BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells. METHODS: We generated a non-shed CA IX mutant by deletion of amino acids 393-402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice. RESULTS: We demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs. CONCLUSIONS: Our results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cell-associated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.

• MeSH
• Phenotype MeSH
• Neoplasm Invasiveness pathology   MeSH
• Carbonic Anhydrase IX metabolism   MeSH
• Carcinogenesis metabolism   pathology   MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Neoplasms metabolism   pathology   MeSH
• ADAM17 Protein metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Carbonic Anhydrase IX MeSH
• ADAM17 Protein MeSH

The systemic application of highly potent drugs such as cytostatics poses the risks of side effects, which could be reduced by using a carrier system able to specifically deliver the encapsulated drug to the target tissue. Essential components of a nanoparticle-based drug delivery system include the drug carrier itself, a targeting moiety, and a surface coating that minimizes recognition by the immune system. The present work reports on the preparation, in vitro characterization and in vivo testing of a new delivery system consisting of fluorescent silica nanoparticles functionalised with a non-immunogenic stealth polymer poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA) and a monoclonal antibody IgG M75 that specifically binds to Carbonic Anhydrase IX (CA IX). CA IX is a promising therapeutic target, as it is a hallmark of several hypoxic tumours including colorectal carcinoma. Uniquely in this work, the monoclonal antibody was covalently coupled to the surface of fluorescently labelled silica nanoparticles via a multivalent amino-reactive co-polymer rather than a traditional bivalent linker. The pHPMA-M75 functionalised SiO2 nanoparticles exhibited excellent colloidal stability in physiological media. Their in vitro characterisation by flow cytometry proved a highly specific interaction with colorectal carcinoma cells HT-29. In vivo study on athymic NU/NU nude mice revealed that the SiO2-pHPMA-M75 nanoparticles are capable of circulating in the blood after intravenous administration and accumulate in the tumour at tenfold higher concentration than nanoparticles without specific targeting, with a considerably longer retention time. Additionally, it was found that by reducing the dose administered in vivo, the selectivity of the nanoparticle biodistribution could be further enhanced in favour of the tumour.

• Publication type
• Journal Article MeSH

Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery.

• MeSH
• Biological Assay * MeSH
• DNA * MeSH
• Enzymes metabolism   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Humans MeSH
• Drug Discovery * MeSH
• Reproducibility of Results MeSH
• Sensitivity and Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA * MeSH
• Enzymes MeSH
• Enzyme Inhibitors MeSH

BACKGROUND: Carbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin. METHODS: Presence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay. RESULTS: We found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness. CONCLUSIONS: These findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.

• Keywords
• Apoptosis, Carbonic anhydrase IX, Chemotherapy, Ectodomain, Hypoxia, Metalloproteinase, Shedding,
• MeSH
• Apoptosis drug effects   genetics   MeSH
• Cycloheximide administration & dosage   MeSH
• Epithelial-Mesenchymal Transition genetics   MeSH
• HeLa Cells MeSH
• Cell Hypoxia genetics   MeSH
• Carbonic Anhydrase IX administration & dosage   genetics   metabolism   MeSH
• Humans MeSH
• Antibodies, Monoclonal administration & dosage   MeSH
• Neoplasms genetics   pathology   MeSH
• ADAM17 Protein genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ADAM17 protein, human MeSH Browser
• Cycloheximide MeSH
• Carbonic Anhydrase IX MeSH
• Antibodies, Monoclonal MeSH
• ADAM17 Protein MeSH

This work is concerned with the surface modification of fluorescent silica nanoparticles by a monoclonal antibody (M75) and the specific bioadhesion of such particles to surfaces containing the PG domain of carbonic anhydrase IX (CA IX), which is a trans-membrane protein specifically expressed on the surfaces of several tumor cell lines. The adhesion strength of antibody-bearing silica nanoparticles to antigen-bearing surfaces was investigated under laminar flow conditions in a microfluidic cell and compared to the adhesion of unmodified silica nanoparticles and nanoparticles coupled with an unspecific antibody. Adhesion to cancer cells using flow cytometry was also investigated and in all cases the adhesion strength of M75-modified nanoparticles was significantly stronger than for the unmodified or unspecific nanoparticles, up to several orders of magnitude in some cases. The specific modification of nano- and microparticles by an antibody-like protein therefore appears to be a feasible approach for the targeting of tumor cells.

• MeSH
• Antigens, Neoplasm chemistry   immunology   metabolism   MeSH
• Cell Adhesion MeSH
• NIH 3T3 Cells MeSH
• Antigen-Antibody Complex MeSH
• Carbonic Anhydrase IX MeSH
• Carbonic Anhydrases chemistry   immunology   metabolism   MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Microfluidic Analytical Techniques MeSH
• Antibodies, Monoclonal chemistry   immunology   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Nanoparticles chemistry   MeSH
• Silicon Dioxide chemistry   MeSH
• Surface Properties MeSH
• Protein Structure, Tertiary MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Neoplasm MeSH
• CA9 protein, human MeSH Browser
• Antigen-Antibody Complex MeSH
• Carbonic Anhydrase IX MeSH
• Carbonic Anhydrases MeSH
• M75 monoclonal antibody MeSH Browser
• Antibodies, Monoclonal MeSH
• Silicon Dioxide MeSH

Tumour-associated protein carbonic anhydrase IX (CA IX) has two major forms. One is a cell-associated, transmembrane protein seen on Western blots as a twin band of 54/58 kDa, expressed in gastric mucosa and in several types of cancer. The other is a soluble protein s-CA IX of 50/54 kDa, which is released into the culture medium or into the body fluids, most likely by proteolytic cleavage of the extracellular part from transmembrane and intracellular sequences. While TC media of CA IX-positive tumour cell lines or short-term cultures of tumour explants contain a relatively high concentration of s-CA IX (20-50 ng ml(-1)), the level of this antigen in blood serum and urine of renal clear cell carcinoma patients is about 1000 x lower. The concentration of CA IX in the blood and in urine varies within wide limits and there is no obvious correlation with tumour size. After nephrectomy, s-CA IX is cleared from the blood within a few days. Only an extremely low concentration of CA IX was detectable in the sera and in urine of control individuals.

• MeSH
• Antigens, Neoplasm blood   urine   MeSH
• Cell Membrane metabolism   MeSH
• Adult MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Carbonic Anhydrase IX MeSH
• Carbonic Anhydrases blood   urine   MeSH
• Carcinoma, Renal Cell enzymology   metabolism   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Biomarkers, Tumor blood   urine   MeSH
• Tumor Cells, Cultured MeSH
• Neoplasm Proteins blood   urine   MeSH
• Kidney Neoplasms enzymology   metabolism   pathology   MeSH
• Blotting, Northern MeSH
• Prognosis MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Antigens, Neoplasm MeSH
• CA9 protein, human MeSH Browser
• Carbonic Anhydrase IX MeSH
• Carbonic Anhydrases MeSH
• Biomarkers, Tumor MeSH
• Neoplasm Proteins MeSH