Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo

Abstract

Bladder cancer is the fifth most common in incidence and one of the most expensive cancers to treat. Early detection greatly improves the chances of survival and bladder preservation. The pH low insertion peptide (pHLIP) conjugated with a near-infrared fluorescent dye [indocyanine green (ICG)] targets low extracellular pH, allowing visualization of malignant lesions in human bladder carcinoma ex vivo. Cystectomy specimens obtained after radical surgery were immediately irrigated with nonbuffered saline and instilled with a solution of the ICG pHLIP construct, incubated, and rinsed. Bladders were subsequently opened and imaged, the fluorescent spots were marked, and a standard pathological analysis was carried out to establish the correlation between ICG pHLIP imaging and white light pathological assessment. Accurate targeting of bladder lesions was achieved with a sensitivity of 97%. Specificity is 100%, but reduced to 80% if targeting of necrotic tissue from previous transurethral resections or chemotherapy are considered as false positives. The ICG pHLIP imaging agent marked high-grade urothelial carcinomas, both muscle invasive and nonmuscle invasive. Carcinoma in situ was accurately diagnosed in 11 cases, whereas only four cases were seen using white light, so imaging with the ICG pHLIP peptide offers improved early diagnosis of bladder cancers and may also enable new treatment alternatives.

Keywords: NIR imaging; acidity; bladder cancer; fluorescence-guided surgery; human tissue.

Fig. 1.

Normalized absorbance (A) and fluorescence (B) spectra are shown of the ICG pHLIP construct measured in PBS, pH 7.4, containing 10 mM d-glucose. The fluorescence (with an excitation wavelength of 790 nm) of the ICG pHLIP construct is increased about 25-fold in the presence of POPC liposomes compared with the emission in buffer.

Fig. 2.

Representative white light (A, D, G, and J), NIR fluorescence (B, E, H, and K) ex vivo imaging of bladder specimens and H&E stained tumor sections (C, F, I, and L) are shown, demonstrating targeting of invasive high-grade urothelial carcinoma (A–C), noninvasive high grade urothelial carcinoma (D–F), carcinoma in situ (G–I), and dysplasia (J–L) by the ICG pHLIP imaging agent. The diagnosis was confirmed by pathological analysis. The fluorescent lesions were marked in case 11 to identify locations for pathology analysis.