Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects.

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

Unraveling the mechanism of carbonic anhydrase IX inhibition by alkaloids from Ruta chalepensis: A synergistic analysis of in vitro and in silico data.

Due to the pivotal role of carbonic anhydrase IX (CA IX) in pathological conditions, there's a pressing need for novel inhibitors to improve patient outcomes and clinical management. Herein, we investigated the inhibitory efficacy of six alkaloids from Ruta chalepensis against CA IX through in vitro inhibition assay and computational modeling. Skimmianine and maculosidine displayed significant inhibitory activity in vitro, with low IC50 values of 105.2 ± 3.2 and 295.7 ± 14.1 nM, respectively. Enzyme kinetics analyses revealed that skimmianine exhibited a mixed inhibition mode, contrasting with the noncompetitive inhibition mechanism observed for the reference drug (acetazolamide), as indicated by intersecting lines in the Lineweaver-Burk plots. The findings of docking calculations revealed that skimmianine and maculosidine exhibited extensive polar interactions with the enzyme. These alkaloids demonstrate substantial binding interactions and occupy identical binding site as acetazolamide, thereby enhancing their efficacy as inhibitors of CA IX. Utilizing a 100 ns molecular dynamics (MD) simulation, the dynamic interactions between isolated alkaloids and CA IX were intensively assessed. Analysis of diverse MD parameters revealed that skimmianine and maculosidine displayed consistent trajectories and notable energy stabilization during their interaction with CA IX. The findings of MM/PBSA analysis depicted the minimum binding free energy for skimmianine and maculosidine. In addition, the Potential Energy Landscape (PEL) analysis revealed distinct and stable conformational states for the CA IX-ligand complexes, with Skimmianine showing the most stable and lowest energy configuration. These computational findings align with experimental results, emphasizing the potential efficacy of skimmianine and maculosidine as inhibitors of CA IX.

Diagnostic efficacy of [68Ga]Ga-NY104 PET/CT to identify clear cell renal cell carcinoma.

PURPOSE: Most clear cell renal cell carcinoma (ccRCC) overexpresses carbonic anhydrase IX (CAIX). [68Ga]Ga-NY104 is a small-molecule PET agent selectively targeting CAIX. This study aims to assess the efficacy of [68Ga]Ga-NY104 PET/CT to identify ccRCC. MATERIALS AND METHODS: Participants were prospectively recruited in the study (ClinicalTrials.gov: NCT05902377). They were further divided into two groups: group 1, patients with primary renal mass who were scheduled for surgery, group 2, patients with suspected/confirmed metastatic ccRCC. All patients underwent [68Ga]Ga-NY104 PET/CT. RESULTS: A total of 47 patients (mean age, 58.8 years ± 13.5, 34 men) were recruited, including 20 patients in group 1 and 27 patients in group 2. The patient-level sensitivity, specificity, and accuracy of [68Ga]Ga-NY104 PET scan was 62%, 33%, 58% for group 1 and 95%, 100%, 96% for group 2. [68Ga]Ga-NY104 PET identified additional 26 disease regions in 67% (14/21) of patients that were previously unknown. The tumor uptake was correlated with immunohistochemical staining results. CONCLUSIONS: [68Ga]Ga-NY104 PET/CT has a high diagnostic efficacy for patients with metastatic ccRCC, while it might be of limited value in the diagnosis of primary ccRCC.

Development of carbonic anhydrase IX-targeting molecular-targeted photodynamic therapy.

The efficacy of molecular-targeted photodynamic therapy (MT-PDT) targeting carbonic anhydrase (CA) IX, a cancer-specific molecule, was demonstrated. CA ligand-directed photosensitizers 1-3 were evaluated for their ability to deactivate CAIX protein in cells. Compounds 2 and 3 selectively deactivated CAIX protein under 540 nm light without affecting internal standard proteins. Mechanistic studies revealed that compound 3 not only induced CAIX-selective light inactivation via singlet oxygen but also induced cell membrane damage, resulting in an anti-tumor effect. In vivo studies of CAIX-targeting MT-PDT revealed that treatment with compound 3 followed by light irradiation exhibited remarkable anti-tumor activity, leading to tumor degeneration and necrosis.

Discovery of novel 1,8-naphthalimide piperazinamide based benzenesulfonamides derivatives as potent carbonic anhydrase IX inhibitors and ferroptosis inducers for the treatment of triple-negative breast cancer.

A novel series of 1,8-naphthalimide piperazinamide based benzenesulfonamides derivatives were designed and synthesized as carbonic anhydrase IX (CA IX) inhibitors and ferroptosis inducers for the treatment of triple-negative breast cancer (TNBC). The representative compound 9o exhibited more potent inhibitory activity and selective against CA IX over off-target CA II, compared with positive control SLC-0111. Molecular docking study was also performed to gain insights into the binding interactions of 9o in the binding pocket of CAIX. Moreover, compound 9o exhibited superior antitumor activities against breast cancer cells under hypoxia than that of normoxia conditions. Mechanism studies revealed that compound 9o could act as DNA intercalator and effectively suppressed cell migration, arrested the cell cycle at G1/S phase and induced apoptosis in MDA-MB-231 cells, while inducing ferroptosis accompanied by the dissipation of MMP and the elevation intracellular levels of ROS. Notably, in vivo studies demonstrated that 9o effectively inhibited tumor growth and metastasis in a highly metastatic murine breast cancer 4 T1 xenograft model. Taken together, this study suggests that compound 9o represents a potent and selective CA IX inhibitor and ferroptosis inducer for the treatment of TNBC.

Novel benzenesulfonamide derivatives as potential selective carbonic anhydrase IX, XII inhibitors with anti-proliferative activity: Design, synthesis and in silico studies.

As inhibitors of carbonic anhydrases (CAs) IX and XII, a novel series of 1,2,3-triazole benzenesulfonamide derivatives 17a-l containing pyrazolyl-thiazole moiety was designed, synthesized, and tested for anti-proliferative activity. Compounds 17e-h demonstrated more effective inhibitory activity than acetazolamide (IC50 63 nM CA IX and IC50 92 nM CA XII), with IC50 range of 25-52 nM against CA IX and IC50 range of 31-80 nM against CA XII. To verify selectivity against CA IX and CA XII, carbonic anhydrase inhibitory activity of compounds 17e-h against the physiological CA I and CA II isoforms was carried out. The results showed that compounds 17e-h induced lower inhibitory activity against CA I and CA II with IC50 range of 0.428-0.638 µM (CA I) and 0.095-0.164 µM (CA II), in addition to higher selectivity indices (CA I/CA IX S.I. 8.9-19.92, CA I/CA XII S.I. 5.78-16.06) and (CA II/CA IX S.I. 2.83-4.35, CA II/CA XII S.I. 2.05-3.15) when compared to that of acetazolamide, IC50 of 0.199 µM (CA I), 0.133 µM (CA II) (CA I/CA IX S.I. 3.15, CA I/CA XII S.I. 2.16) and (CA II/CA IX S.I. 2.11, CA II/CA XII S.I. 1.44). Concerning anti-proliferative activity of compounds 17e-h, investigations were done on HEPG-2 cell line with IC50 ranges of 3.44-15.03 µM in comparison, 5-FU and doxorubicin showed IC50 values of 11.80 and 9.53 µM, respectively. Furthermore IC50 of MCF-7 and MDA-MB-231 were determined under both normoxic and hypoxic conditions with IC50 values ranging from 3.18-8.26 µM MCF-7 (normoxic), 1.39-6.05 µM MCF-7 (hypoxic), 7.13-26.3 µM MDA-MB-231 (normoxic), 0.76-16.3 µM MDA-MB-231 (hypoxic) using acetazolamide and SLC-0111 as selective CA inhibition references. Moreover, compounds 17e-h demonstrated greater safety against the normal cell line, MCF-10A, with IC50 of 23.06-99.50 µM in comparison to 5-FU and doxorubicin IC50 of 59.8 and 71.8 µM respectively. They also demonstrated (MCF-7 S.I. range of 3.77-31.28) in contrast to doxorubicin (S.I. 13.72) and (HepG-2 S.I. range of 3.60-6.95) in comparison to doxorubicin (S.I. 7.53). In relation to CA IX, XII inhibition, molecular docking of and ADME studies of sulfonamide derivatives 17a-l with CA IX (PDB: 5FL6) and CA XII (PDB: 1JD0) was carried out. Additionally, molecular dynamic simulation was carried out for compounds 17e and 17g which maintained good stability inside the active sites of both enzymes, with average RMSDs of 2.3 Å and 2.1 Å, respectively.

Preclinical and pilot clinical evaluation of a small-molecule carbonic anhydrase IX targeting PET tracer in clear cell renal cell carcinoma.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) highly expresses carbonic anhydrase IX (CAIX). The purpose of this study was to evaluate 68Ga-NY104, a small-molecule CAIX-targeting PET agent, in tumor models of ccRCC and patients diagnosed with confirmed, or suspicious, ccRCC. METHODS: The in vivo and ex vivo biodistribution of 68Ga-NY104 was investigated in CAIX-positive OS-RC-2 xenograft-bearing models. The binding of the tracer was further validated using autoradiography for human ccRCC samples. In addition, three patients with confirmed or suspicious ccRCC were studied. RESULTS: NY104 can be labeled with high radiochemical yield and purity. It quickly cleared through kidney with α-half-life of 0.15 h. Discernible uptake is noted in the heart, lung, liver, stomach, and kidney. The OS-RC-2 xenograft demonstrated intense uptake 5 min after injection and gradually increased until 3 h after injection with ID%/g of 29.29 ± 6.82. Significant binding was detected using autoradiography on sections of human ccRCC tumor. In the three patients studied, 68Ga-NY104 was well-tolerated and no adverse events were reported. Substantial accumulation was observed in both primary and metastatic lesions in patient 1 and 2 with SUVmax of 42.3. Uptake in the stomach, pancreas, intestine, and choroid plexus was noted. The lesion in third patient was correctly diagnosed as non-metastatic for negative 68Ga-NY104 uptake. CONCLUSION: 68Ga-NY104 can efficiently and specifically bind to CAIX. Given the pilot nature of our study, future clinical studies are warranted to evaluate 68Ga-NY104 for detection of CAIX-positive lesions in patients with ccRCC. TRIAL REGISTRATION: The clinical evaluation part of this study was retrospectively registered at ClinicalTrial.gov (NCT05728515) as NYPILOT on 6 Feb, 2023.

Carbonic anhydrase IX inhibitor S4 triggers release of DAMPs related to immunogenic cell death in glioma cells via endoplasmic reticulum stress pathway.

BACKGROUND: Immunogenic cell death (ICD), which releases danger-associated molecular patterns (DAMP) that induce potent anticancer immune response, has emerged as a key component of therapy-induced anti-tumor immunity. The aim of this work was to analyze whether the carbonic anhydrase IX inhibitor S4 can elicit ICD in glioma cells. METHODS: The effects of S4 on glioma cell growth were evaluated using the CCK-8, clonogenic and sphere assays. Glioma cell apoptosis was determined by flow cytometry. Surface-exposed calreticulin (CRT) was inspected by confocal imaging. The supernatants of S4-treated cells were concentrated for the determination of HMGB1and HSP70/90 expression by immunoblotting. RNA-seq was performed to compare gene expression profiles between S4-treated and control cells. Pharmacological inhibition of apoptosis, autophagy, necroptosis and endoplasmic reticulum (ER) stress was achieved by inhibitors. In vivo effects of S4 were evaluated in glioma xenografts. Immunohistochemistry (IHC) was performed to stain Ki67 and CRT. RESULTS: S4 significantly decreased the viability of glioma cells and induced apoptosis and autophagy. Moreover, S4 triggered CRT exposure and the release of HMGB1 and HSP70/90. Inhibition of either apoptosis or autophagy significantly reversed S4-induced release of DAMP molecules. RNA-seq analysis indicated that the ER stress pathway was deregulated upon exposure to S4. Both PERK-eIF2α and IRE1α- XBP1 axes were activated in S4-treated cells. Furthermore, pharmacological inhibition of PERK significantly suppressed S4-triggered ICD markers and autophagy. In glioma xenografts, S4 significantly reduced tumor growth. CONCLUSIONS: Altogether, these findings suggest S4 as a novel ICD inducer in glioma and might have implications for S4-based immunotherapy. Video Abstract.

Quantitative Imaging of Hypoxic CAIX-Positive Tumor Areas with Low Immune Cell Infiltration in Syngeneic Mouse Tumor Models.

Limited diffusion of oxygen in combination with increased oxygen consumption leads to chronic hypoxia in most solid malignancies. This scarcity of oxygen is known to induce radioresistance and leads to an immunosuppressive microenvironment. Carbonic anhydrase IX (CAIX) is an enzyme functioning as a catalyzer for acid export in hypoxic cells and is an endogenous biomarker for chronic hypoxia. The aim of this study is to develop a radiolabeled antibody that recognizes murine CAIX to visualize chronic hypoxia in syngeneic tumor models and to study the immune cell population in these hypoxic areas. An anti-mCAIX antibody (MSC3) was conjugated to diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with indium-111 (111In). CAIX expression on murine tumor cells was determined using flow cytometry, and in vitro affinity of [111In]In-MSC3 was analyzed in a competitive binding assay. Ex vivo biodistribution studies were performed to determine in vivo radiotracer distribution. CAIX+ tumor fractions were determined by mCAIX microSPECT/CT, and the tumor microenvironment was analyzed using immunohistochemistry and autoradiography. We showed that [111In]In-MSC3 binds to CAIX-expressing (CAIX+) murine cells in vitro and accumulates in CAIX+ areas in vivo. We optimized the use of [111In]In-MSC3 for preclinical imaging such that it can be applied in syngeneic mouse models and showed that we can quantitatively distinguish between tumor models with varying CAIX+ fractions by ex vivo analyses and in vivo mCAIX microSPECT/CT. Analysis of the tumor microenvironment identified these CAIX+ areas as less infiltrated by immune cells. Together these data demonstrate that mCAIX microSPECT/CT is a sensitive technique to visualize hypoxic CAIX+ tumor areas that exhibit reduced infiltration of immune cells in syngeneic mouse models. In the future, this technique may enable visualization of CAIX expression before or during hypoxia-targeted or hypoxia-reducing treatments. Thereby, it will help optimize immuno- and radiotherapy efficacy in translationally relevant syngeneic mouse tumor models.

PET/MRI of hypoxia and vascular function in ER-positive breast cancer: correlations with immunohistochemistry.

OBJECTIVES: To explore the relationship between indices of hypoxia and vascular function from 18F-fluoromisonidazole ([18F]-FMISO)-PET/MRI with immunohistochemical markers of hypoxia and vascularity in oestrogen receptor-positive (ER +) breast cancer. METHODS: Women aged > 18 years with biopsy-confirmed, treatment-naïve primary ER + breast cancer underwent [18F]-FMISO-PET/MRI prior to surgery. Parameters of vascular function were derived from DCE-MRI using the extended Tofts model, whilst hypoxia was assessed using the [18F]-FMISO influx rate constant, Ki. Histological tumour sections were stained with CD31, hypoxia-inducible factor (HIF)-1α, and carbonic anhydrase IX (CAIX). The number of tumour microvessels, median vessel diameter, and microvessel density (MVD) were obtained from CD31 immunohistochemistry. HIF-1α and CAIX expression were assessed using histoscores obtained by multiplying the percentage of positive cells stained by the staining intensity. Regression analysis was used to study associations between imaging and immunohistochemistry variables. RESULTS: Of the lesions examined, 14/22 (64%) were ductal cancers, grade 2 or 3 (19/22; 86%), with 17/22 (77%) HER2-negative. [18F]-FMISO Ki associated negatively with vessel diameter (p = 0.03), MVD (p = 0.02), and CAIX expression (p = 0.002), whilst no significant relationships were found between DCE-MRI pharmacokinetic parameters and immunohistochemical variables. HIF-1α did not significantly associate with any PET/MR imaging indices. CONCLUSION: Hypoxia measured by [18F]-FMISO-PET was associated with increased CAIX expression, low MVD, and smaller vessel diameters in ER + breast cancer, further corroborating the link between inadequate vascularity and hypoxia in ER + breast cancer. KEY POINTS: • Hypoxia, measured by [18F]-FMISO-PET, was associated with low microvessel density and small vessel diameters, corroborating the link between inadequate vascularity and hypoxia in ER + breast cancer. • Increased CAIX expression was associated with higher levels of hypoxia measured by [18F]-FMISO-PET. • Morphologic and functional abnormalities of the tumour microvasculature are the major determinants of hypoxia in cancers and support the previously reported perfusion-driven character of hypoxia in breast carcinomas.

Clinical and Prognostic Impact of the Warburg Effect in Esophageal Carcinoma: Monocarboxylate Transporters as Candidates for Therapeutic Targeting.

INTRODUCTION: Esophageal cancer (EC) seems to display increased glycolytic activity, but clinical studies on the expression/prognostic significance of glycometabolism-related proteins, as well as functional assays, are missing. METHODS: Expression of 10 glycolytic biomarkers was evaluated by immunohistochemistry in tissue sections from 95 patients. Two esophageal squamous cell carcinoma (ESCC) cell lines were used to assess the effect of monocarboxylate transporter (MCT) downregulation on cell viability and extracellular lactate/glucose accumulation. RESULTS: Expression of MCT1, MCT4, CD147, and GLUT1 was significantly associated with an ESCC histopathology, while a poor clinicopathological profile was seen in GLUT1- and LDHA-positive EC cases. In the ESCC group, MCT1 immunoreactivity is associated with high TNM stage and metastasis. The 3-year overall survival (OS) rate was significantly influenced by MCT4 and CAIX positivity and HKII negativity. Those biomarkers were considered independent prognostic factors of OS in multivariate analysis. Dual inhibition of MCT1/4 expression decreased cell viability and extracellular lactate accumulation in ESCC cells. CONCLUSION: Elevated glycolytic rates correlate with a poor clinicopathological profile in EC patients. MCT4 and CAIX positivity independently predict a worse prognosis. Due to the lack of information on treatment modalities, we could not further infer the role of these biomarkers in predicting response to therapy, which needs to be assessed in future studies. In addition, MCT1/4 targeting should be performed both "in vitro" and "in vivo" to further explore its impact on tumor growth and response to classical therapies. HKII expression and function, particularly in the tumor stroma, should be investigated.

CA9 and PRELID2; hypoxia-responsive potential therapeutic targets for pancreatic ductal adenocarcinoma as per bioinformatics analyses.

A strong hypoxic environment has been observed in pancreatic ductal adenocarcinoma (PDAC) cells, which contributes to drug resistance, tumor progression, and metastasis. Therefore, we performed bioinformatics analyses to investigate potential targets for the treatment of PDAC. To identify potential genes as effective PDAC treatment targets, we selected all genes whose expression level was related to worse overall survival (OS) in The Cancer Genome Atlas (TCGA) database and selected only the genes that matched with the genes upregulated due to hypoxia in pancreatic cancer cells in the dataset obtained from the Gene Expression Omnibus (GEO) database. Although the extracted 107 hypoxia-responsive genes included the genes that were slightly enriched in angiogenic factors, TCGA data analysis revealed that the expression level of endothelial cell (EC) markers did not affect OS. Finally, we selected CA9 and PRELID2 as potential targets for PDAC treatment and elucidated that a CA9 inhibitor, U-104, suppressed pancreatic cancer cell growth more effectively than 5-fluorouracil (5-FU) and PRELID2 siRNA treatment suppressed the cell growth stronger than CA9 siRNA treatment. Thus, we elucidated that specific inhibition of PRELID2 as well as CA9, extracted via exhaustive bioinformatic analyses of clinical datasets, could be a more effective strategy for PDAC treatment.

PET radiotracers and fluorescent probes for imaging human carbonic anhydrase IX and XII in hypoxic tumors.

Positron emission tomography (PET) and fluorescent imaging play a pivotal role in medical diagnosis, biomedical oncologic research, and drug development process, which include identification of target location, target engagement, but also prove on mechanism of action or pharmacokinetics of new drug candidates. PET estimates physiological changes at the molecular level using specific radiotracers containing a short-lived positron emitting radionuclide such as fluorine-18 or carbon-11, whereas fluorescent imaging techniques use fluorescent probes labeled with suitable drug candidates for detection at the molecular level. The human carbonic anhydrase (hCA) isoforms IX and XII are overexpressed in hypoxic cancer cells, promoting tumor growth by regulating extra/intracellular pH, ferroptosis, and metabolism, being recognized as promising targets for anticancer theranostic agents. In this review, we have focused on PET radiotracers as well as fluorescent probes for diagnosis and treatment of tumors expressing hCA IX and hCA XII.

2H-chromene and 7H-furo-chromene derivatives selectively inhibit tumour associated human carbonic anhydrase IX and XII isoforms.

Tumour associated carbonic anhydrases (CAs) IX and XII have been recognised as potential targets for the treatment of hypoxic tumours. Therefore, considering the high pharmacological potential of the chromene scaffold as selective ligand of the IX and XII isoforms, two libraries of compounds, namely 2H-chromene and 7H-furo-chromene derivatives, with diverse substitution patterns were designed and synthesised. The structure of the newly synthesised compounds was characterised and their inhibitory potency and selectivity towards human CA off target isoforms I, II and cancer-associated CA isoforms IX and XII were evaluated. Most of the compounds inhibit CA isoforms IX and XII with no activity against the I and II isozymes. Thus, while the potency was influenced by the substitution pattern along the chromene scaffold, the selectivity was conserved along the series, confirming the high potential of both 2H-chromene and 7H-furo-chromene scaffolds for the design of isozyme selective inhibitors.

Discovery of new 6-ureido/amidocoumarins as highly potent and selective inhibitors for the tumour-relevant carbonic anhydrases IX and XII.

A series of 6-ureido/amidocoumarins (5a-p and 7a-c) has been designed and synthesised to develop potent and isoform- selective carbonic anhydrase hCA XI and XII inhibitors. All coumarin derivatives were investigated for their CA inhibitory effect against hCA I, II, IX, and XII. Interestingly, target coumarins potently inhibited both tumour-related isoforms hCA IX (KIs: 14.7-82.4 nM) and hCA XII (KIs: 5.9-95.1 nM), whereas the cytosolic off-target hCA I and II isoforms have not inhibited by all tested coumarins up to 100 µM. These findings granted the target coumarins an excellent selectivity profile towards both hCA IX and hCA XII isoforms, supporting their development as promising anticancer candidates. Moreover, all target molecules were evaluated for their anticancer activities against HCT-116 and MCF-7 cancer cells. The 3,5-bis-trifluoromethylphenyl ureidocoumarin 5i, exerted the best anticancer activity. Overall, ureidocoumarins, particularly compound 5i, could serve as a promising prototype for the development of potent anticancer CAIs.

Selective carbonic anhydrase IX and XII inhibitors based around a functionalized coumarin scaffold.

Inhibition of specific carbonic anhydrase (CA) enzymes is a validated strategy for the development of agents to target cancer. The CA isoforms IX and XII are overexpressed in various human solid tumors wherein they play a critical role in regulating extracellular tumor acidification, proliferation, and progression. A series of novel sulfonamides based on the coumarin scaffold were designed, synthesized and characterized as potent and selective CA inhibitors. Selected compounds show significant activity and selectivity over CA I and CA II to target the tumor-associated CA IX and CA XII with high inhibition activity at the single digit nanomolar level. Twelve compounds were identified to be more potent compared with acetazolamide (AAZ) control to inhibit CA IX while one was also more potent than AAZ to inhibit CA XII. Compound 18f (Ki's = 955 nM, 515 nM, 21 nM and 5 nM for CA's I, II, IX, and XII, respectively) is highlighted as a novel CA IX and XII inhibitor for further development.

Propranolol, Promising Chemosensitizer and Candidate for the Combined Therapy through Disruption of Tumor Microenvironment Homeostasis by Decreasing the Level of Carbonic Anhydrase IX.

Resistance to chemotherapy represents a persisting medical problem, ranking among main causes of chemotherapy failure and cancer mortality. There is a possibility to utilize and repurpose already existing therapeutics which were not primarily intended for oncological treatment. Overactivation of adrenergic receptors and signaling dysregulation promotes tumor progression, metastatic potential, immune system evasion, tumor angiogenesis and drug resistance. The non-selective beta-blocker propranolol, approved in infantile haemangioma treatment, has a high potential for use in cancer therapy. We analyzed the effects of propranolol and 5-fluorouracil combination on sensitive and resistant cells derived from colorectal carcinoma in monolayers, single-component and co-culture spheroids and in vivo mouse models. Our results revealed that propranolol is able to exert its effect not only in chemosensitive colorectal cells, but also in 5-fluorouracil resistant cells. Propranolol disrupts the hypoxic adaptation machinery by inhibiting HIF1α, carbonic anhydrase IX, and activates apoptosis, which may be important in the management of chemo-resistant patients. We showed that propranolol slows down the growth of xenografts formed from colorectal cancer cells, even from cells already adapted to the ß-blocker. We provide clear evidence that blockade of ß-adrenergic receptors affects essential signaling pathways modulating tumor microenvironment and thus the response to anticancer therapy. Our findings indicate that propranolol could be repurposed to serve as chemosensitizer in combined therapy aimed at disrupting homeostasis of tumor microenvironment.

CA-IX-Expressing Small Extracellular Vesicles (sEVs) Are Released by Melanoma Cells under Hypoxia and in the Blood of Advanced Melanoma Patients.

Cutaneous melanoma is a highly aggressive skin cancer, with poor prognosis. The tumor microenvironment is characterized by areas of hypoxia. Carbonic anhydrase IX (CA-IX) is a marker of tumor hypoxia and its expression is regulated by hypoxia-inducible factor-1 (HIF-1). CA-IX has been found to be highly expressed in invasive melanomas. In this study, we investigated the effects of hypoxia on the release of small extracellular vesicles (sEVs) in two melanoma in vitro models. We demonstrated that melanoma cells release sEVs under both normoxic and hypoxic conditions, but only hypoxia-induced sEVs express CA-IX mRNA and protein. Moreover, we optimized an ELISA assay to provide evidence for CA-IX protein expression on the membranes of the sEVs. These CA-IX-positive sEVs may be exploited as potential biomarkers for liquid biopsy.

Expression Dynamics of CA IX Epitope in Cancer Cells under Intermittent Hypoxia Correlates with Extracellular pH Drop and Cell Killing by Ureido-Sulfonamide CA IX Inhibitors.

Carbonic anhydrase IX (CA IX) is a membrane-bound CA isozyme over-expressed in many hypoxic tumor cells, where it ensures pH homeostasis and has been implicated in tumor survival, metastasis and resistance to chemotherapy and radiotherapy. Given the functional importance of CA IX in tumor biochemistry, we investigated the expression dynamics of CA IX in normoxia, hypoxia and intermittent hypoxia, which are typical conditions experienced by tumor cells in aggressive carcinomas. We correlated the CA IX epitope expression dynamics with extracellular pH acidification and with viability of CA IX-expressing cancer cells upon treatment with CA IX inhibitors (CAIs) in colon HT-29, breast MDA-MB-231 and ovarian SKOV-3 tumor cell models. We observed that the CA IX epitope expressed under hypoxia by these cancer cells is retained in a significant amount upon reoxygenation, probably to preserve their proliferation ability. The extracellular pH drop correlated well with the level of CA IX expression, with the intermittent hypoxic cells showing a similar pH drop to fully hypoxic ones. All cancer cells showed higher sensitivity to CA IX inhibitors (CAIs) under hypoxia as compared to normoxia. The tumor cell sensitivity to CAIs under hypoxia and intermittent hypoxia were similar and higher than in normoxia and appeared to be correlated with the lipophilicity of the CAI.

Carbonic Anhydrase IX in Tumor Tissue and Plasma of Breast Cancer Patients: Reliable Biomarker of Hypoxia and Prognosis.

Carbonic anhydrase IX (CA IX) is recognized as an excellent marker of hypoxia and an adverse prognostic factor in solid tumors, including breast cancer (BC). Clinical studies confirm that soluble CA IX (sCA IX), shed into body fluids, predicts the response to some therapeutics. However, CA IX is not included in clinical practice guidelines, possibly due to a lack of validated diagnostic tools. Here, we present two novel diagnostic tools-a monoclonal antibody for CA IX detection by immunohistochemistry and an ELISA kit for the detection of sCA IX in the plasma-validated on a cohort of 100 patients with early BC. We confirm that tissue CA IX positivity (24%) correlates with tumor grading, necrosis, negative hormone receptor status, and the TNBC molecular subtype. We show that antibody IV/18 can specifically detect all subcellular forms of CA IX. Our ELISA test provides 70% sensitivity and 90% specificity. Although we showed that this test could detect exosomes in addition to shed CA IX ectodomain, we could not demonstrate a clear association of sCA IX with prognosis. Our results indicate that the amount of sCA IX depends on subcellular CA IX localization, but more strictly on the molecular composition of individual molecular subtypes of BC, particularly on metalloproteinases inhibitor expression.