Functional consequences of altered glycosylation of tumor-associated hypoxia biomarker carbonic anhydrase IX.

Glycosylation is a posttranslational modification of proteins affecting numerous cellular functions. A growing amount of evidence confirms that aberrant glycosylation is involved in pathophysiological processes, including tumor development and progression. Carbonic anhydrase IX (CAIX) is a transmembrane protein whose expression is strongly induced in hypoxic tumors, which makes it an attractive target for anti-tumor therapy. CAIX facilitates the maintenance of intracellular pH homeostasis through its catalytic activity, which is linked with extracellular pH acidification promoting a more aggressive phenotype of tumor cells. The involvement of CAIX in destabilizing cell-cell contacts and the focal adhesion process also contributes to tumor progression. Previous research shows that CAIX is modified with N-glycans, O-glycans, and glycosaminoglycans (GAG). Still, the impact of glycosylation on CAIX functions has yet to be fully elucidated. By preparing stably transfected cells expressing mutated forms of CAIX, unable to bind glycans at their defined sites, we have attempted to clarify the role of glycan structures in CAIX functions. All three types of prepared mutants exhibited decreased adhesion to collagen. By surface plasmon resonance, we proved direct binding between CAIX and collagen. Cells lacking glycosaminoglycan modification of CAIX also showed reduced migration and invasion, indicating CAIX glycosaminoglycans' involvement in these processes. Analysis of signaling pathways affected by the loss of GAG component from CAIX molecule revealed decreased phosphorylation of c-Jun, of p38α kinase, focal adhesion kinase, and reduced level of heat shock protein 60 in cells cultured in hypoxia. Cells expressing CAIX without GAG exhibited increased metabolon formation and increased extracellular pH acidification. We also observed reduced CAIX GAG glycans in the inflammatory environment in hypoxia, pathophysiological conditions reflecting in vivo tumor microenvironment. Understanding the glycan involvement in the characteristics and functions of possible targets of cancer treatment, such as cell surface localized CAIX, could improve the therapy, as many drugs target glycan parts of a protein.

ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non­redundantly to ADAM17.

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme participating in adaptive responses of tumors to hypoxia and acidosis. CA IX regulates pH, facilitates metabolic reprogramming, and supports migration, invasion and metastasis of cancer cells. Extracellular domain (ECD) of CA IX can be shed to medium and body fluids by a disintegrin and metalloproteinase (ADAM) 17. Here we show for the first time that CA IX ECD shedding can be also executed by ADAM10, a close relative of ADAM17, via an overlapping cleavage site in the stalk region of CA IX connecting its exofacial catalytic site with the transmembrane region. This finding is supported by biochemical evidence using recombinant human ADAM10 protein, colocalization of ADAM10 with CA IX, ectopic expression of a dominant­negative mutant of ADAM10 and RNA interference­mediated suppression of ADAM10. Induction of the CA IX ECD cleavage with ADAM17 and/or ADAM10 activators revealed their additive effect. Similarly, additive effect was observed with an ADAM17­inhibiting antibody and an ADAM10­preferential inhibitor GI254023X. These data indicated that ADAM10 is a CA IX sheddase acting on CA IX non­redundantly to ADAM17.

Seroprevalence of SARS-CoV-2 antibodies in the county town of Slovakia - a pilot study from the Trencin city.

Slovakia is a country with only 5.45 million inhabitants. However, the past two years of the COVID-19 pandemic have shown huge inter-regional differences. These were represented by different numbers of diagnosed SARS-CoV-2 cases and the vaccination rates in the regions, as well as by the willingness of the inhabitants to comply with anti-pandemic measures or to undergo testing. The occurrence of such regional disparities provided a rational basis for monitoring the epidemic situation within smaller areas, e.g. at city level. Trencin is a medium-sized Slovak county town with about 55 000 inhabitants. The city administration gave its residents the opportunity to assess their current level of antibodies against the SARS-CoV-2 virus, and received an additional benefit in the form of data on the real epidemic situation in the city, which helped in further management of anti-pandemic measures. The primary aim of the study, conducted in January and February 2022, was to determine the levels of antibodies against the SARS-CoV-2 virus in the inhabitants of Trencin. The results showed that 75% of the study participants, representing the adult population of the city, had detectable IgG antibodies against the SARS-CoV-2 spike protein. Noteworthy, at the time of the study, 13% of the Trencin city population who were unaware of overcoming COVID-19 had specific antibodies against the virus. Furthermore, the antibody levels in recovered unvaccinated subjects increased not only with the severity of their COVID-19 symptoms, but also after multiple recoveries from the disease. On the other hand, the severity of side effects after vaccination did not influence the antibody levels. The results of the study are in line with the current view that hybrid immunity (vaccination plus SARS-CoV-2 infection in any order) offers greater protection than immunity elicited by vaccination or COVID-19 separately. Keywords: SARS-CoV-2 coronavirus; COVID-19; ELISA; seroprevalence; antibodies; vaccination.

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B.

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.

Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX.

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 antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, affinity, extracellular pH measurement, multicellular aggregation analysis, and real-time monitoring of invasion with the xCELLigence system. RESULTS: Based on the results from in vivo experiments, we have selected mouse monoclonal antibodies VII/20 and IV/18. The first one is directed at the conformational epitope of the catalytic domain, internalizes after binding to the antigen, and halts tumor growth while blocking extracellular acidification. The second targets the sequential epitope of the proteo-glycan domain, does not internalize, and is able to block the attachment of cancer cells to the matrix preventing metastasis formation. In vitro experiments prove that humanized versions of the parental murine antibodies, CA9hu-1 and CA9hu-2, have preserved these characteristics. They can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME, and both are able to induce an immune response and have high affinity, as well as ADCC and CDC activity. CONCLUSION: CA9hu-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.

Seroprevalence of SARS-CoV-2 IgG antibodies in the staff of the Slovak Academy of Sciences in response to COVID-19 and/or vaccination: situation in August 2021.

Cross-sectional seroprevalence study of SARS-CoV-2 IgG antibodies was accomplished in the Slovak Academy of Sciences to inform authorities of research institutions about the situation at their workplaces, to assess the risk of next exposure to SARS-CoV-2, and to guide decisions on institutional measures sustaining essential research in evolving epidemic situation. Study participants provided informed consent, anamnestic information, and self-collected dry blood spot samples that were analyzed by ELISA for SARS-CoV-2 S protein-specific IgG antibodies. Relative antibody levels detected in 1928 subjects showed seroprevalence of 84.13% and led to the following main findings consistent with the current knowledge: (1) mRNA-based vaccines induce better humoral response compared to adenovirus vaccines, (2) antibody levels reflect severity of COVID-19 symptoms, (3) post-COVID vaccination results in marked elevation of IgG levels particularly in asymptomatic and mild cases, (4) antibody levels decrease with increasing time elapsed from vaccination or COVID-19. In addition, data sorting to distinct research institutes and their clustering to three principal scientific sections of the Slovak Academy of Sciences revealed marked differences in seroprevalence, and allowed to identify workplaces with relatively high seropositivity and response rate that can potentially provide a safer working environment than those, where seroprevalence was low or unknown due to low participation. Thus, findings of this study can have direct implications on management decisions during the next pandemic development, with the necessity to keep in mind the phenomenon of time-dependent immunity waning and current spread of more contagious Delta variant of SARS-CoV-2. Keywords: SARS-CoV-2 coronavirus; COVID-19; spike protein; seroprevalence; antibodies; vaccination.

Targeting of carbonic anhydrase IX-positive cancer cells by glycine-coated superparamagnetic nanoparticles.

Antibody-modified magnetic nanoparticles were prepared to study their cellular uptake in 3D multicellular spheroidal cell cultures. For this purpose, carbonic anhydrase IX specific monoclonal antibody VII/20 was selected to conjugate on the surface of positively charged glycine coated magnetic nanoparticles in a form of a stable magnetic fluid. In this work, glycine-functionalized magnetic nanoparticles were characterized by different methods. X-ray photoelectron analysis confirmed the binding of glycine to the magnetic nanoparticles, and quantification of the glycine coating on the surface of the magnetic nanoparticles was conducted by thermogravimetric analysis. The optimal weight ratio of glycine to magnetic nanoparticles was determined to be 5 showing good colloid stability due to the high surface charge density of protonated glycine coating shown by the great zeta potential (⁓40 mV). The antibody conjugation to the functionalized magnetic nanoparticles was performed at an antibody to magnetic nanoparticles weight ratio equal to 0.5. Applications of antibody-modified magnetic nanoparticles in cancer therapy rely on their ability to specifically target cancer tissues and enter the tumour intracellular space. Here, we show that antibody coupled nanoparticle internalization was triggered by selective binding to tumour cells expressing hypoxic marker carbonic anhydrase IX. Moreover, our results confirmed specific penetration of conjugated nanoparticles into the tumour cell spheroids.

Understanding metabolic alterations and heterogeneity in cancer progression through validated immunodetection of key molecular components: a case of carbonic anhydrase IX.

Cancer metabolic heterogeneity develops in response to both intrinsic factors (mutations leading to activation of oncogenic pathways) and extrinsic factors (physiological and molecular signals from the extracellular milieu). Here we review causes and consequences of metabolic alterations in cancer cells with focus on hypoxia and acidosis, and with particular attention to carbonic anhydrase IX (CA IX). CA IX is a cancer-associated enzyme induced and activated by hypoxia in a broad range of tumor types, where it participates in pH regulation as well as in molecular mechanisms supporting cancer cells' invasion and metastasis. CA IX catalyzes reversible conversion of carbon dioxide to bicarbonate ion plus proton and cooperates with a spectrum of molecules transporting ions or metabolites across the plasma membrane. Thereby CA IX contributes to extracellular acidosis as well as to buffering intracellular pH, which is essential for cell survival, metabolic performance, and proliferation of cancer cells. Since CA IX expression pattern reflects gradients of oxygen, pH, and other intratumoral factors, we use it as a paradigm to discuss an impact of antibody quality and research material on investigating metabolic reprogramming of tumor tissue. Based on the validation, we propose the most reliable CA IX-specific antibodies and suggest conditions for faithful immunohistochemical analysis of molecules contributing to heterogeneity in cancer progression.

PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity.

Human carbonic anhydrase IX (CAIX), a unique member of the α carbonic anhydrase family, is a transmembrane glycoprotein with high enzymatic activity by which CAIX contributes to tumorigenesis through pH regulation. Due to its aberrant expression, CAIX is considered to be a marker of tumor hypoxia and a poor prognostic factor of several human cancers. Hypoxia-activated catalytic function of CAIX is dependent on posttranslational modification of its short intracellular domain. In this work, we have identified that C-terminal Ala459 residue, which is common across CAIX of various species as well as additional transmembrane isoforms, plays an important role in CAIX activation and in pH regulation. Moreover, structure prediction I-TASSER analysis revealed involvement of Ala459 in potential ligand binding. Using tandem mass spectrometry, Protein-L-isoaspartyl methyltransferase (PIMT) was identified as a novel interacting partner, further confirmed by an in vitro pulldown assay and an in situ proximity ligation assay. Indeed, suppression of PIMT led to increased alkalinization of culture media of C33a cells constitutively expressing CAIX in hypoxia. We suggest that binding of PIMT represents a novel intracellular signal required for enzymatic activity of CAIX with a potential unidentified downstream function.

Molecular targeting of bioconjugated graphene oxide nanocarriers revealed at a cellular level using label-free Raman imaging.

Two-dimensional materials as graphene oxide (GO) are able to accommodate labels as well as toxins for diagnostics and therapy, respectively. The transmembrane protein carbonic anhydrase (CA IX) is one of the molecules selectively expressed by tumor cells. Here, we demonstrate bioconjugation of GO to biotinylated M75 antibody highly selective towards CA IX. Based on a model system, binding between the bioconjugated GO-M75 and Madin-Darby Canine Kidney (MDCK) cells was evaluated. As proven by fluorescence-activated cell sorting, higher intake was observed for GO-M75 towards MDCK cells ectopically expressing CA IX protein on their surface when compared to control MDCK. In particular, we were able to localize GO nanocarrier crossing the membrane during endocytosis, thanks to the optical cross-sectioning of living cells in real-time employed the label-free confocal Raman microscopy. The increased affinity of the prepared GO-M75 molecular complexes validates the use of two-dimensional materials for future strategies of targeted cancer treatment.

Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells.

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.

CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins.

Tumor metastasis is tightly linked with invasive membrane protrusions, invadopodia, formed by actively invading tumor cells. Hypoxia and pH modulation play a role in the invadopodia formation and in their matrix degradation ability. Tumor-associated carbonic anhydrase IX (CAIX), induced by hypoxia, is essential for pH regulation and migration, predisposing it as an active component of invadopodia. To investigate this assumption, we employed silencing and inhibition of CA9, invadopodia isolation and matrix degradation assay. Quail chorioallantoic membranes with implanted tumor cells, and lung colonization assay in murine model were used to assess efficiency of in vivo invasion and the impact of CAIX targeting antibodies. We showed that CAIX co-distributes to invadopodia with cortactin, MMP14, NBCe1, and phospho-PKA. Suppression or enzymatic inhibition of CAIX leads to impaired invadopodia formation and matrix degradation. Loss of CAIX attenuated phosphorylation of Y421-cortactin and influenced molecular machinery coordinating actin polymerization essential for invadopodia growth. Treatment of tumor cells by CAIX-specific antibodies against carbonic or proteoglycan domains results in reduced invasion and extravasation in vivo. For the first time, we demonstrated in vivo localization of CAIX within invadopodia. Our findings confirm the key role of CAIX in the metastatic process and gives rationale for its targeting during anti-metastatic therapy.

A Multifunctional Graphene Oxide Platform for Targeting Cancer.

Diagnosis of oncological diseases remains at the forefront of current medical research. Carbonic Anhydrase IX (CA IX) is a cell surface hypoxia-inducible enzyme functionally involved in adaptation to acidosis that is expressed in aggressive tumors; hence, it can be used as a tumor biomarker. Herein, we propose a nanoscale graphene oxide (GO) platform functionalized with magnetic nanoparticles and a monoclonal antibody specific to the CA IX marker. The GO platforms were prepared by a modified Hummers and Offeman method from exfoliated graphite after several centrifugation and ultrasonication cycles. The magnetic nanoparticles were prepared by a chemical precipitation method and subsequently modified. Basic characterization of GO, such as the degree of oxidation, nanoparticle size and exfoliation, were determined by physical and chemical analysis, including X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDX), and atomic force microscopy (AFM). In addition, the size and properties of the poly-L-lysine-modified magnetic nanoparticles were characterized. The antibody specific to CA IX was linked via an amidic bond to the poly-L-lysine modified magnetic nanoparticles, which were conjugated to GO platform again via an amidic bond. The prepared GO-based platform with magnetic nanoparticles combined with a biosensing antibody element was used for a hypoxic cancer cell targeting study based on immunofluorescence.

Carbonic Anhydrase IX-Mouse versus Human.

In contrast to human carbonic anhydrase IX (hCA IX) that has been extensively studied with respect to its molecular and functional properties as well as regulation and expression, the mouse ortholog has been investigated primarily in relation to tissue distribution and characterization of CA IX-deficient mice. Thus, no data describing transcriptional regulation and functional properties of the mouse CA IX (mCA IX) have been published so far, despite its evident potential as a biomarker/target in pre-clinical animal models of tumor hypoxia. Here, we investigated for the first time, the transcriptional regulation of the Car9 gene with a detailed description of its promoter. Moreover, we performed a functional analysis of the mCA IX protein focused on pH regulation, cell-cell adhesion, and migration. Finally, we revealed an absence of a soluble extracellular form of mCA IX and provided the first experimental evidence of mCA IX presence in exosomes. In conclusion, though the protein characteristics of hCA IX and mCA IX are highly similar, and the transcription of both genes is predominantly governed by hypoxia, some attributes of transcriptional regulation are specific for either human or mouse and as such, could result in different tissue expression and data interpretation.