Abstract 3949: In vivo imaging of tumor hypoxia by a new near-infrared fluorescent carbonic anhydrase IX-targeted agent

Abstract

Carbonic anhydrase IX (CA IX), a cell surface enzyme involved in tumor cell acidification, is induced during tumor hypoxia (lack of oxygen) in many different types of tumors. CA IX expression is correlated to tumor cell proliferation and metastasis, poor prognosis, and resistance to therapeutic intervention, making CA IX an important biomarker in the study of hypoxia, tumor cell proliferation, and therapy. Therefore, determination of CAIX expression and identification of hypoxic tumors in vivo are critical for cancer research and treatments. We have designed and synthesized a near-infrared (NIR) fluorescent agent (VM3219) for in vivo detection and quantification of a cell surface biomarker of hypoxia, CA IX based on a well-known CA IX inhibitor, acetazolamide. VM3219 was characterized extensively in HeLa cells and in mouse HeLa tumor xenografts. Preliminary chemical and biochemical characterization of VM3219 showed excellent CA IX selectivity and affinity. In vitro, VM3219 detected 4-7-fold up-regulation of CA IX in live hypoxic HeLa cells as visualized by fluorescence microscopy and quantified by flow cytometry. VM3219 fluorescence signals were blocked by 80% when cells were pre-incubated with unlabeled acetazolamide. Fluorescent signal of VM3219 was low in normoxic HeLa cells and was comparable to the signal that were detected by using a non-binding control agent (VM3182) incubated with normoxic or hypoxic cells. In vivo imaging by fluorescence molecular tomography (FMT^TM) showed that approximately 5% of the injected dose (2 nmol VM3219) was retained in the tumor tissues after 24 h (100 -120 pmol), 10-20-fold more than measured with a non-binding control agent. Tumor fluorescence was blocked >65% by prophylactic acetazolamide treatment. Maintenance of tumor-bearing mice on low oxygen (8%), as compared to normally housed tumor bearing mice (20% oxygen), induced a >2X (∼250 pmol) increase in VM3219 tumor fluorescence signal suggesting that CAIX expression was up-regulated within tumors by low oxygen. In tissue sections, VM3219 signal was shown to be specifically co-localized in the tumor hypoxic regions with CA IX antibody and Hypoxyprobe (pimonidazole), and the signal was away from non-hypoxic regions as determined by Hoechst stain. In conclusion, VM3219 is a new non-invasive tumor hypoxia imaging agent that offers a tool for specific imaging of a biologically important hypoxia biomarker, CA IX.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3949. doi:1538-7445.AM2012-3949