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.
Highlights
Carbonic anhydrase IX (CA IX) is a transmembrane cell surface enzyme which catalyzes the reversible interconversion of CO2 to bicarbonate and a proton
We report that HS680 has high in vitro and in vivo specificity for CA IX, accumulates preferentially in hypoxic regions of CA IX positive tumors and can be used to non-invasively detect and quantify CA IX in tumors as well as changes in CA IX expression induced by manipulation of oxygen levels
The induction of cell hypoxia in the hypoxic chamber and subsequent expression of CA IX relative to normoxia was validated in HT-29 and HeLa cells using pimonidazole staining and binding (Figure S1A), measurement of CA IX protein levels by CA IX ELISA of HT-29 anf HeLa cell lysates (Figure S1B) or by flow cytometry of anti-CA IX antibody binding to four different cell lines (Figure S1C), and detection of a decrease in media pH (Figure S1A)
Summary
Carbonic anhydrase IX (CA IX) is a transmembrane cell surface enzyme which catalyzes the reversible interconversion of CO2 to bicarbonate and a proton. A result of rapid cell proliferation combined with disordered vascular development [9,10] and subsequent expression of CA IX, is correlated to metastasis, poor prognosis and resistance to therapeutic intervention making CA IX an important biomarker in the study of hypoxia, tumor cell proliferation and therapy [11,12,13,14,15,16,17,18,19]. While near-infrared (NIR) labeled CA IX antibodies for detection of CA IX in breast cancer have been reported [36,39], none of these fluorescent inhibitors are in the NIR range, which would allow efficient penetration of photons through living tissue and minimize interference from tissue autofluorescence, necessary for deep tissue imaging and optical tomographic quantification [40,41,42,43]
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