intensity beneath a 0.3-mm occluder at an offset distance of 1 mm, establishing the optimal offset for deep-tissue detection. In an orthotopic bladder cancer mouse model, strong Raman signals were detected through the bladder 3 h after intravesical administration at an offset distance of 2 mm. Under open surgical scenarios, microscopic SERS imaging precisely delineated tumor margins in the bladder and correlated well with H&E histopathology. The orthotopic model was generated by mechanically disrupting the bladder mucosa, followed by intravesical instillation of bladder cancer cells, with tumor growth longitudinally monitored by BLI. Both DV and AuNS@DV significantly decreased tumor burden with comparable efficacy while maintaining normal body weight gain. Histopathological analyses further confirmed marked suppression of intravesical tumor progression, particularly in the DV and AuNS@DV groups. Collectively, these results establish AuNS@DV as a promising intravesical precision theranostic agent for HER2-expressing BCa.
Conclusion:
BCa management is hindered by high recurrence rates, suboptimal intravesical treatment efficacy, and the absence of convenient non-invasive approaches for tumor surveillance. Here, we developed theranostic AuNS@DV for Raman image-guided cystoscopy, integrating diagnosis and treatment into a single agent. AuNS@DV harnesses the high sensitivity and deep penetration of SESORRS to enable a streamlined, clinically translatable workflow for BCa. Its HER2-targeting capability further prolongs intravesical retention, thereby enhancing both imaging performance and therapeutic efficacy. Together, these results show that intravesical AuNS@DV addresses key limitations of current BCa therapies and monitoring strategies, supporting its potential for clinical translation as a precision theranostic agent.
Image/Figure Caption:
Figure 1. HER2 is highly expressed in non‑muscle‑invasive bladder cancer (NMIBC) and several bladder cancer (BCa) cell lines. (a) Representative images of cell‑surface HER2 immunohistochemical (IHC) staining in human NMIBC tissues, evaluated according to American Society of Clinical Oncology (ASCO) guidelines. (b) HER2 IHC expression scores relative to pathological tumor (pT) stage, histological grade, and disease status. (c) Relative HER2 gene expression assessed by Western blotting and (d) qRT‑PCR in BCa cell lines and breast cancer cell lines. SK‑BR‑3 (HER2‑high) and MDA‑MB‑231 (HER2‑low) were used as reference controls. (e) Immunofluorescence (IF) staining confirmed HER2 expression in 5637, T24, and UM‑UC‑3 BCa cells, with breast cancer cell lines as controls.
Figure 2. Characterization of the AuNS@DV probe. (a) Hydrodynamic size distribution, (b) zeta-potentials, (c) UV−vis absorption spectra of AuNS@DV probe. (d) Raman spectra of AuNS@GMP in 10 minutes and (e) time stability mapping of Raman intensity. (f) The lowest detection sensitivity for AuNS@GMP reaches the sub-pM level.
Figure 3. In vitro SESORRS imaging of the phantom. (a) Photograph of the mold loaded with the probe. (b) SERRS image acquired under uncovered conditions; (c) corresponding Raman spectrum. (d) Schematic illustration of SESORRS imaging. (e) SESORRS image at a thickness of 0.5 mm; the optimal Raman signal was achieved at an offset distance of 1 mm. (f) Corresponding Raman spectrum for the conditions in (e).
Figure 4. SESORRS imaging of an orthotopic bladder cancer mouse model. (a–f) SESORRS imaging of mice with orthotopic bladder cancer. (a) Bioluminescence imaging (BLI) of a mouse with orthotopic bladder cancer. (b) SESORRS image of the orthotopic bladder cancer mouse; strong Raman information was obtained at an offset distance of 2 mm. (c) Corresponding Raman spectrum to b. (d) Gross pathological image of the bladder tissue from the orthotopic bladder cancer mouse. (e) SERRS image of the bladder from the orthotopic bladder cancer mouse. (f) Corresponding H&E-stained pathological image to e. (g) Corresponding Raman spectrum to e. (g–m) SESORRS imaging of healthy mice. (g) BLI of a healthy mouse. (h) SESORRS image of the healthy mouse model; no obvious Raman signal was observed. (i) Corresponding Raman spectrum to h. (j) Gross pathological image of the bladder tissue from the healthy mouse. (k) SERRS image of the bladder from the healthy mouse. (l) Corresponding Raman spectrum to k. (m) Corresponding H&E-stained pathological image to k.
Figure 5. Investigation of the anti-tumor effect of probe therapy in an orthotopic bladder cancer mouse model. （a）Schematic diagram of orthotopic bladder cancer mouse model establishment and the therapeutic timeline. (b) Bioluminescence imaging (BLI) of mice before and after treatment. (c) Body weight changes of orthotopic bladder cancer mice in different groups. (d) Gross pathological images of the bladder. (e) Corresponding statistical graph of bladder weight. (f) H&E-stained pathological images of the bladder in different groups.
Author
Jiang Yang, Prof.
Sun Yat-sen University Cancer Center