Indium-111-labeled Dual CA9-targeted Probe as a Potential Nuclear Imaging Agent for Hypoxic Colorectal Cancer Detection in a Xenograft Mouse Model

Purpose: Colorectal cancer (CRC) is known as a common malignant neoplasm worldwide. Tumor hypoxia is common features in CRC and involves in radiotherapy and chemotherapy resistance. Therefore, to develop specifically diagnostic targeted probe at hypoxic CRC for providing correct treatment methods and improving survival and quality of life is urgent needed. Previous studies have indicated that the carbonic anhydrase 9 (CA9) protein is considered as a specific marker for hypoxic colorectal cancer (CRC) detection. In this study, we attend to develop a nuclear imaging agent probe, CA9 targeted-peptide (CA9tp) conjugated with CA9 inhibitor, acetazolamide (AAZ), to detect the CA9 expression in a hypoxic CRC xenograft mouse model.

Methods: We designed and product a dual CA9-targeted probe (AAZ-CA9tp) with automatic microwave peptide synthesizer for cell binding, stability, and toxicity assay in hypoxic CRC in vitro. The 1, 4, 7, 10-Tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA)-conjugated AAZ-CA9tp further was labeled with indium-111 (¹¹¹In-DOTA-AAZ-CA9tp) as nuclear imaging reagent for imaging and bio-distribution analysis in hypoxic and CA9 overexpressed HCT15-bearing mice. Finally, the ¹¹¹In-DOTA-AAZ-CA9tp was performed tissue binding ability assay in clinical human CRC section by using autoradiography.

Results: The CA9ap-AAZ was confirmed the CA9protein targeting ability in hypoxic HCT15 cells. The dual CA9-targeted probe was showed high serum stability and non-toxicity in vitro. We further observed that the ¹¹¹In-DOTA-AAZ-CA9tp was significantly enhanced in hypoxic tumor tissues of HCT15-bearing xenograft mice as compared to the non-AAZ-CA9tp control. In addition, we demonstrated that the ¹¹¹In-DOTA-AAZ-CA9tp can targeted at CA9 overexpress position in human hypoxic CRC tissue section.

Conclusions: Our results showed that the isotope-labeled imaging agent (¹¹¹In-DOTA-AAZ-CA9tp) may be served as a powerful tool for hypoxic CRC detection in clinical.

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