Introduction and Objectives: Up to 50% of patients with non-muscle invasive bladder cancer suffer from recurrence or progression into a muscle-invasive disease despite multiple resections and long-term chemo and immunotherapy. Possible reasons for that are incomplete resection and reimplantation of cancer cells, which could be prevented by early detection and improved resection of tumor margins. Fluorescence-guided surgery is beneficial in some patients with flat tumors; however, it is currently based on metabolic differences of cancer cells and lacks specificity in many cases. Molecular imaging could improve the specificity of fluorescence-guided surgery since it can target biomarkers expressed by individual tumors. We aimed to select a biomarker and develop a probe for molecular imaging of urothelial carcinoma (UC).
Methods: One of the biomarkers present in UC is a heparan sulphate proteoglycan Glypican-1 (GPC-1). The anti-GPC-1 monoclonal antibody Miltuximab (Glytherix Ltd. Sydney, Australia) was used to analyze the expression of GPC-1 in UC cell lines T24 and UM-UC-6. Miltuximab was then labelled by a fluorescent dye IRDye800CW and tested in vivo. Luciferase-transfected UM-UC-6 cells were injected subcutaneously into the hind leg of ten nude mice. When the tumors reached 1000 mm3 in volume, the mice were intravenously injected with 5.7 mg/kg Miltuximab-IRDye800 or isotype IgG-IRDye800. They were then imaged daily for ten days and sacrificed for ex vivo imaging to study the distribution of Miltuximab within the body and its accumulation in the tumors.
Results: Flow cytometry and Western blotting demonstrated high expression of GPC-1 in cells UM-UC-6 and T24. UM-UC-6 cells formed subcutaneous tumors that were histologically representative of tumors of human bladder in all animals. From one to ten days after the intravenous injection of the fluorescently labeled Miltuximab, we detected its high accumulation in the tumors by in vivo and ex vivo imaging. One day after the injection, tumors could be visualized in both groups with tumor-to-noise (T/N) ratio of 2. On the 5th day, this value went up to 6.7 for Miltuximab and 3.5 for the Isotype IgG group. By day 10, non-bound antibody molecules further cleared from the mice, resulting in T/N ratio of 14.1 in the mice injected with Miltuximab, while in the control group the value reached only 4.8.
Conclusions: Overexpression of GPC-1 in UC shows its high potential as a target for molecular imaging. The animal model developed in this study demonstrated accumulation of the antibody in the tumor and its strong visualization, which could be helpful in tumor detection and resection. Another advantage of this platform is its high potential for clinical translation. IRDye800 can be visualized by existing equipment for indocyanine green imaging and has been proven to be safe in several clinical trials. The antibody Miltuximab has also been used in clinic and has an excellent safety profile.
Citation Format: Dmitry Polikarpov, Douglas Campbell, Lucinda McRobb, Alexander Zaslavsky, Andrei Zvyagin, Bradley Walsh, Ganesh Palapattu, David Gillatt. Application of Miltuximab®-IRDye800 for near-infrared fluorescence imaging of urothelial carcinoma in vivo [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A08.