Evaluation of CAIX and CAXII Expression in Breast Cancer at Varied O2 Levels: CAIX is the Superior Surrogate Imaging Biomarker of Tumor Hypoxia.
OBJECTIVE
Hypoxia is commonly observed in regions of primary tumors and metastases, and is associated with resistance to treatment, more aggressive tumor phenotypes and poor prognosis. Reliable and validated imaging biomarkers of hypoxia are needed for pre-clinical studies and clinical use. Expression of cell-surface carbonic anhydrases IX and XII (CAIX and CAXII) in tumor cells has been associated with tumor hypoxia. CAIX and CAXII specific antibodies conjugated to fluorescent dye were evaluated for the non-invasive detection of hypoxia in vivo.


METHODS
Human breast cancer cell lines (MCF10A, DCIS, MCF7, ZR-75.1 and MDA-mb231) were characterized for CAIX and CAXII expression by real-time RT-PCR and immunocytochemistry (ICC) under normoxic and hypoxic conditions. Immunohistochemical (IHC) staining of CAIX, CAXII and the commercially available exogenous hypoxia marker, pimonidazole, was performed using sections of ZR-75.1 and MDA-mb-231 orthotopic breast cancer xenograft tumors from nude mice. In vivo fluorescence imaging of ZR-75.1 tumors in animals housed at varied levels of oxygen was used to quantify the relative uptake of the CAIX and CAXII agents and a commercially available sulfonamide-based agent. Corresponding tumor sections were IHC stained for CAIX, CAXII and pimonidazole.


RESULTS
CAIX mRNA expression was significantly higher (p < 0.05) in hypoxia for all cell lines, which was in agreement with protein expression by ICC. CAXII expression was mixed, with a modest hypoxia-related increase in two cell lines (p < 0.05) and no change in others. Quantified IHC staining of ZR-75.1 and MDA-mb-231 tumor sections showed that CAIX and CAXII expression was elevated in regions with pimonidazole staining, but CAXII levels were lower than CAIX. Tumor uptake of the CAIX targeted agent, and IHC staining of CAIX and pimonidazole in corresponding tumor sections were correlated, and co-registered, and shown to be significantly elevated by level of oxygenation (p < 0.001): hypoxia > normoxia > hyperoxia. However, the CAXII and sulfonamide agents were not significantly correlated with hypoxia.


CONCLUSIONS
These studies suggest that the fluorescently labeled CAIX-specific agent is a more robust indicator of hypoxia in vivo compared to the CAXII-specific agent or the agent specific to the CA active site.
Department of Cancer Imaging & Metabolism, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.
Narges K
NK
Tafreshi
Analytic Microscopy Core Facility, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.
Mark C
MC
Lloyd
Analytic Microscopy Core Facility, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.
Joshua B
JB
Proemsey
Analytic Microscopy Core Facility, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.|Breast Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.|Department of Anatomic Pathology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.
Marilyn M
MM
Bui
Department of Biostatistics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.
Jongphil
J
Kim
Department of Cancer Imaging & Metabolism, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.
Robert J
RJ
Gillies
Department of Cancer Imaging & Metabolism, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA. david.morse@moffitt.org.
David L
DL
Morse
D000704:Analysis of Variance; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D000071231:Carbonic Anhydrase IX / Q000235:genetics / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D005260:Female; D005453:Fluorescence; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods*; D010100:Oxygen / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014054:Tomography; D000072258:Tumor Hypoxia / Q000187:drug effects* / Q000235:genetics*; D023041:Xenograft Model Antitumor Assays
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D012333:RNA, Messenger; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; C477378:carbonic anhydrase XII; D010100:Oxygen
Breast cancer; Carbonic anhydrase IX; Carbonic anhydrase XII; Non-invasive imaging of hypoxia; Targeted agents