Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo

The acidic tumour microenvironment is now recognized as a tumour phenotype that drives cancer somatic evolution and disease progression, causing cancer cells to become more invasive and to metastasise. This property of solid tumours reflects a complex interplay between cellular carbon metabolism and acid removal that is mediated by cell membrane carbonic anhydrases and various transport proteins, interstitial fluid buffering, and abnormal tumour-associated vessels. In the past two decades, a convergence of advances in the experimental and mathematical modelling of human cancers, as well as non-invasive pH-imaging techniques, has yielded new insights into the physiological mechanisms that govern tumour extracellular pH (pHe). In this review, we examine the mechanisms by which solid tumours maintain a low pHe, with a focus on carbonic anhydrase IX (CAIX), a cancer-associated cell surface enzyme. We also review the accumulating evidence that suggest a role for CAIX as a biological pH-stat by which solid tumours stabilize their pHe. Finally, we highlight the prospects for the clinical translation of CAIX-targeted therapies in oncology.

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In vivo Molecular Prediction of Carbonic Anhydrase IX-G250MN Expression on Immunotherapy Outcome in Renal Cancer

Clinical Cancer Research ◽

10.1158/1078-0432.ccr-05-1512 ◽

2005 ◽

Vol 11 (24) ◽

pp. 8886-8886 ◽

Cited By ~ 3

Author(s):

Adrienne H. Brouwers ◽

Otto C. Boerman ◽

Wim J.G. Oyen

Keyword(s):

Carbonic Anhydrase ◽

Renal Cancer ◽

Carbonic Anhydrase Ix

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Time course of exchanges between red cells and extracellular fluid during CO2 uptake

Journal of Applied Physiology ◽

10.1152/jappl.1975.38.4.710 ◽

1975 ◽

Vol 38 (4) ◽

pp. 710-718 ◽

Cited By ~ 75

Author(s):

R. E. Forster ◽

E. D. Crandall

Keyword(s):

Carbonic Anhydrase ◽

Time Course ◽

Extracellular Fluid ◽

Extracellular Ph ◽

Co2 Uptake ◽

Sudden Increase ◽

Red Cell ◽

Ph Change ◽

Red Cell Membrane

A stopped-flow rapid-reaction apparatus was used to follow the time course of extracellular pH in a human red cell suspension following a sudden increase in PCO2. The extracellular pH change was slow (t1/2 similar to 3.5 s) considering the presence of carbonic anhydrase in the cells. When carbonic anhydrase was added to the extracellular fluid, the half-time was reduced to less than 20 ms. The explanation for these phenomena is that the equilibration of H+ across the red cell membrane is rate-limited by the uncatalyzed reaction CO2 plus H2O formed from H2CO3 outside the cells. A theoretical model was developed which successfully reproduced the experimental results. When the model was used to simulate CO2 exchange in vivo, it was determined that blood PCO2 and pH require long times (greater than 50 s) to approach equilibrium between cells and plasma after leaving an exchange capillary. We conclude that cell-plasma equilibrium may never be reached in vivo, and that in vitro measurements of these quantities may not represent their true values at the site of sampling.

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Sortase-mediated site-specific modification of interleukin-2 for the generation of a tumor targeting acetazolamide-cytokine conjugate

10.1101/2020.07.20.211870 ◽

2020 ◽

Author(s):

Baptiste Gouyou ◽

J Millul ◽

A Villa ◽

S Cazzamalli ◽

D Neri ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Tumor Targeting ◽

Interleukin 2 ◽

Industrial Applications ◽

Carbonic Anhydrase Ix ◽

Site Specific ◽

Tumor Bearing ◽

Bioactive Proteins

1.AbstractSmall ligands specific to tumor-associated antigens can be used as alternatives to antibodies for the delivery of small payloads such as radionuclides, cytotoxic drugs and fluorophores. Their use as delivery moiety of bioactive proteins like cytokines remains largely unexplored. Here, we describe the preparation and in vivo characterization of the first small molecule-cytokine conjugate targeting carbonic anhydrase IX (CAIX), a marker of renal cell carcinoma and hypoxia. Site-specific conjugation between interleukin-2 and acetazolamide was obtained by Sortase A-mediated transpeptidation. Binding of the conjugate to the cognate CAIX antigen was confirmed by surface plasmon resonance. The in vivo targeting of structures expressing carbonic anhydrase IX was assessed by biodistribution experiments in tumor bearing mice. Optimization of manufacturability and tumor targeting performance of acetazolamide-cytokine products will be required in order to enable industrial applications.Graphical abstract

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A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells

Biochemical Journal ◽

10.1042/bcj20190177 ◽

2019 ◽

Vol 476 (10) ◽

pp. 1497-1513 ◽

Cited By ~ 8

Author(s):

Mam Y. Mboge ◽

Zhijuan Chen ◽

Daniel Khokhar ◽

Alyssa Wolff ◽

Lingbao Ai ◽

...

Keyword(s):

Breast Cancer ◽

Carbonic Anhydrase ◽

Cancer Cells ◽

Cancer Progression ◽

Expression Patterns ◽

Extracellular Ph ◽

Carbonic Anhydrase Ix ◽

Monocarboxylate Transporter ◽

Proton Efflux ◽

Ca Ix

AbstractThe most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+ shuttle system in CA IX to facilitate proton efflux through MCT4.

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Dual-functional Liposomes with Carbonic Anhydrase IX Antibody and BR2 Peptide Modification Effectively Improve Intracellular Delivery of Cantharidin to Treat Orthotopic Hepatocellular Carcinoma Mice

Molecules ◽

10.3390/molecules24183332 ◽

2019 ◽

Vol 24 (18) ◽

pp. 3332 ◽

Cited By ~ 3

Author(s):

Zhang ◽

Lin ◽

Chan ◽

Liu ◽

Lu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Carbonic Anhydrase ◽

Therapeutic Potential ◽

Intracellular Delivery ◽

Carbonic Anhydrase Ix ◽

Ca Ix ◽

Dual Functional ◽

Peptide Modification

Liposomal nanotechnology has a great potential to overcome the current major problems of chemotherapy. However, the lack of penetrability and targetability retards the successful delivery of liposomal carriers. Previously, we showed that BR2 peptide modification endowed cantharidin-loaded liposomes with intracellular penetration that enhanced the drug cytotoxic effects. Here, we aimed to improve the targeting delivery of drugs into cancer cells via highly expressed carbonic anhydrase IX (CA IX) receptors by modifying our previous catharidin-loaded BR2-liposomes with anti-CA IX antibody. A higher cellular uptake of dual-functional liposomes (DF-Lp) than other treatments was observed. Induction of CA IX over-expressing resulted in a higher cellular binding of DF-Lp; subsequently, blocking with excess antibodies resulted in a decreased cancer-cell association, indicating a specific targeting property of our liposomes towards CA IX expressed cells. After 3h tracking, most of the liposomes were located around the nucleus which confirmed the involvement of targeting intracellular delivery. Cantharidin loaded DF-Lp exhibited enhanced cytotoxicity in vitro and was most effective in controlling tumor growth in vivo in an orthotopic hepatocellular carcinoma model compared to other groups. Collectively, our results presented the advantage of the BR2 peptide and CA IX antibody combination to elevate the therapeutic potential of cantharidin loaded DF-liposomes.

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Novel Humanized Monoclonal Antibodies for Targeting Hypoxic Human Tumors via Two Distinct Extracellular Domains of Carbonic Anhydrase IX

10.21203/rs.3.rs-827745/v1 ◽

2021 ◽

Author(s):

Miriam Zatovicova ◽

Ivana Kajanova ◽

Monika Barathova ◽

Martina Takacova ◽

Martina Labudova ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Cancer Progression ◽

Standard Therapy ◽

Catalytic Domain ◽

Conformational Epitope ◽

Carbonic Anhydrase Ix ◽

Acidic Environment ◽

Primary Tumors ◽

Ca Ix

Abstract Background Hypoxia in the tumor microenvironment (TME) is often the main factor in the cancer progression. Moreover, low levels of oxygen in tumor tissue may signal that the first or second-line therapy will not be successful. This knowledge triggers the inevitable search for different kinds of treatment that will successfully cure aggressive tumors. Due to its exclusive expression on cancer cells, carbonic anhydrase IX belongs to the group of the most precise targets in hypoxic tumors. CA IX possesses several exceptional qualities that predetermine its crucial role in targeted therapy. Its expression on the cell membrane makes it an easily accessible target, while its absence in healthy corresponding tissues makes the treatment practically harmless. The presence of CA IX in solid tumors causes an acidic environment that may lead to the failure of standard therapy. Methods Parental mouse hybridomas (IV/18 and VII/20) were humanized to antibodies which were subsequently named CA9hu-1 and CA9hu-2. From each hybridoma we obtained 25 clones. Each clone was tested for ADCC and CDC activity, affinity, extracellular pH measurement, multicellular aggregation analysis and real-time monitoring of invasion with xCELLigence system. ResultsBoth CA9hu-1 and CA9hu-2 are IgG1 antibodies and they were both examined in vivo. Here we describe anti-CAIX antibodies that can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME. CA9hu-1 is directed at the conformational epitope of the catalytic domain, while CA9hu-2 targets the sequential epitope of the proteo-glycan domain. They are both able to induce an immune response, have high affinity, as well as ADCC and CDC activity. While the first one internalizes after binding to the antigen, the second one is able to reduce metastases formation. More importantly, they have both proved the ability to block the acidification of the extracellular environment. ConclusionCA9hu-1 and CA9hu-2 are the very first humanized antibodies against CA IX that are likely to become suitable therapies for hypoxic tumors. These antibodies can be applied in the treatment therapy of primary tumors and suppression of metastases formation.

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