Carbonic anhydrase IX (CA IX) is traditionally considered to be an immunomarker of clear cell renal cell carcinoma (RCC). However, CA IX expression has also been documented in other RCCs subtypes. Discrimination between clear cell RCC and non-clear cell RCC is crucial for further patient management. The aim of this study was to assess CA IX immunoreactivity across the spectrum of RCC with sarcomatoid differentiation. The expression of CA IX was evaluated in 32 cases of RCCs with sarcomatoid differentiation (12 clear cell RCC, 7 papillary RCC and 13 chromophobe RCC). Seven urothelial carcinomas (UC) with sarcomatoid differentiation (originally from renal pelvis) and 23 soft tissue tumors were also included as a control cohort. The sensitivity for CA IX in sarcomatoid component of clear cell RCC was 91.7 % (moderate/strong CA IX staining in >60 % of sarcomatoid component). However, the CA IX specificity was rather low (45 %), as a significant proportion of sarcomatoid components in different renal cell carcinoma subtypes also stained with CA IX (2/7 papillary RCC, 9/14 chromophobe RCC). When urothelial carcinoma and soft tissue tumors were included in the evaluation, the specificity of CA IX staining for sarcomatoid clear cell RCC reached 60 %. In conclusion, CA IX shows decent sensitivity for sarcomatoid clear cell RCC, but with low specificity, hence limiting its diagnostic utility as a reliable marker of in tumors with predominant sarcomatoid component.

The present study outlines the design and synthesis of dual-tail analogues of SLC-0111 as carbonic anhydrase inhibitors (CAIs) targeting tumor isoforms IX and XII 4a-h and 5a-h, along with pharmacokinetic studies. The synthesized compounds were evaluated for their inhibitory activity against four carbonic anhydrase isoforms (hCA I, II, IX, and XII), revealing potent activity, particularly against hCA IX and XII. Notably, compounds 4b, 5a, and 5b demonstrated strong inhibition of hCA IX with Ki values of 20.4, 12.9, and 18.2 nM, respectively, compared to acetazolamide (AAZ), which has a Ki of 25 nM. Additionally, compounds 5a, 5b, 5c, and 5d showed selective inhibition of hCA XII, with Ki values of 26.6, 8.7, 17.2, and 10.9 nM, respectively, relative to AAZ (Ki = 5.7 nM). Moreover, both series were tested for their anti-proliferative activity following the US-NCI protocol against a panel of more than fifty cancer cell lines. Compound 5h met the activity criteria and was automatically scheduled for further evaluation at five concentrations with 10-fold dilutions, revealing high toxicity toward leukemia and lower toxicity against melanoma. In addition, the MTT cytotoxicity assay was performed on 5f, 5d and acetazolamide using WI-38 cells. Furthermore, an in vivo pharmacokinetic study was conducted using UPLC-MS/MS on the most potent derivative, 5d, demonstrating a comparable pharmacokinetic profile compared to the reference drug acetazolamide. Furthermore, molecular docking prediction studies were conducted for the most active compounds, 5d and 5h, to elucidate their interactions with the active site hot spots of the CA isoform.

Numerous studies have highlighted the anti-cancer effects of nonsteroidal anti-inflammatory drugs (NSAIDs), although the underlying mechanisms remain unclear. This study focuses on elucidating the impact of the NSAID ibuprofen (IBU) on cancer cells exposed to hypoxia, as the hypoxic microenvironment significantly influences tumor progression, metastatic potential, and therapy resistance. Given that carbonic anhydrase IX (CA IX) is a key hypoxia-associated protein and a promising therapeutic target due to its tumor-specific expression, we primarily examined the impact of IBU on CA IX and the transcription factors regulating CA IX expression. We found that IBU downregulates expression and protein level of CA IX in hypoxic colon carcinoma and head and neck cancer cells, resulting in a reduction of membranous CA IX. To elucidate the mechanism of this phenomenon, we analyzed the key CA IX-regulating transcription factor HIF-1 and found decreased levels of the HIF-1α subunit in IBU-treated cells, leading to its impaired binding to the CA9 promotor. Analysis of another transcription factor involved in CA IX expression, NFκB, showed suppressed NFκB pathway under IBU treatment. Moreover, we demonstrated IBU-mediated induction in apoptosis in cancer cells, as well as a decrease in their ability to migrate. Our study is the first to demonstrate that ibuprofen downregulates carbonic anhydrase IX expression in hypoxic colon and head and neck tumor cells by decreasing HIF-1α levels. Additionally, ibuprofen impairs key transcription factors NFκB and STAT3, leading to reduced adaptation to hypoxic stress, decreased tumor cell viability, and migration. This indicates its potential as a therapeutic agent in combination therapy for colon carcinoma or head and neck cancer.

Lung cancer remains the leading cause of cancer-related mortality globally, with lung adenocarcinoma (LUAD) being the most prevalent subtype. Despite extensive research efforts, the role of transcription factors in LUAD progression remains largely uncharacterized. In this study, we focused on ZNF266, a transcription factor whose impacts on LUAD have not been investigated.

Using high-throughput sequencing data, we observed a significant downregulation of ZNF266 expression in LUAD tissues. To validate this finding, we conducted a retrospective analysis of nearly three thousand LUAD patients' data from public databases and our institution. Functional studies were performed using cell lines, organoids, and xenograft models to assess the role of ZNF266 in LUAD progression. RNA sequencing, chromatin immunoprecipitation, DNA pull-down assays, and dual-luciferase reporter assays were employed to elucidate the underlying mechanism. Additionally, adeno-associated virus (AAV)-mediated overexpression of ZNF266 was used to evaluate its therapeutic potential.

Patients with low ZNF266 expression had poorer prognosis compared to those with high expression. ZNF266 inhibits the malignant phenotypes of LUAD, including proliferation, migration, and invasion. Mechanistically, ZNF266 binds to the promoter region of CA9, suppressing its transcription. This leads to a reduction in intracellular pH and subsequent inhibition of the mTOR signaling pathway, which is crucial for cancer cell growth and survival. Furthermore, AAV-mediated overexpression of ZNF266 significantly inhibited tumor growth in patient-derived xenograft models.

Our study demonstrated that ZNF266 inhibits LUAD progression in a pH-dependent manner via modulating CA9 expression, uncovering its therapeutic significance for LUAD treatment.

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, for which targeted treatment is currently lacking. Carbonic anhydrase IX (CAIX) is a known cancer target due to its selective overexpression in hypoxia, a hallmark of many solid cancers including TNBC. This study aimed to develop a robust murine TNBC cell line 4T1-based model system that could be used in the comprehensive preclinical evaluation of targeting CAIX. The model is based on the original 4T1 breast cancer cell line and two genetically edited versions of it-one with biallelic CRISPR/Cas9-mediated Car9 inactivation and another with constitutively expressed Car9, thus ensuring negative and positive controls for CAIX production in the model system, respectively. The generated cell lines were validated for CAIX production and characterised functionally in vitro and in vivo after orthotopic implantation in syngeneic BALB/c mice. Results demonstrated significantly reduced primary tumour growth and metastatic progression rates in animals with CAIX-deficient tumours, while the CAIX-expressing tumours had vascularised phenotypes with prominent central areas of coagulative necrosis. The differential CAIX expression levels in the model were preserved during tumour growth in syngeneic mice, as verified by in vivo imaging using a novel high-affinity CAIX-specific near-infrared (NIR) fluorescent imaging probe, GZ22-4. Constitutive overexpression of autologous CAIX did not elicit specific autoantibody responses in vivo, demonstrating the suitability of this model for evaluating the efficacy of anti-CAIX vaccination as a therapeutic strategy. The in vivo study was repeated as an independent experiment and demonstrated good robustness of the developed model.

Fluorescence tumor-targeted imaging is a sensitive technique that may assist surgeons to remove residual cancer tissue during resection. Carbonic anhydrase IX (CAIX) is a tumor-associated cell-surface glycoprotein, upregulated in hypoxic environments, and a suitable biomarker to develop targeted dyes for fluorescence-guided surgery. This study describes design, synthesis, in vitro and in vivo assessment of novel CAIX-targeting fluorescent probes based on the well-known drug acetazolamide, addressing the contribution of both targeting moiety and fluorophore structure on imaging efficacy.

All the CAIX-targeting heptamethine cyanines synthesized and described in the present work were characterized in terms of their optical properties in different media. The affinity to human serum albumin was evaluated by UV-VIS spectrophotometry. The affinity to the CA catalytic site was determined on a recombinant bovine CAII enzyme (bCAII), with a fluorescent-based assay. Human colon adenocarcinoma HT-29 cells, highly expressing CAIX, were used for the in vitro characterization, including cell binding, uptake and competition assays, by flow cytometry. Finally, the in vivo tumor targeting efficacy of a selected group of probes was assessed by Optical Imaging in a mouse subcutaneous tumor from HT-29 cells, characterized by both expression of CAIX and a hypoxic tumor microenvironment.

First, a family of CAIX-targeting probes was prepared by functionalizing a novel glucamine-bearing heptamethine cyanine (Dye1) with a modified acetazolamide moiety, whose acetyl group was replaced with i) aminooctanoic acid C8, ii) phenylalanine, iii) amino-PEG2-acid and iv) the longer linker 4a, or 2 commercially available benzenesulfonamides. From the in vitro screening of this first group of compounds, the C8-AZA targeting moiety was selected due to its highest affinity. Indeed, Dye1-C8-AZA exhibited the lowest KD values for both bCAII (6.1 ± 1.6 nM) and CAIX-expressing HT-29 cells (58 ± 9 nM), even lower than HypoxyFluor-1 (HF-1), a CAIX-targeted dye already reported in the literature. Then, other heptamethine cyanines (Dye2-Dye5, linear or cyclic, with different substituents on the indolenines and different conjugation position of the targeting vector) were functionalized with C8-AZA and fully characterized both in vitro and in vivo, to evaluate the combinatory effect of vector and fluorophore on the performance of the resulting probes. The different chemical features of the cyanines influenced the optical properties, solubility, binding with albumin, biodistribution, and imaging efficacy of the probes, while leaving unaffected the high affinity to the target. When tested in vivo for the visualization of CAIX-expressing HT-29 tumors, all C8-AZA probes showed high and specific tumor accumulation, often superior to HF-1.

Several CAIX-targeting probes were synthesized to test the combinatory effect of different molecular vectors and dyes on the biological properties. All probes containing the C8-AZA targeting moiety displayed higher affinity and specificity to the target, while imaging efficacy in vivo was strongly influenced also by the structure of the labelling dye. All probes, and among them especially Dye1-C8-AZA, displayed efficient in vivo tumor accumulation. These results support further studies toward clinical testing of CAIX as suitable target for tumor fluorescence imaging and pave the way for future clinical applications.

DDX11 antisense RNA 1 (DDX11-AS1) has been recognized for its strong correlation with hepatocellular carcinoma (HCC). Nevertheless, the exact biological functions and fundamental molecular processes of DDX11-AS1 in HCC require further in-depth investigation.

A comprehensive bioinformatics analysis was carried out to explore the expression of DDX11-AS1 and its clinical implication in HCC utilizing the TCGA data. qRT-PCR was employed to validate the expression of DDX11-AS1 in HCC tissues/cell lines. RNA fluorescence in situ hybridization (RNA-FISH) was used to observe the subcellular localization of DDX11-AS1 in HCC cells. Loss-of-function experiments, both in vitro and in vivo, were executed to elucidate the biological functions of DDX11-AS1 in HCC. RNA sequencing (RNA-seq) was employed to identify genes and signaling pathways potentially regulated by DDX11-AS1. Rescue experiments were conducted to validate that carbonic anhydrase IX (CA9) mediates DDX11-AS1 promoting HCC progression. The influence of nuclear respiratory factor 1 (NRF1) on the transcription of DDX11-AS1 was investigated through dual-luciferase reporter assays and ChIP-qPCR.

The increased expression of DDX11-AS1 is positively associated with several aggressive clinical characteristics (pathologic T stage, histologic grade, AFP level, and vascular invasion), and is closely linked to unfavorable outcomes in HCC patients, acting as a separate hazardous factor for overall survival. DDX11-AS1 is predominantly situated in the nucleus of HCC cells. DDX11-AS1 knockdown impeded the growth, migration, and invasion capabilities of HCC cells in vitro, and reduced the tumor enlargement in a subcutaneous mouse model. RNA-Seq unveiled that silencing DDX11-AS1 lessened the expression of CA9 and suppressed the activity of the MEK/ERK signaling cascade in HCC cells. Rescue experiments uncovered that CA9 acts as a downstream target facilitating the cancer-causing roles of DDX11-AS1 in HCC. Furthermore, DDX11-AS1 was revealed to be transcriptionally regulated by NRF1.

DDX11-AS1, a NRF1-induced lncRNA, facilitates HCC development by upregulating CA9 expression and activating the MEK/ERK signaling cascade.

Tumor microenvironment governs various therapeutic tolerability of cancer such as ferroptosis and immunotherapy through rewiring tumor metabolic reprogramming like Warburg metabolism. Highly expressed carbonic anhydrases (CA) in tumor that maintaining the delicate metabolic homeostasis is thus the most potential target to be modulated to resolve the therapeutic tolerability. Hence, in this article, a self-healable and pH-responsive spermidine/ferrous ion hydrogel loaded with CA inhibitor (acetazolamide, ACZ) and glucose oxidase (ACZ/GOx@SPM-HA Gel) was fabricated through the Schiff-base reaction between spermidine-dextran and oxidized hyaluronic acid, along with ferrous coordination. Investigation on cancer cell lines (MOC-1) demonstrated ACZ/GOx@SPM-HA Gel may induce cellular oxidative stress and mitochondrial dysfunction through disrupting the cellular homeostasis. Moreover, with the facilitation of autophagy induced by spermidine, ACZ/GOx@SPM-HA Gel may trigger a positive feedback loop to maximally amplify cellular ferroptosis and promote DAMPs release. The anti-tumor evaluation on xenograft mice models furtherly proved the local injection of such hydrogel formulation could efficiently inhibit the tumor growth and distinctively promote the immunogenicity of tumor bed to provide a more favorable environment for immunotherapy. Overall, ACZ/GOx@SPM-HA Gel, with such feasible physiochemical properties and great biocompatibility, holds great potential in treating solid tumors with acidosis-mediated immunotherapy tolerance.

Tumor acidosis causes resistance to immune checkpoint blockade (ICB). We hypothesized that a "pH-sensitizer" can increase tumor extracellular pH (pHe) and improve tumor control following ICB. We also hypothesized that pHe measured with acidoCEST MRI can predict improved tumor control with ICB.

We tested the effects of pH-sensitizers on proton efflux rate (PER), cytotoxicity, T cell activation, tumor immunogenicity, tumor growth and survival using 4T1 and B16-F10 tumor cells. We measured in vivo tumor pHe of 4T1 and B16-F10 models with acidoCEST MRI.

Among the pH-sensitizers tested, someprazole caused the greatest reduction in PER without exhibiting cytotoxicity or reducing T cell activation. Esomeprazole improved 4T1 tumor control with ICB administered one day after the pH-sensitizer. Tumor pHe positively correlated with TCF-1 + CD4 effector and CD8 T cell intratumoral frequencies and predicted improved 4T1 tumor control with ICB. For comparison, esomeprazole had a mild effect on B16-F10 tumor pHe, and worsened tumor control with ICB and increased intratumoral myeloid and dendritic cell (DC) frequencies.

A pH-sensitizer can improve tumor control with ICB, and acidoCEST MRI can be used to measure pHe and predict tumor control, but only in the 4T1 model and not the B16-F10 model.

Tumor hypoxia indicates a worse prognosis in brain malignancies; however, current gold-standard methods for assessing tumor hypoxia are invasive and often inaccessible. Magnetic Resonance Imaging (MRI) is widely available, but its validity for identifying tumor hypoxia or hypoxia-related neoangiogenesis is not well characterized. A systematic literature search was performed across PubMed and Embase Databases. The search query identified MRI studies that validated hypoxia-surrogate imaging sequences against gold-standard hypoxia or neoangiogenesis detection methods in patients with brain malignancies. Literature screen identified 23 manuscripts published between 2007 and 2022. Among conventional MRI sequences, peritumoral edema and signal change after contrast administration were associated with gold-standard oxygen-assessment methods. T2*- and T2'-derived measures were associated with gold-standard methods, while reports on quantitative measures of oxygen extraction fraction were conflicting. Fiber density, tissue cellularity, blood volume, vascular transit time, and permeability measurements were associated with gold-standard methods, whereas blood flow measurements yielded conflicting results. MRI measures are promising surrogates for tumor hypoxia or hypoxia-related neoangiogenesis. Additional studies are needed to reconcile disparate findings. Future sensitivity analyses are needed to establish the MRI methods most accurate at identifying tumor hypoxia.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer that often displays resistance to conventional cancer therapies, including chemotherapy and radiation therapy. Targeted treatments, including immunotherapies and small molecular inhibitors, have been associated with improved outcomes. However, variations in the patient response and the development of resistance suggest that more models that better recapitulate the pathogenesis and metastatic mechanisms of ccRCC are required to improve our understanding and disease management. Here, we examined the transcriptional landscapes of in vitro cell culture as well as in vivo orthotopic and metastatic NOD/SCID-γ mouse models of ccRCC using a single patient-derived RCC243 cell line to allow unambiguous comparison between models. In our mouse model assays, RCC243 cells formed metastatic tumors, and all tumors retained clear cell morphology irrespective of model type. Notably, gene expression profiles differed markedly between the RCC243 tumor models-cell culture, orthotopic tumors, and metastatic tumors-suggesting an impact of the experimental model system and whether the tumor was orthotopic or metastatic. Furthermore, we found conserved prognostic markers between RCC243 tumor models and human ccRCC patient datasets, and genes upregulated in metastatic RCC243 were associated with worse patient outcomes.

Epilepsy is a neurological disorder characterized by repeated convulsions. Antiepileptic drugs (AEDs) are the main course of therapy for epilepsy. These medications are given according to each patient's personal medical history and the types of seizures they suffer. They have been employed for decades to manage epilepsy, thus delivering relief from seizures through numerous mechanisms of action. Aside from their anticonvulsant attributes, current evidence suggests that certain AEDs may display potential inhibitory effects against cancer invasion and metastasis. This review explored the complicated interactions between the modes of action of AEDs and the pathways causing cancer, and the potential impact of AEDs on the invasion and metastasis of various forms of cancer, while addressing their associated side effects. For example, valproic acid inhibits histone deacetylase, causing hyperacetylation of genes, especially those regulating cell cycle, culminating in cell cycle arrest. Topiramate inhibits carbonic anhydrase, thus disrupting the acidic microenvironment needed for cancer cells to thrive. Lacosamide increases the slow inactivation of the voltage gated Na+ channel, thus inhibiting the growth, proliferation, and metastasis of many cancers. Although drug development is a complex task due to regulatory, intellectual property, and economic challenges, researchers are exploring drug repurposing tactics to overcome these challenges and to find new therapeutic alternatives for diseases like cancer. Thus, drug repurposing is considered among the most effective ways to develop drug candidates using novel properties and therapeutic characteristics, and this review also discusses these issues.

A small library of novel thiazolidinone-based sulfonamide derivatives was designed, synthesized and evaluated for their ability to target human carbonic anhydrase (hCA) isoforms IX and XII, which are overexpressed in malignant cells and play a key role in metastasis and therapeutic response of cancer cells. A molecular hybridization approach was employed to design the molecules by combining different moieties identified as having antitumor activity. The thiazolidinone core was functionalized with benzenesulfonamide as a zinc-binding group and different isatin derivatives to enhance the chemical profile and optimize the hydrophilic/lipophilic balance. Biological evaluation against hCA I, II, IX and XII isoforms showed promising inhibitory activities, and some compounds exhibited selectivity and high inhibitory activity against hCA IX and hCA XII while not affecting off-target hCA I and hCA II. In particular, compound 3h demonstrated high selectivity with Ki values of 57.8 nM for hCA IX and 44.3 nM for hCA XII.

Introduction: Carbonic anhydrase IX (CA IX) is a tumor-associated enzyme involved in cancer progression and survival. Targeting CA IX with selective inhibitors like SLC-0111 has shown therapeutic potential. This study aimed to develop a novel 4-pyridyl analog (Pyr) of SLC-0111 with enhanced anticancer activity. Methods: Pyr was synthesized using a tail-based design and characterized by NMR. Its cytotoxicity was tested against cancer and normal cell lines. CA inhibition, cell cycle effects, apoptosis induction, and protein expression changes were evaluated. Molecular docking and ADMET predictions assessed binding and drug-like properties. Results and Discussion: Pyr showed selective cytotoxicity toward cancer cells and potent CA IX inhibition. It induced G0/G1 arrest, apoptosis, and modulated p53, Bax, and Bcl-2 levels. Docking confirmed strong CA IX binding, and ADMET analysis indicated good oral bioavailability. These results support Pyr as a promising anticancer candidate.

Advances in molecular diagnostics have ushered in a new era for patients with prostate, renal, and urothelial cancers, with novel radiographic and molecular modalities for the assessment of disease burden and minimal residual disease (MRD). Conventional imaging has a limited threshold for disease detection and is often unable to discern clinically occult disease with varying risks of false-negative or false-positive findings depending on the disease state and type of imaging.

We provide an overview of emerging radiographic and molecular tools in development within the genitourinary (GU) disease space. A literature review of contemporary basic, translational, and clinical research studies was performed, covering the timeframe of 1980-2024 through the MEDLINE (via PubMed) and Scopus databases. We highlight select examples of emerging technologies and biomarker-informed clinical trials, which aim to quantify disease at lower thresholds and have the potential for integrating MRD in clinical practice for GU patients.

The development of novel radiotracers, such as prostate-specific membrane antigen or carbonic anhydrase IX, is being evaluated in both clinical practice and trial setting, aiming to change the management of these tumors. Molecular tools including circulating tumor cells and byproducts such as plasma and urine cell-free circulating tumor DNA provide the opportunity for MRD detection. MRD capture on single-cell or cellular byproducts can serve as a conduit for genomic and transcriptomic analyses, providing insight into the molecular underpinnings and clonal evolution of disease.

While the full potential for MRD applications has yet to be realized, we are witnessing the emergence of novel techniques aimed at MRD detection and the rapid development of elegantly designed studies implementing iterative detection of MRD as means to provide biological rationale and tailor therapeutic options in GU tumors.

Tumour hypoxia frequently presents a major challenge in the treatment of neuroblastoma (NBL). The neuroblastoma cells produce carbonic anhydrase IX (CA IX), an enzyme crucial for the survival of cancer cells in low-oxygen environments.

We designed and synthesised a novel high-affinity inhibitor of CA IX. The highest to-date. The affinities were determined for all human catalytically active CA isozymes showing significant selectivity for CA IX over other isozymes. The inhibitor effect on neuroblastoma cancer cell growth was determined in vitro and in vivo via a mice xenograft model.

The novel designed inhibitor effectively mitigated the acidification induced by CA IX and reduced spheroid growth under hypoxic conditions in the SK-N-AS cell line. It also diminished the secretion of pro-tumour chemokines IL-8 (CXCL2) and CCL2. When we combined this novel CA IX inhibitor with a compound that inhibits the chemokine receptor CCR2 protein activity, we observed a reduction in mouse tumour growth. The combined treatment also prompted tumours to exhibit adaptive resistance by producing higher levels of vascular endothelial growth factor receptors (VEGFR) and other compensatory signals.

This research underscores the pivotal role of CA IX in cancer and the potential of a novel CA IX inhibitor-based combination intervention therapy for neuroblastoma treatment.

The role of human Papillomavirus (HPV) in metabolic reprogramming is implicated in the development and progression of cervical cancer. During carcinogenesis, cancer cells modify various metabolic pathways to generate energy and sustain their growth and development. Cervical cancer, one of the most prevalent malignancies affecting women globally, involves metabolic alterations such as increased glycolysis, elevated lactate production, and lipid accumulation. The oncoproteins, primarily E6 and E7, which are encoded by high-risk HPVs, facilitate the accumulation of several cancer markers, promoting not only the growth and development of cancer but also metastasis, immune evasion, and therapy resistance. HPV oncoproteins interact with cellular MYC (c-MYC), retinoblastoma protein (pRB), p53, and hypoxia-inducible factor 1α (HIF-1α), leading to the induction of metabolic reprogramming and favour the Warburg effect. Metabolic reprogramming enables HPV to persist for an extended period and accelerates the progression of cervical cancer. This review summarizes the role of HPV oncoproteins in metabolic reprogramming and their contributions to the development and progression of cervical cancer. Additionally, this review provides insights into how metabolic reprogramming opens avenues for novel therapeutic strategies, including the discovery of new and repurposed drugs that could be applied to treat cervical cancer.

The malignant tumor is a serious disease threatening human life. Increasing studies have confirmed that the tumor microenvironment (TME) is composed of a variety of complex components that precisely regulate the interaction of tumor cells with other components, allowing tumor cells to continue to proliferate, resist apoptosis, evade immune surveillance and clearance, and metastasis. However, the characteristics of each component and their interrelationships remain to be deeply understood. To target TME, it is necessary to deeply understand the role of various components of TME in tumor growth and search for potential therapeutic targets. Herein, we innovatively classify the TME into physical microenvironment (such as oxygen, pH, etc.), mechanical microenvironment (such as extracellular matrix, blood vessels, etc.), metabolic microenvironment (such as glucose, lipids, etc.), inflammatory microenvironment and immune microenvironment. We introduce a concise but comprehensive classification of the TME; depict the characteristics of each component in TME; summarize the existing methods for detecting each component in TME; highlight the current strategies and potential therapeutic targets for TME; discuss current challenges in presenting TME and its clinical applications; and provide our prospect on the future research direction and clinical benefits of TME.

The tumor microenvironment (TME) of triple-negative breast cancer (TNBC) is a highly heterogeneous and very aggressive form of the disease that has few suitable treatment options; however, spatial transcriptomics (ST) is a powerful tool for elucidation of the TME in TNBC. Because of its spatial context preservation, ST has a unique capability to map tumor-stroma interactions, immune infiltration, and therapy resistance mechanisms (which are key to understanding TNBC progression), compared with conventional transcriptomics. This review shows the use of ST in TNBC, its utilization in spatial biomarker identification, intratumoral heterogeneity definition, molecular subtyping refinement, and prediction of immunotherapy responses. Recent insight from ST-driven insights has explained the key spatial patterns on immune evasion, chemotherapy resistance, racial disparities of TNBC, and aspects for patient stratification and therapeutic decision. With the integration of ST with the subjects of proteomics and imaging mass cytometry, this approach has been enlarged and is now applied in precision medicine and biomarker discovery. Recently, advancements in AI-based spatial analysis for tumor classification, identification of biomarkers, and creation of therapy prediction models have occurred. However, continued developments in ST technologies, computational tools, and partnerships amongst multiple centers to facilitate the integration of ST into clinical routine practice are needed to unlock novel therapeutic targets.

Multiple studies have demonstrated the benefit of engineering hybrid ligands that combine the unique benefits of small molecules and proteins or peptides. However, the molecular complexity of hybrid ligands generates a parameter space so large it cannot be exhaustively explored. We systematically evaluated the impact of one molecular design element, conjugation site, on the discovery of functional protein-small molecule hybrids (PriSMs). We utilized a library of yeast-displayed fibronectin domain variants with amino acid and loop length diversity in the paratope and a single cysteine at one of 18 possible conjugation sites. The protein variants were coupled with maleimide-functionalized acetazolamide and sorted via competitive flow cytometry to discover potent and selective inhibitors of three isoforms of carbonic anhydrase. Deep sequencing of the resultant populations of functional PriSMs revealed an isoform-dependent distribution of conjugation site preferences. The top PriSMs showed potency and selectivity gains up to 23- and 100-fold (in this case, for CA-II vs CA-XII, with a 43-fold selectivity gain for CA-II vs CA-IX) relative to PEG2-acetazolamide alone. The presented study expands our fundamental understanding of the role of conjugation site in PriSM function and informs future PriSM engineering efforts by highlighting the benefit of conjugation site diversity in PriSM libraries.

Human carbonic anhydrase (CA) isoenzymes IX and XII are overexpressed in cancer cells, contributing to tumor microenvironment acidification and representing important targets for cancer therapy. In this study, we identified compound V35 (ZINC09419065) as a selective inhibitor of CA IX and CA XII with enhanced binding stability and selectivity compared to standard inhibitors. We analyzed conserved regions in CA I, CA II, CA IX, and CA XII to investigate their isozyme selectivity, revealing critical selectivity determinants at positions 95, 141, and 203. Molecular docking results indicated that V35 interacts robustly with CA XII, forming a metal ion coordination complex with Zn via HIS94, HIS96, HIS119, and THR199, similar to the interaction pattern of standard inhibitor SLC-0111. Molecular dynamics (MD) simulations conducted over 500 ns under hypoxic conditions showed that V35 has high binding stability, with root mean square deviation (RMSD) and fluctuation (RMSF) values comparable to SLC-0111, demonstrating its conformational stability in CA XII. Binding free energy calculations using the MMGBSA method showed that V35 achieves binding free energy of -44.17 kcal/mol with CA XII, closely matching SLC-0111 (-49.41 kcal/mol). Density functional theory (DFT) calculations further highlighted V35's electrostatic potential distribution, supporting its isozyme selectivity. Post-dynamics analysis indicated that the ester functional groups and the inward movement of HIS64 stabilize V35's interactions in CA XII, a feature absent in CA I.

Dramatic advances in biological discoveries, since the 1990s, have continued to reshape the treatment paradigm of metastatic renal cell carcinoma (RCC). Von Hippel Lindau (VHL) gene alterations are associated with pro-angiogenic activity and are central to the pathogenesis of clear cell RCC (ccRCC), the most predominant histologic subtype of RCC. Antiangiogenic strategies revolving around this VHL/HIF/VEGF axis have been shown to improve survival in metastatic ccRCC. The discovery of immune checkpoints and agents that target their inhibition introduced a new treatment paradigm for patients with RCC. While initially approved as monotherapy, studies investigating immune checkpoint inhibitor combinations have led to their approval as the new standard of care, providing durable responses and unprecedented improvements in clinical outcome. Despite these advances, the projected 14 390 deaths in 2024 from RCC underscore the need to continue efforts in expanding and optimizing treatment options for patients with metastatic RCC. This article reviews key findings that have transformed the way we understand and treat metastatic RCC, in addition to highlighting novel treatment strategies that are currently under development.

Background-Aggressive solid tumors are commonly characterized by both basic intracellular pH and acidic extracellular pH, which increase cell survival and proliferation. As carbonic anhydrases IX/XII are involved in this pH regulation, their inhibition is an appealing approach in cancer therapy, avoiding cancer cell survival and proliferation. Substituted coumarins are selective non-classical CA IX and CA XII inhibitors. Methods-In this study, new 7-hydroxycoumarinamides were synthesized and assayed for CA inhibition and antiproliferative activity. Results-All of the coumarinamides showed human CA IX and CA XII selective inhibition over the off-target CA I and CA II isoforms. Coumarin acts as a suicide inhibitor because its heterocyclic ring can be hydrolyzed by CA esterase activity to give the corresponding 2-hydroxycinnamic acid derivative which blocks the entrance of the active site. The 2-hydroxycinnamic acid derivatives deriving from the most potent and selective coumarinamides were docked into CA IX and XII to better understand the activity and selectivity against the two CA isoforms. The most active coumarinamides also produced a decrease of A549 cell proliferation and were able to arrest cells at the G1/S checkpoint. Conclusions-These results may open new perspectives for developing coumarin-based CA IX/XII inhibitors.

This first-in-human study aimed to evaluate the radiation dosimetry and whole-body biodistribution of [18F]AlF-NYM005, a novel small-molecule carbonic anhydrase IX (CAIX) targeting agent, and to investigate its ability to detect CAIX-positive tumors using PET scans in a cohort of clear cell renal cell carcinoma (ccRCC) patients.

[18F]AlF-NYM005 was synthesized using a fully automatic cassette module Mortenon M1 (Nuoyu, China). Thirty-five patients with a suspicious lesion considered primary renal malignancy or a history of ccRCC were prospectively recruited and studied. All patients underwent [18F]AlF-NYM005 PET/CT examinations and the maximum standardized uptake value (SUVmax) was measured on conventional [18F]AlF-NYM005 PET/CT images. Among these patients, five patients underwent dynamic [18F]AlF-NYM005 PET/CT scanning (120 min) of the lower abdomen. Another subset of five ccRCC patients underwent sequential whole-body PET scans at 30, 60, 90, and 120 min (one of the five patients underwent additional 150 min and 180 min scans) after [18F]AlF-NYM005 injection to assess biodistribution and dosimetry. The influx constant (Ki) was calculated from the dynamic [18F]AlF-NYM005 PET/CT data using the Patlak model. Whole-body biodistribution was calculated as time-activity curves (TACs) describing dynamic uptake patterns in the patients' major organs, followed by calculation of tracer kinetics and cumulative organ activity. Effective doses of [18F]AlF-NYM005 and individual organ doses were also calculated.

[18F]AlF-NYM005 was successfully synthesized with a radiochemical purity of > 95% and an average labeling yield of 36.5 ± 8.3%. All patients tolerated the PET examinations well, and no adverse side effects were observed. The total body effective dose was 7.6E-03 mSv/MBq. The highest agent uptake was observed in the kidneys, stomach, and liver, contributing to an effective dose of 0.0126 ± 0.0029 mSv/MBq. The TACs showed optimal normal organ uptake with high tumor uptake and long retention of up to 2 h post-injection. Notably, a rapid increase of the tracer followed by a rapid decrease in the blood pool, kidney, liver, and tumor lesions was observed, indicating that [18F]AlF-NYM005 was rapidly eliminated from blood and urine. For the kinetic data analysis, the Ki for the primary kidney lesions had a mean of 0.082 ± 0.057 ml/g/min. The CAIX-positive tumors displayed rapid uptake, and all lesions were detectable within 30 min, with no additional lesions observed in the subsequent multi-time point scans. The patient-level sensitivity, specificity, and accuracy of [18F]AlF-NYM005 PET/CT were 93.8%, 75.0%, and 90% for group 1 and 92.3%, 100%, and 93.3% for group 2, respectively. For per-lymph node analysis, [18F]AlF-NYM005 PET/CT demonstrated 92.9% sensitivity, 90.5% specificity, and 91.8% accuracy in diagnosing metastatic lymph nodes. For per-distant metastasis analysis, it showed 90.5% sensitivity, 91.3% specificity, and 90.6% accuracy. The SUVmax of [18F]AlF-NYM005 PET/CT for primary ccRCC lesions was 15.5 ± 7.35. Tumor uptake was positive correlated with immunohistochemical staining findings.

This pilot study in ccRCC patients has demonstrated the safety, acceptable radiation dosimetry, favorable biodistribution, and exceptional tumor uptake of [18F]AlF-NYM005. The preliminary diagnostic study indicated the potential utility of [18F]AlF-NYM005 PET/CT, showing promising results in the diagnosis of primary or metastatic ccRCC.

This study was registered at ClinicalTrial.gov (ChiCTR2200058108) as NYPILOT on 29 March, 2022.

Carbonic anhydrases constitute a family of metalloenzymes vital for maintaining acid-base balance and regulating pH in physio-pathological processes. These findings suggest carbonic anhydrases as potential therapeutic targets for treating pH-associated disorders, including cerebral ischemia, to mitigate hypoxia- and reoxygenation-induced neuronal damage. A focus on carbonic anhydrase IX showed that ischemic stress altered subcellular distributions of this enzyme in rodent neuronal populations. Given the enzyme's canonical membrane localization, we implemented pharmacological inhibition using a membrane-impermeant sulfonamide inhibitor in neuronal models of brain ischemia. The treatments exerted neuroprotective effects on neurons from Car9 knockout mice. Moreover, administration of the sulfonamide inhibitor to rats subjected to transient middle cerebral artery occlusion decreased infarct volumes and improved neurological deficits. Our results support the involvement of carbonic anhydrase IX in postischemic damage and pave the way for possible pharmacological interventions with selective inhibitors in the management of brain ischemia.

Human carbonic anhydrases (hCAs) are involved in many physiological processes including respiration, pH control, ion transport, bone resorption, and gastric fluid secretion. Recently, CA IX and CA XII have been studied for their role in cancer diseases, motivating the design of inhibitors of these isoforms.

Here, we used the tail approach to design a new series of monoaryl (1a-i) and bicyclic (1j-n) benzensulfonamide derivatives CA IX and CA XII inhibitors. All synthesized compounds were investigated toward a panel of hCAs, and most of them exhibited potent CA inhibitory activity for CA II, CA IX and CA XII with Ki values. In silico studies were performed to investigate the binding mode between inhibitors and CA.

The best compound was 1i that showed a low nanomolar range of Ki value as CA inhibitor (Ki = 9.4, 5.6 and 6.3 nM hCA II, IX and XII, respectively).

Autistic individuals with disproportionate megalencephaly (ASD-DM), characterized by enlarged brains relative to body height, have higher rates of intellectual disability and face more severe cognitive challenges than autistic children with average brain sizes. The cellular and molecular mechanisms underlying this neurophenotype remain poorly understood. To investigate these mechanisms, we generated human induced pluripotent stem cells from non-autistic typically developing children and autistic children with and without disproportionate megalencephaly. We assessed these children longitudinally from ages two to twelve years using magnetic resonance imaging and comprehensive cognitive and medical evaluations. We show that neural progenitor cells (NPCs) derived from ASD-DM children exhibit increased rates of cell survival and suppressed cell death, accompanied by heightened oxidative stress and ferrous iron accumulation. Despite these stressors, ASD-DM NPCs actively suppress apoptosis and ferroptosis by regulating proteins such as caspase-3 (CASP3), poly(ADP-ribose) polymerase 1 (PARP1), and glutathione peroxidase 4 (GPX4). Cellular ferroptotic signatures are further supported by elevated expression of selenocysteine genes, including GPX4 , in the blood of ASD-DM children and their mothers, suggesting potential hereditary or environmental influences. Furthermore, we show that peripheral expression of GPX4 and other selenocysteine genes correlate with cognitive outcomes (IQ). These findings underscore the role of ferroptosis in autism, pointing to potential diagnostic biomarkers and targets for intervention.

The field of cancer therapeutics has witnessed significant advancements over the past decades, particularly with the emergence of immunotherapy. This chapter traces the transformative journey from traditional antibody-based therapies to the innovative use of nanobodies in the treatment and diagnosis of solid tumors. Nanobodies are the smallest fragments of antibodies derived from camelid immunoglobulins and have redefined the possibilities in cancer theranostics due to their unique structural and functional properties. We provide an overview of the biochemical characteristics of nanobodies that make them particularly suitable for theranostic applications, such as their small size, high stability, enhanced infiltration into the complex tumor microenvironment (TME) and ability to bind with high affinity to epitopes that are inaccessible to conventional antibodies. Further, their ease of modification and functionalization has enabled the development of nanobody-based drug conjugates/toxins and radiolabeled compounds for precise imaging and targeted radiotherapy. We elucidate how nanobodies are being served as valuable tools for prognostic assessment, enabling clinicians to predict disease aggressiveness, monitor treatment response, and stratify patients for personalized therapeutic interventions.

The acidic tumor microenvironment, a hallmark of many solid tumors, is primarily induced by the high glycolytic rate of tumor cells. To avoid acidosis, tumor cells ingeniously maintain an acidic extracellular pH while keeping a relatively alkaline intracellular pH. Overturning the unique pH gradient of tumor cells has exhibited to be a viable approach for cancer therapy. In this review, the formation and regulatory mechanisms of the acidic microenvironment in solid tumors will be firstly outlined. Subsequently, we will comprehensively summarize the latest advancements in anti-tumor therapy via using nanomedicines to manipulate the tumor pH gradient, including acidifying intracellular environment and alkalizing extracellular environment. Following this, we will discuss the future potential of strategies employing nanomedicines to reverse tumor pH gradient. This review aims to foster research on therapeutic approaches targeting the pH regulation of solid tumors and holds an optimistic outlook for the future development of this field.

Background: The aim of this study was to evaluate biomarkers and biological characteristics of tumor biopsies from patients with head and neck cancer (HNC) to assess the risk of early death. Furthermore, we analyzed whether any combination of markers could be used for the prognostication of death within six months after cancer diagnosis. Materials and Methods: Patients diagnosed with HNC, receiving curative treatment decision at a multidisciplinary tumor board meeting, and who died within six months of diagnosis were included in this study. Nine patients who died within six months from diagnosis were identified and matched according to the tumor site and stage to seventeen patients who survived for at least two years. Results: The expression of markers was compared between the early-death patients and survivors. There was significantly higher Ki-67 expression in patients who died within six months than in those surviving for two years, with a mean difference of 21% (p = 0.038). A significant difference in cytoplasmic survivin expression was noted where early-death patients had increased expression compared to the survivors (p = 0.021). Furthermore, the intensity of survivin staining differed between the groups (p = 0.006). Conclusions: The results of this pilot study indicate that Ki67 and survivin could be potential prognostic biomarkers for early death in patients with HNC and possibly included in a panel of prognostic markers of value for treatment decision making.

Triple negative breast carcinoma (TNBC) is characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 receptor expression. Carbonic anhydrase IX (CA IX) is a tumor-associated cell surface glycoprotein that is involved in adaptation to hypoxia-induced acidosis and plays a role in cancer progression. The aim of this study was to investigate CA IX expression in TNBC and its relationship with treatment effect.

Immunohistochemical staining was performed on tru-cut biopsy materials with CA IX antibody. Positive staining was graded as low (<10%) and high (>10%). In addition, the relationship between tumor diameter, histological grade and the treatment effect on mastectomy materials performed after neoadjuvant treatment was evaluated.

TNBCs with positive staining for CA IX exhibited higher histological grade, and higher Ki-67 index compared to TNBCs with negative staining (p < 0.05). The response to treatment decreased as the degree of CA IX staining increased. There was no significant difference between the high staining group and low staining group in terms of patient age, tumor diameter and breast localisation.

CA IX enzyme is a poor prognostic marker in TNBC cases. However, overexpression of CA IX was associated with reduced response to treatment.

Treatment of osteosarcoma is hampered by tumor hypoxia and requires alternative approaches. Although the CCL2-CCR2 axis is indispensable in tumor-induced inflammation and angiogenesis, its blockade has not been effective to date. This study aimed to characterize how CCR2 inhibition affects the crosstalk of osteosarcoma cells with immune cells to better delineate tumor resistance mechanisms that help withstand such treatment. In this study, 143B cells were exposed to healthy donor PBMC supernatants in a transwell assay lacking direct cell-to-cell contact and subjected to different oxygen concentrations. In addition, mice bearing orthotopic 143B tumors were subjected to CCR2 antagonist treatment. Our findings show that hypoxic conditions alter cytokine and cancer- related protein expression on cells and impair CCR2 antagonist effects in the experimental osteosarcoma model. CCL2-CCR2 axis blockade in the 143B xenografts, which are positive for hypoxia marker CAIX, did not slow 143B tumor growth or metastasis but altered tumor microenvironment by VEGFR downregulation and shift in the CD44-positive cell population towards high CD44 expression. This study highlights differential responses of tumor cells to CCR2 antagonists in the presence of different oxygen saturations and expands our knowledge of compensatory mechanisms leading to CCL2-CCR2 treatment resistance.

This study is focused on the design, synthesis, and evaluation of some sulfonamide derivatives for their inhibitory effects on human carbonic anhydrase (hCA) enzymes I, II, IX, and XII as well as for their antioxidant activity. The purity of the synthesized molecules was confirmed by the HPLC purity analysis and was found in the range of 93%-100%. The inhibition constant (Ki) against hCA I ranged from 0.75 nM to 1972 nM. The sulfonamides inhibited isoform hCA II significantly, with a Ki ranging from 0.09 to 56 nM. Similarly, the inhibitory effects on hCA IX and XII were found with Ki spanning from 27.8 to 2099 nM and 9.43 to 509 nM, respectively. Most of the synthesized compounds showed significant inhibition in comparison to standard drugs such as acetazolamide, ethoxzolamide, zonisamide, methazolamide, dorzolamide, and SLC-0111. Antioxidant activity was assessed using the DPPH assay, with compound 13 showing better antioxidant activity with an IC50 of 54.8 µg/mL, as compared to the standard ascorbic acid (IC50 64.7 µg/mL). The molecular docking studies provided insights into the binding modes of these compounds. The in silico physicochemical properties, pharmacokinetic/ADME, and toxicity properties evaluations confirmed favorable drug-likeness properties, complying with Lipinski's rule. These findings underscore the therapeutic potential of these compounds for the treatment of retinal/cerebral edema, glaucoma, edema, epilepsy management, high-altitude sickness, and cancer.

Lactones, a diverse and abundant class of molecules found in nature, exhibit a wide range of bioactivities, including anti-inflammatory, anticancer, and antibacterial effects. Among them, acyl homoserine lactones (AHSLs) play a crucial role in quorum sensing, influencing bacterial pathogenicity and biofilm formation in Gram-negative bacteria. Paraoxonases (PONs), calcium-containing enzymes known for their lactonase activity, have been shown to hydrolyze AHSLs and reduce the biofilm formation of several pathogenic bacteria. In this study, we explored the potential lactonase activity of a class of zinc(II) enzymes, the carbonic anhydrases (CAs), aiming to uncover new insights into their catalytic versatility. Using LC-MS and MS/MS analyses, we investigated the lactonase activity of CAs and assessed several lactones through a stopped-flow kinetic assay as substrates/inhibitors. Our findings reveal that lactones are novel "prodrug" inhibitors of CAs, with lactones DHC and 6 showing the most promising inhibition constants (KIs) in the low micromolar range against both human and bacterial isozymes.

Membrane proteins, especially extracellular domains, are key therapeutic targets due to their role in cell communication and associations. Yet, their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently to deorphanize their respective receptors. We developed a comprehensive recombinant protein library of extracellular domains of human transmembrane proteins and proteins found in the ER-Golgi-lysosomal systems. Using this library, we conducted a flow-cytometric screen that identified several cell surface binding events, including an interaction between carbonic anhydrase 9 (CAH9/CA9/CAIX) and CD14high cells. Further analysis revealed this interaction was indirect and mediated via platelets bound to the monocytes. CA9, best known for its diverse roles in cancer, is a promising therapeutic target. We utilized our library to develop an AlphaLISA high-throughput screening assay, identifying CLEC2 as one robust CA9 binding partner. A five-amino-acid sequence (EDLPT) in CA9, identical to a CLEC2 binding domain in Podoplanin (PDPN), was found to be essential for this interaction. Like PDPN, CA9-induced CLEC2 signaling is mediated via Syk. A Hodgkin's lymphoma cell line (HDLM-2) endogenously expressing CA9 can activate Syk-dependent CLEC2 signaling, providing enticing evidence for a novel function of CA9 in hematological cancers. In conclusion, we identified numerous interactions with monocytes and platelets and validated one, CA9, as an endogenous CLEC2 ligand. We provide a new list of other putative CA9 interaction partners and uncovered CA9-induced CLEC2 activation, providing new insights for CA9-based therapeutic strategies.

This study aimed to investigate the diagnostic efficacy of [68Ga]Ga-NYM046 PET/CT in animal models and patients with clear cell renal cell carcinoma (ccRCC) and to compare its performance with that of 18F-FDG PET/CT. Methods: The in vivo biodistribution of [68Ga]Ga-NYM046 was evaluated in mice bearing OS-RC-2 xenografts. Twelve patients with ccRCC were included in the study; all completed paired [68Ga]Ga-NYM046 PET/CT and 18F-FDG PET/CT. The diagnostic efficacies of these 2 PET tracers were compared. Moreover, the positive rate of carbonic anhydrase IX in the pathologic tissue sections was compared with the SUVmax obtained by PET/CT. Results: The tumor accumulation of [68Ga]Ga-NYM046 at 1 h after injection in OS-RC-2 xenograft tumor models was 7.21 ± 2.39 injected dose per gram of tissue. Apart from tumors, the kidney and stomach showed high-uptake distributions. In total, 9 primary tumors, 96 involved lymph nodes, and 147 distant metastases in 12 patients were evaluated using [68Ga]Ga-NYM046 and 18F-FDG PET/CT. Compared with 18F-FDG PET/CT, [68Ga]Ga-NYM046 PET/CT detected more primary tumors (9 vs. 1), involved lymph nodes (95 vs. 92), and distant metastases (137 vs. 127). In quantitative analysis, the primary tumors' SUVmax (median, 13.5 vs. 2.4; z = -2.668, P = 0.008) was significantly higher in [68Ga]Ga-NYM046 PET/CT. Conversely, the involved lymph nodes' SUVmax (median, 5.9 vs. 7.6; z = -3.236, P = 0.001) was higher in 18F-FDG PET/CT. No significant differences were found for distant metastases (median SUVmax, 5.0 vs. 5.0; z = -0.381, P = 0.703). Higher [68Ga]Ga-NYM046 uptake in primary tumors corresponded to higher expression of carbonic anhydrase IX, with an R 2 value of 0.8274. Conclusion: [68Ga]Ga-NYM046 PET/CT offers a viable strategy for detecting primary tumors, involved lymph nodes, and distant metastases in patients with ccRCC.

Human carbonic anhydrase IX (CAIX) plays a key role in maintaining pH homeostasis of malignant neoplasms, thus creating a favorable microenvironment for the growth, invasion, and metastasis of tumor cells. Recent studies have established that inhibition of CAIX expressed on the surface of tumor cells significantly increases the efficacy of classical chemotherapeutic agents and makes it possible to suppress the resistance of tumor cells to chemotherapy, as well as to increase their sensitivity to drugs (in particular, to reduce the required dose of cytostatic agents). In this work, we studied the ability of new CAIX inhibitors based on substituted 1,2,4-oxadiazole-containing primary aromatic sulfonamides, to potentiate the cytostatic effect of gefitinib (selective inhibitor of epidermal growth factor receptor tyrosine kinase domain) under hypoxic conditions. We investigated a combined effect of gefitinib and CAIX inhibitors 4-(3-phenyl-1,2,4-oxadiazol-5-yl)thiophene-2-sulfonamide (1), 4-(5-(thiophene-3-yl)-1,2,4-oxadiazol-3-yl)benzenesulfonamide (2), 4-(3-(pyridin-2-yl)-1,2,4-oxadiazol-5-yl)thiophene-2-sulfonamide (3), and 4-(5-methyl-1,2,4-oxadiazol-3-yl)benzenesulfonamide (4) on gefitinib cytotoxicity, cell proliferation, activation of caspases-3/7, and cell cycle control in human lung adenocarcinoma A549 cells. It was found that the combinations of compounds 1 and 2 with gefitinib suppressed the invasive potential of A549 cells. Compound 1 had the greatest effect and can be considered as a promising candidate for further research.