In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Abstract

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.

Evaluation of a Carbonic Anhydrase IX-Targeted Near-Infrared Dye for Fluorescence-Guided Surgery of Hypoxic Tumors

“…However, despite the availability of ligand-targeted NIR dyes that bind the epidermal growth factor receptor (48), folate receptor alpha (49), CCK2R (50) and PSMA (51), the majority of human cancers still cannot be imaged with a tumor-targeted fluorescent dye. Several reports of CA IX-targeted fluorescent and radioimaging agents have shown the utility of carbonic anhydrase IX targeting ligands for visualization of cancers (38,52,53). Using a different high affinity CA IX ligand, we demonstrate here that a novel CA IX-targeted NIR dye that can illuminate CA IX over-expressing tumors with similar or better results than previous CA IX-fluorescent conjugates.…”

Early Detection of Acute Drug-Induced Liver Injury in Mice by Noninvasive Near-Infrared Fluorescence Imaging

“…Imaging probes are designed to detect tissue biomarkers and accumulate directly at the local site of tissue injury, offering rapid multiplexing imaging of various biologic readouts. NIR fluorescent probes have been used effectively to image various disease conditions modeled in preclinical species, including cancer (Weissleder and Ntziachristos, 2003; Montet et al, 2005; Kossodo et al, 2010; Bao et al, 2012), atherosclerosis (Chen et al, 2002; Deguchi et al, 2006), cardiac pathologies (Nahrendorf et al, 2007; Sosnovik et al, 2007), arthritis (Wunder et al, 2004; Peterson et al, 2010), central nervous system demyelination (Eaton et al, 2013), and osteoarthritis (Vermeij et al, 2014). Many of these same fluorescent imaging probes are currently being examined for their utility in toxicology/safety screening (Peterson, 2016).…”

“…Recent advances in optical imaging and near-infrared (NIR) probes (Weissleder and Ntziachristos, 2003; Korideck and Peterson, 2009; Krautz-Peterson et al, 2009; Kossodo et al, 2010; Peterson et al, 2010; Bao et al, 2012; Lin et al, 2012; Zhang et al, 2012; Eaton et al, 2013; Daghighi et al, 2014) have shown the benefit of biologic, rather than phenotypic or anatomic, readouts in preclinical drug efficacy research. Although imaging of preclinical models of safety/toxicology has not been pursued extensively to date, the ability to use fluorescent imaging probes that detect and quantify a variety of biologic activities has considerable potential in this area as well (Amoozegar et al, 2012; Shuhendler et al, 2014; Peterson, 2016).…”

Prognostic marker analysis in pediatric intracranial ependymomas

“…CA IX has proven useful as a biomarker of hypoxic regions in formalin fixed, paraffin embedded tissues [18][19][20][21]. The identification by immunohistochemistry of hypoxia-related tissue factors CA IX and VEGF have been shown to be useful in characterizing patient outcome in various tumor types [14,17], but we are aware of only one previous study reporting CA IX expression in ependymomas.…”