Claudin-1 Research Articles

Abstract 1239: SAIL66, a next generation T cell engager targeting CLDN6, potentiates efficacy by binding to CD3/CD137

Abstract Background: Claudin-6 (CLDN6), one of over 20 known human CLDN family members, is upregulated in many solid tumors but shows minimal or no expression in healthy adult tissues. This makes it an ideal target for cancer therapy. Although several therapeutic modalities targeting CLDN6 are currently under investigation—including T cell engagers (TCE), CAR-T, and antibody drug conjugates—a more potent therapeutic option is still desirable. TCEs are a promising avenue within cancer immunotherapy, but their current efficacy and safety must be improved, particularly against solid tumors. Methods and Results: To enhance immune activation and achieve successful clinical outcomes against solid tumors expressing CLDN6, we generated SAIL66, a CLDN6-targeting next generation TCE, using a new technological format called Dual-Ig®. SAIL66 is a tri-specific monoclonal antibody designed to bind to CLDN6 on cancer cells with one Fab arm and to both CD3 and CD137 with the other Fab arm. The preclinical characterization of SAIL66 was performed in a series of in vitro and in vivo studies which included comparisons to a conventional TCE targeting CLDN6 and CD3. SAIL66 demonstrated high specificity for CLDN6 despite its similarity to CLDN3, CLDN4, and CLDN9, suggesting a lack of off-target toxicity in patients. Using a Jurkat cell system harboring NFAT or NF-κB reporter genes, we confirmed that SAIL66 exerts CD3 and CD137 signal induction activity depending on CLDN6 expression. SAIL66 induced activation of T cells, release of cytokines, and lysis of CLDN6-positive cancer cells. In vivo studies with a humanized mouse (huNOG) model showed that SAIL66 had better anti-tumor efficacy in eliminating CLDN6-positive cancer cells than a conventional TCE. Flow cytometry analysis in the huNOG model revealed that SAIL66 also more significantly increased CD3+ T cell infiltration of tumor tissue and resulted in a smaller percentage of exhausted T cells in tumor. Moreover, transcriptome analysis in the huNOG model showed that both SAIL66 and the conventional TCE increase expression of genes associated with immune activation, with a durable increase observed especially with SAIL66. Further, we clarified that T cells repeatedly stimulated with SAIL66 had a greater ability to inhibit the growth of CLDN6-positive cancer cells than T cells stimulated with the conventional TCE in vitro. Conclusion and Discussion: Our study shows that SAIL66 has better anti-tumor efficacy against CLDN6-positive cancer cells than a conventional TCE. This is due to effective immune activation by SAIL66 which triggers CD3 signaling (signal 1) combined with CD137 co-stimulatory signaling (signal 2) in T cells. Thus, SAIL66 represents a promising therapeutic option for solid tumors expressing CLDN6. A clinical trial for CLDN6-positive cancer patient is now ongoing (NCT05735366). Citation Format: Moe Yoshimoto, Naoki Kimura, Takayuki Kamikawa, Shinya Ishii, Masaru Muraoka, Kenji Taniguchi, Ryo Uchikawa, Momoko Okuda-Miura, Sho Akai, Tatsushi Kodama, Hirofumi Sakumoto, Shigeto Kawai, Mei Shimada, Mika Kamata-Sakurai, Hiroyuki Aburatani, Takehisa Kitazawa, Tomoyuki Igawa. SAIL66, a next generation T cell engager targeting CLDN6, potentiates efficacy by binding to CD3/CD137 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1239.

Abstract 7497: Advancing multi-biomarker CTC assay and CDx development through the automated GenoCTC system

Abstract Introduction: The successful outcomes observed in clinical trials emphasize the effectiveness of therapeutic approaches relying on biomarker tests, particularly in the development of cancer drugs. The parallel development of a drug and its associated predictive biomarker is considered an ideal strategy for achieving rapid and successful drug development in precision medicine. In this context, we employ an automated circulating tumor cell (CTC) isolation platform, known as GenoCTC, to facilitate the development of biomarker-driven CTC assays, with the ultimate goal of developing companion diagnostic (CDx) tests. Method: This study employed the 5th generation GenoCTC device, a fully automated system designed for marker-based CTC enrichment using immune magnetophoresis. We confirmed the isolation of CTCs through spiking tests, utilizing cancer biomarkers such as cMET, Mesothelin (MSLN), and Claudin 3 (CLDN3). cMET is a biomarker extensively studied in various cancers, particularly non-small cell lung cancer. We previously showed the prognostic significance of cMET expressing CTCs in HR+ and HER2- metastatic breast cancer patients. MSLN and CLDN3 are tumor-associated proteins showing overexpression across diverse cancer types, with a potential for the development of antibody-based cytotoxic drugs. The GenoCTC device allows efficient separation of CTCs expressing these biomarkers, ensuring high accuracy, precision, specificity, and sensitivity. Simultaneously, therapeutic drugs directed at these markers are progressing through phase trials or preclinical studies. Conclusion: Our results show that cMET, MSLN, and CLDN3 are potential markers for the development of biomarker tests, that can improve the patient selection process for personalized therapy targeting these biomarkers. Citation Format: Chaithanya Chelakkot, Jieun Park, Chae Rin Kim, Yeonu Lee, Yu Rim Lee, Saehyung Lee, Jun Young Choi, Young Ho Moon, Yoon-La Choi, Young Kee Shin, Hun Seok Lee. Advancing multi-biomarker CTC assay and CDx development through the automated GenoCTC system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7497.

Abstract 7643: Prognostic significance and immune infiltration patterns related to Claudin heterogeneity in pancreatic ductal adenocarcinoma patients

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) stands out for its aggressive nature, often manifesting through invasion, metastasis, and recurrence. These characteristics are partly attributable to the Epithelial-mesenchymal transition (EMT), a critical phenomenon that follows the disintegration of epithelial barriers, including tight junctions (TJs), where Claudin (CLDN) proteins are integral components. Objective: This study focused on elucidating the differential expression of claudins in PDAC and exploring their correlations with disease progression, invasiveness, and immune infiltration. Methodology: Utilizing UCSC TOIL RNA-seq, TCGA-PAAD genomic, and clinical data, we investigated the genomic alterations and differential expression of claudins in PDAC. Conducted survival analysis using log-rank tests. Assessed immune infiltration patterns using the EPIC deconvolution algorithm. Results: Of 177 PDAC samples examined, 23 (13%) exhibited at least one form of claudin gene alteration, including mutations, amplifications, or deep deletions. Remarkably, 10 claudins (CLDN1, 2, 4, 5, 7, 11, 12, 15, 18, and 23) were found to be significantly overexpressed in PDAC samples, meeting a log2FC threshold of ≥1 (p < 0.05). Further, we identified a direct correlation between mRNA expression and copy numbers, alongside an inverse relationship with methylation levels, particularly in CLDN1, CLDN12, CLDN15, CLDN23, and CLDN4. Intriguingly, claudin expression escalated from grades G1 to G3 but diminished in G4, with CLDN1, CLDN4, and CLDN7 showing significant variability (Kruskal-Wallis rank test p-values of 1.2E-5, 0.00064, and 0.023, respectively). Survival analysis indicated that high expression levels of CLDN1 and CLDN4 were linked to poorer patient outcomes, while higher levels of CLDN5 and CLDN15 correlated with improved survival (log-rank p-values of 0.007, 0.016, 0.0035, and 0.0012, respectively), findings consistent across both univariate and multivariate analyses. Furthermore, immune infiltration patterns, assessed via the EPIC deconvolution algorithm, varied significantly between high and low CLDN expression groups. Specifically, low CLDN1 expression was associated with increased presence of B-cells, CAF, and CD8+ T-cells but fewer endothelial cells, a pattern reversed in high CLDN1 expression cases. A similar trend was observed for CLDN4. Conclusion: Our study underscores the significant prognostic value of claudin proteins in PDAC, highlighting their potential as indicators of disease progression and survival and their influence on the tumor's immune environment. Citation Format: Ajaz A. Bhat, Shahabuddin Usmani, Hana Q. Sadida, Sheema Hashem, Tariq Masoodi, Ikhlak Ahmed, Rakesh Kumar, Mayank Singh, Punita Dhawan, Shahab Uddin, Muzafar A. Macha, Ammira S. Al-Shabeeb Akil. Prognostic significance and immune infiltration patterns related to Claudin heterogeneity in pancreatic ductal adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7643.

Abstract 4153: PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model

Abstract There is a critical need to develop new imaging and therapeutic strategies for pancreatic cancer. Current positron emission tomography (PET) imaging assessments of pancreatic ductal adenocarcinoma (PDAC) rely on 18F-FDG as a radiopharmaceutical. To increase the sensitivity of PDAC detection, we identified Claudin-4 (CLDN4), a tight junction transmembrane protein, as a PDAC biomarker through spatial transcriptomic and proteomic analyses. CLDN4 expression has previously been reported to be enhanced in 99% of primary and 100% of metastatic pancreatic cancer tissue samples and in 10 of 11 precancerous pancreatic intraepithelial neoplasia (PanIN) specimens. A small fragment of naturally occurring Clostridium perfringens enterotoxin (CPE) was previously reported to bind to the extracellular loop domain of CLDN4 protein (PDB:7kp4) with nanomolar (nM) affinity (Kd). A CLDN4 binding peptide (C4BP) was developed as a radioligand by modifying the n-terminus of C4BP which does not impact receptor binding. Here, we designed and synthesized a DOTA-PEG1-C4BP ligand on a rink amide resin using a microwave-assisted peptide synthesizer. Radiolabeling of DOTA-PEG1-C4BP (2 nmol) with 64CuCl2 (70 MBq) conferred 64Cu-C4BP (28 MBq/nmol) with 80% non-decay corrected yield and >99% radiochemical purity. PET/CT imaging of 64Cu-C4BP (~0.1 μg/mouse) performed in an inducible PDAC (KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato(KT);Ptf1aCreER;Trp53fl/fl) model at 84 day after tamoxifen injection resulted in ~25% injected activity per cubic centimeter (IA/cc) accumulation in metastases and at ~18% IA/cc retention in pancreatic tumors within 30 minutes of tail vein injection with minimal background accumulation. Ex vivo PET imaging confirmed the localized accumulation of 64Cu-C4BP in transgenic mice within the colon, pancreas, and liver. Blocking of 64Cu-C4BP accumulation with the cold compound in a xenograft model (NOD/SCID) bearing Capan-1 PDAC tumors demonstrated the specificity of 64Cu-C4BP binding. Here we report that CLDN4 is a promising target and 64Cu-C4BP is a promising ligand for the detection of pancreatic cancer. Citation Format: Jai Woong Seo, James Wang, Aris J. Kare, Spencer K. Tumbale, Bo Wu, Marina N. Raie, Mallesh Pandrala, Katherine W. Ferrara. PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4153.

Abstract 1863: α-ABN501, a novel antibody targeting claudin-3, demonstrates the potential to be a new therapeutic option for small cell lung cancer

Abstract Small Cell Lung Cancer (SCLC) is the most aggressive and lethal type of lung cancer, characterized by a poor prognosis, with a 5-year survival rate of only 7% for overall SCLC patients. Limited treatment options such as chemotherapy and immune-checkpoint inhibitor therapy are available for SCLC patients, very often followed by rapid recurrence and metastasis. Thus, new effective treatment options for SCLC remain high unmet medical needs. Overexpression of claudin-3 (CLDN3), one of the tight junction proteins, is observed in various types of cancers. The abnormal conformation of tumor cell growth leads to the external exposure of CLDN3, in contrast, the normal cells that maintain high polarity do not expose CLDN3 externally. Therefore, CLDN3 could be a potential therapeutic target and biomarker. Based on the analysis of the expression pattern of CLDN3 using a single-cell RNA sequencing dataset derived from SCLC patients, we found that CLDN3 could be a superior and unique target of SCLC compared to others (e.g., DLL3 and B7H3). Hence, we developed a fully human monoclonal antibody, afucosylated ABN501 (α-ABN501), which has high specificity and strong affinity to CLDN3-expressing cells. The α-ABN501 antibody showed CLDN3-dependent killing effects against the SCLC cells, expressing different levels of CLDN3, as assessed via the antibody-dependent cellular cytotoxicity (ADCC) assay wherein using NK cells expressing CD16a (NK-92MI-CD16a), peripheral blood mononuclear cells (PBMC), and primary NK cells. Further, the efficacy study in the CLDN3-expressing SCLC cell derived xenograft mouse model revealed that α-ABN501 was highly efficacious in inhibition of tumor growth. Collectively, we elucidated that the ADCC activity of α-ABN501 was dependent on the expression level of CLDN3 and especially specific to the SCLC cells, which underlines significant suppression of tumor growth. Present study data sets suggest that the α-ABN501 can be a potential new therapeutic option for SCLC. In addition, the α-ABN501 antibody is currently under way of development into new drug platforms for further applications. Citation Format: Jae Choi, Sunghyun Hong, Ha Yeon Park, Jaemun Kim, Sangin Lee, Myeung-Ryun Seo, Sang-Beom Bang, Hyo-Jin Shin, Kwang-il Jeong, Hobin Yang, Sungyoul Hong, Jun Young Choi, Na Young Kim, Young Kee Shin, Saehyung Lee. α-ABN501, a novel antibody targeting claudin-3, demonstrates the potential to be a new therapeutic option for small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1863.

Abstract 5798: Preclinical safety assessment for specificity of biotherapeutics using the membrane proteome array

Abstract Rigorous specificity analysis is critical for the development of antibody-based therapies, as even minimal off-target binding can lead to toxicity and clinical failures. Long believed to be exquisitely specific, recent preclinical data and our own work indicate that monoclonal antibodies (MAbs) frequently (~25%) display cross-reactivity. In many cases, off-target interactions occur with unrelated proteins that cannot be predicted by protein sequence homology. Cross-reactivity can lead to serious or even life-threatening consequences especially when MAbs are configured as bispecifics, antibody-drug conjugates (ADC) or CAR-T cell therapies, and IND applications for biotherapeutics require cross-reactivity assessment to prevent adverse events. Tissue cross-reactivity (TCR) studies have traditionally been used to screen for off-target binding, however, with poor predictive value for in vivo safety and toxicity. We developed the Membrane Proteome Array (MPA) platform to de-risk MAb-based therapeutics by testing for specificity across 6,000 human membrane proteins expressed in unfixed cells. The MPA assesses binding interactions by high-throughput flow cytometry allowing for high sensitivity detection and rapid analysis. All targets identified on the MPA screen are validated by secondary titration analysis. In contrast to TCR studies, proteins in the MPA exist in their native conformations and are not altered by fixation. Recently, we used the MPA to select a lead antibody candidate targeting the oncotherapeutic target Claudin 6 (CLDN6). Through MPA screening, we successfully identified a lead candidate devoid of cross-reactivity against other CLDN family members or any other membrane protein across the human genome. The MPA has also been used to rapidly evaluate the specificity of novel CAR-T cell therapies for cancer and autoimmunity. The MPA has been used for lead selection and data from the MPA has been accepted by the FDA as a part of IND applications for antibody-based therapies. The MPA is currently under consideration for being developed as an FDA-qualified Drug Development Tool. Citation Format: Kristopher S. Raghavan, Jonathan T. Sullivan, Carmen Navia, Rachel H. Fong, Benjamin J. Doranz. Preclinical safety assessment for specificity of biotherapeutics using the membrane proteome array [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5798.

Abstract 5494: Spatial organization between claudin and the tumor microenvironment in head and neck squamous cell carcinomas

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth leading form of cancer worldwide and one of the most difficult and challenging malignancies, with a high level of heterogeneity and a wide range of treatment responses. Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer and accounts for over 90% of cancers that develop within the mucosal epithelium of the oral cavity. Additional carcinogenic risk factors, including tobacco, alcohol and HPV infection are also associated with the pathogenesis of oral carcinogenesis. Unfortunately, today the failure of standard treatment modalities, such as surgery, radiotherapy, and chemotherapy underscore the need to identify better biomarkers of this disease. Advances, especially in spatial technology have created unprecedented opportunities to identify cell types and biomarkers of disease processes, revealing important relationships within the tumor microenvironment (TME) that allow a detailed characterization of specific cell phenotypes defined by co- or lack of expression of multiple markers that may help in predicting clinical responses and mechanisms of resistance to therapy. Recent findings indicate that abnormal expression of claudins (CLDNs) is closely related to the occurrence, progression and prognosis of many solid tumors. Specifically, CLDN6 is expressed in numerous tumors but rarely found in adult normal tissues making it a potential therapeutic target in head and neck tumor types. To investigate this further, expression of CLDN6 and the its relationship with changes within the TME of cohort OSCC tumors was determined using a proprietary comprehensive spatial workflow and gene expression analysis. Methods: A retrospective analysis was performed on biopsied Formalin-Fixed Paraffin-Embedded (FFPE) samples from OSCC patients with and without a history of smoking and treated +/- chemotherapy. Patient samples representing matched normal tissue to serve as controls were compared to samples derived from OSCC tumors. In order to perform a comprehensive expression profiling of the TME from OSCC tumors we used the spatial MultiOmyx™ immunofluorescence (IF) multiplexing assay with transcriptomic information from bulk mRNA gene expression acquired using the NanoString nCounter® PanCancer IO 360™ panel that includes the Tumor Inflammation Signature (TIS), as well as targets that characterize immune hot, desert, and cold regions in the stroma and tumor epithelium. Results: We characterized the expression of CLDN6 across the different cohorts of OSCC samples and further investigated the relationship within the tumor immune microenvironment coupled to genes related to the prognosis of OSCC. A better understanding of the TME and gene expression profiling of OSCC tumors and the effect of smoking will allow us to tailor the treatment of patients to continue to improve outcomes. Citation Format: Kirsteen Maclean, Lakshmi Chandramohan, Maryam Rohafza, Harry Nunns, Erinn Parnell, Qingyan Au, Anna Juncker-Jensen. Spatial organization between claudin and the tumor microenvironment in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5494.

Identification and validation of IRF6 related to ovarian cancer and biological function and prognostic value

BackgroundOvarian cancer (OC) is a severe gynecological malignancy with significant diagnostic and therapeutic challenges. The discovery of reliable cancer biomarkers can be used to adjust diagnosis and improve patient care. However, serous OC lacks effective biomarkers. We aimed to identify novel biomarkers for OC and their pathogenic causes.MethodsThe present study used the differentially expressed genes (DEGs) obtained from the “Limma” package and WGCNA modules for intersection analysis to obtain DEGs in OC. Three hub genes were identified—claudin 3 (CLDN3), interferon regulatory factor 6 (IRF6), and prostasin (PRSS8)—by searching for hub genes through the PPI network and verifying them in GSE14407, GSE18520, GSE66957, and TCGA + GTEx databases. The correlation between IRF6 and the prognosis of OC patients was further confirmed in Kaplan-Miller Plotter. RT-qPCR and IHC confirmed the RNA and protein levels of IRF6 in the OC samples. The effect of IRF6 on OC was explored using transwell invasion and scratch wound assays. Finally, we constructed a ceRNA network of hub genes and used bioinformatics tools to predict drug sensitivity.ResultsThe joint analysis results of TCGA, GTEx, and GEO databases indicated that IRF6 RNA and protein levels were significantly upregulated in serous OC and were associated with OS and PFS. Cell function experiments revealed that IRF6 knockdown inhibited SKOV3 cell proliferation, migration and invasion.ConclusionIRF6 is closely correlated with OC development and progression and could be considered a novel biomarker and therapeutic target for OC patients.

In-house ammonia induced lung impairment and oxidative stress of ducks

Ammonia (NH3) is a toxic gas that in intensive poultry houses, damages the poultry health and induces various diseases. This study investigated the effects of NH3 exposure (0, 15, 30, and 45 ppm) on growth performance, serum biochemical indexes, anti-oxidative indicators, tracheal and lung impairments in Pekin ducks. A total of 288 1-day-old Pekin male ducks were randomly allocated to four groups with 6 replicates and slaughtered after the 21-day test period. Our results showed that 45 ppm NH3 significantly reduced the average daily feed intake (ADFI) of Pekin ducks. NH3 exposure significantly reduced liver, lung, kidney, and heart indexes, and lowered the relative weight of the ileum. With the increase of in-house NH3, serum NH3 and uric acid (UA) concentrations of ducks were significantly increased, as well as liver malondialdehyde (MDA), Superoxide dismutase (SOD), and glutathione peroxidase (GPX-Px) contents. High NH3 also induced trachea and lung injury, thereby increasing levels of tumour necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in the lung, and decreasing the mRNA expressions of zonula occludens 1 (ZO-1) and claudin 3 (CLDN3) in the lung. In conclusion, in-house NH3 decrease the growth performance in ducks, induce trachea and lung injuries and meanwhile increase the compensatory antioxidant activity for host protection.

CLDN6 inhibited cellular biological function of nonsmall cell lung cancer cells through suppressing aerobic glycolysis via the RIP1/ASK1/JNK axis.

Claudin-6 (CLDN6) has been extensively studied in different tumors to date. However, in the case of nonsmall cell lung cancer (NSCLC), CLDN6 has a largely unknown role and molecular mechanism. We detected the expression of CLDN6 in NSCLC tissues and cells using reverse transcription-quantitative polymerase chain reaction (PCR) and western blot assays. A gain-of-function experiment was performed to evaluate the biological effects of CLDN6 on NSCLC cell behaviors. Methylation-specific PCR was utilized to detect the DNA methylation of CLDN6 gene promoter region. The interaction of CLDN6 and receptor interacting protein 1 (RIP1) was determined by coimmunoprecipitation assay. Furthermore, the modulation of CLDN6 on RIP1/apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) axis was confirmed. The results showed that in NSCLC tissues and cells, CLDN6 expression level was declined, and was associated with a high level of DNA methylation. CLDN6 overexpression suppressed the viability, invasion, migration, and promoted cell apoptosis. Besides, the enhanced expression of CLDN6 reduced the glycolysis and the dysfunction of mitochondrial respiration of NSCLC cells. Mechanistic investigation confirmed that CLDN6 interacted with RIP1 and inhibited cellular biological function of NSCLC cells via RIP1/ASK1/JNK axis. Besides, CLDN6 overexpression inhibited tumor growth in vivo. In conclusion, CLDN6 inhibited NSCLC cell proliferation through inactivating aerobic glycolysis via the RIP1/ASK1/JNK axis.

Whole-transcriptome sequencing in advanced gastric or gastroesophageal cancer: A deep dive into its clinical potential.

Advanced gastric and gastroesophageal junction cancers (GC/GEJCs) harbor diverse molecular signatures, highlighting the need for intricate evaluations to identify potential therapeutic targets. Although whole-transcriptome sequencing (WTS) has emerged as a useful tool for understanding these molecular intricacies, its clinical implications have yet to be fully elucidated. This study evaluated the correlation between immunohistochemistry (IHC) and WTS, compared their clinical significance, and identified potential therapeutic targets undetectable through IHC alone. We enrolled 140 patients with advanced GC/GEJC and assessed them using IHC for six pivotal biomarkers: claudin-18 (CLDN18), human epidermal growth factor receptor 2 (HER2), multiple receptor tyrosine kinases (RTKs), and programmed death ligand 1 (PD-L1). Concurrently, WTS was employed as part of the analyses in MONSTAR-SCREEN-2, a multicenter multiomics study. IHC analysis revealed 16.4% HER2, 39.3% CLDN18 (2+/3 + ≥75%), and 15.8% PD-L1 (combined positive score ≥ 10) positivity, among other molecular markers. Significant correlations were observed between IHC and WTS for all six pivotal biomarkers. Among nineteen HER2 IHC-positive patients treated with anti-HER2 therapeutics, ERBB2 status in WTS was significantly associated with progression-free survival (ERBB2-high vs. -low: median 9.0 vs. 5.6 months, log-rank p = 0.046). IHC-based molecular profiling revealed significantly high expression of CLDN18 in RTK-negative patients, with 78.4% positive for either CLDN18 or PD-L1. Additionally, WTS revealed elevated expression of pivotal biomarkers in patients displaying negative targetable biomarkers via IHC. Our findings highlighted the significant correlation between IHC and WTS, reinforcing the clinical utility of WTS. A subset with IHC-negative but WTS-positive status may benefit from specific biomarker-targeted therapies.

Modeling of new markers for the diagnosis and prognosis of pancreatic cancer based on the transition from inflammation to cancer.

Pancreatic adenocarcinoma (PAAD) is a lethal disease with a poor prognosis. Genes involved in acute pancreatitis (AP) or chronic pancreatitis (CP) might be important for PAAD development. This study sought to identify potential PAAD diagnosis markers and to establish a PAAD prognosis prediction model based on AP- and CP-related genes. The significantly differentially expressed genes in both AP or CP and PAAD were obtained by a bioinformatics analysis. A risk-score model for predicting survival was constructed based on The Cancer Genome Atlas (TCGA) data and validated using an International Cancer Genome Consortium (ICGC) cohort. Protein expression and the effects of the genes in the risk models were validated by immunohistochemistry, or Cell Counting Kit-8 (CCK-8) and transwell assays. The study sample data included six AP tissue samples and five normal pancreatic tissue samples, six CP tissue samples and six normal pancreatic tissue samples from the Gene Expression Omnibus (GEO) expression profiling microarrays GSE109227 and GSE41418 data sets, respectively, and fragments per kilobase per million mapped fragments (FPKM) data from four normal controls and 150 PAAD cases from TCGA database, and 182 cancer patient samples with complete survival prognostic data from the ICGC database. In total, 508 significantly differentially expressed genes were found in both AP or CP and PAAD. Trefoil factor 2 (TFF2), tubulointerstitial nephritis antigen (TINAG), trefoil factor 1 (TFF1), aquaporin 5 (AQP5), SAM pointed domain containing ETS transcription factor (SPDEF), anterior gradient protein 2 (AGR2), apolipoprotein B messenger RNA editing enzyme catalytic subunit 1 (APOBEC1), kallikrein-related peptidase 6 (KLK6), dopa decarboxylase (DDC), mucin 13 (MUC13), claudin 18 (CLDN18), annexin A10 (ANXA10), and tetraspanin 1 (TSPAN1) were found to be present in PAAD and had the largest fold change. A risk-score model, comprising 19 genes, was constructed for prognostic prediction. A high-risk score indicated a poor prognosis. TINAG, DDC, SPDEF, and APOBEC1 proteins were increased in PAAD, while TINAG and DDC were correlated with the pathologic grade. Decreased TINAG, APOBEC1, transmembrane protein 94 (TMEM94), and kelch like family member 36 (KLHL36) expression inhibited PAAD cell proliferation, while decreased SPDEF, TMEM94, and KLHL36 expression significantly inhibited PAAD cell migration. The AP and CP co-related genes were significantly correlated with PAAD. TINAG, DDC, SPDEF, and APOBEC1 could serve as new PAAD predictors. The risk model developed in this study could be used to predict the prognosis of PAAD patients.

Association of CLDN molecules with sleep apnea hypopnea syndrome: new biomarker candidates.

Obstructive sleep apnea (OSA) is a common sleep-related breathing disorder, and has become a serious threat to public health. Intermittent hypoxia caused by OSA results in a low-grade inflammatory response that leads to impaired mucosal barrier function. Claudin (CLDN) molecules are important for the permeability of the mucosal epithelium. This study aimed to explore whether CLDN molecules can be a potential biomarker of OSA. A total of 37 healthy controls and 40 OSA patients underwent a physical assessment for OSA and filled out the STOP-Bang Questionnaire (SBQ) and Epworth Sleepiness Scale (ESS). Clinical specimens of plasma and urine were obtained to observe the difference between OSA patients and healthy controls and diagnostic accuracy of CLDN molecules for OSA. CLDN1, CLDN2, and CLDN3 molecules in plasma and urine decreased in OSA patients (both p < 0.05). The areas under the receiver operating characteristic curve (AUCs) of urinary CLDN1, plasma CLDN1, urinary CLDN2, plasma CLDN2, urinary CLDN3, and plasma CLDN3 were 0.887, 0.724, 0.779, 0.676, 0.828, and 0.665, respectively. The AUC of urinary CLDN1 + CLDN2 + CLDN3 was 0.906 (95% confidence interval (CI), 0.831-0.981). The AUC of plasma CLDN1 + CLDN2 + CLDN3 was 0.776 (95% CI, 0.645-0.878). The AUC of urinary CLDN3 + SBQ was 0.899 (95% CI, 0.832-0.967). The AUC of urinary CLDN3 + ESS was 0.896 (95% CI, 0.826-0.966). In addition, Urinary CLDN-3 was negative associated with the severity of OSA. CLDN molecules are promising as useful biomarkers for OSA, which may be related to the impaired barrier function related to OSA.

Claudin-1 as a novel target gene induced in obesity and associated to inflammation, fibrosis, and cell differentiation.

T lymphocytes from visceral and subcutaneous white adipose tissues (vWAT and sWAT, respectively) can have opposing roles in the systemic metabolic changes associated with obesity. However, few studies have focused on this subject. Claudin-1 (CLDN1) is a protein involved canonically in tight junctions and tissue paracellular permeability. We evaluated T-lymphocyte gene expression in vWAT and sWAT and in the whole adipose depots in human samples. A Clariom D-based transcriptomic analysis was performed on T lymphocytes magnetically separated from vWAT and sWAT from patients with obesity (Cohort 1; N = 11). Expression of candidate genes resulting from that analysis was determined in whole WAT from individuals with and without obesity (Cohort 2; patients with obesity: N = 13; patients without obesity: N = 14). We observed transcriptional differences between T lymphocytes from sWAT compared with vWAT. Specifically, CLDN1 expression was found to be dramatically induced in vWAT T cells relative to those isolated from sWAT in patients with obesity. CLDN1 was also induced in obesity in vWAT and its expression correlates with genes involved in inflammation, fibrosis, and adipogenesis. These results suggest that CLDN1 is a novel marker induced in obesity and differentially expressed in T lymphocytes infiltrated in human vWAT as compared with sWAT. This protein may have a crucial role in the crosstalk between T lymphocytes and other adipose tissue cells and may contribute to inflammation, fibrosis, and alter homeostasis and promote metabolic disease in obesity.

Evaluation of kefir grain microbiota, grain viability, and bioactivity from fermenting dairy processing by-products

Four dairy foods processing by-products (acid whey permeate (AWP), buttermilk (BM), sweet whey permeate (SWP), and sweet whey permeate with added milk fat globule ingredient (SWP+MFGM)) were fermented for 4 weeks and compared with traditional kefir milks for production of novel kefir-like dairy products. AU: Sweet whey permeates and SWP supplemented with 1.5% milk fat globule membrane (MFGM) showed to be the most viable by-products for kefir grain fermentation, exhibiting diverse abundance of traditional kefir microorganisms and positive indicators of bioactive properties. Grain viability was assessed with shotgun metagenomics, texture profile analysis, live cell counts, and scanning electron microscopy. Assessed bioactivities of the kefir-like products included antibacterial, antioxidant, potential anticancerogenic properties, and membrane barrier effect on human colorectal adenocarcinoma Caco-2 cells. All kefir grains were most abundant in Lactobacillus kefiranofaciens when analyzed with shotgun metagenomics. When analyzed with live cell counts on selective media, AWP kefir-like product had no countable Lactococcus spp. indicating suboptimal conditions for kefir grain microbiota survival and application for fermented dairy starter culture bacterium. Live cell counts were affirmed with kefir grain surface scanning electron microscopy images. SWP had the most adhesive kefir grain surface while SWP+MFGM had the largest exopolysaccharide (EPS) yield from grain extraction. All kefir and kefir-like products were able to achieve a 6-log reduction against Listeria innocua and Escherichia coli. Traditional milk kefirs had the highest antioxidant capacity for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. AWP had a significantly higher DPPH antioxidant activity and SWP had the lowest Trolox equivalence concentration in the ABTS assay. Sweet whey and supplemented milk fat sweet whey had upregulation of Cldn-1 and Ocln-1 gene expression, which correspond with a significant increase in transepithelial electrical resistance.

Development of tight junction-strengthening compounds using a high-throughput screening system to evaluate cell surface-localized claudin-1 in keratinocytes

Tight junctions (TJs) are important factors constituting the physical barriers of the skin, and their suppression has been described in various conditions, such as aged skin and atopic dermatitis lesions. However, the methods for improving skin TJ function remain insufficient. Therefore, to obtain compounds that can improve TJ function, we developed a novel high-throughput screening system termed live-cell immunostaining to evaluate cell surface-localized claudin-1 (CLDN1) with high selectivity using normal human epidermal keratinocytes (NHEKs). Heparinoid and phospho-pyridoxal (p-Pyr), a metabolite of pyridoxine, were identified as hit compounds. In addition, heparinoid was strongly suggested to increase CLDN1 expression by inhibiting epidermal growth factor receptor signaling. By contrast, p-Pyr did not enhance CLDN1 expression, but it accelerated the translocation of CLDN1 to the cell surface. Finally, we confirmed that heparinoid and p-Pyr improved barrier function in NHEKs in a transepithelial electrical resistance assay. In conclusion, heparinoid and p-Pyr could potentially ameliorate skin conditions by improving TJ function.

Loss of Claudin-1 incurred by DNMT aberration promotes pancreatic cancer progression

Pancreatic cancer (PC) is one of the most malignant and deadly tumors of digestive system with complex etiology and pathogenesis. Dysregulations of oncogenes and tumor suppressors due to epigenetic modifications causally affect tumorogenesis; however the key tumor suppressors and their regulations in PC are only partially defined. In this study, we found that Claudin-1 (encoded by CLDN1 gene) was significantly suppressed in PC that correlated with a poor clinical prognosis. Claudin-1 knockdown enhanced PC cell proliferation, migration, and stemness. Pancreatic specific Cldn1 knockout in KPC (LSLKrasG12D/Pdx1-Cre/Trp53R172H+) and KC (LSLKrasG12D/Pdx1-Cre) mice reduced mouse survival, promoted acinar-to-ductal metaplasia (ADM) process, and accelerated the development of pancreatic intraepithelial neoplasia (PanIN) and PC. Further investigation revealed that Claudin-1 suppression was mainly caused by aberrant DNA methylatransferase 1 (DNMT1) and DNMT3A elevations and the resultant CLDN1 promoter hypermethylation, as a DNMT specific inhibitor SGI-1027 effectively reversed the Claudin-1 suppression and inhibited PC progression both in vitro and in vivo in a Claudin-1 preservation-dependent manner. Together, our data suggest that Claudin-1 functions as a tumor suppressor in PC and its epigenetic suppression due to DNMT aberrations is a crucial event that promotes PC development and progression.

Polystyrene microplastic-induced oxidative stress triggers intestinal barrier dysfunction via the NF-κB/NLRP3/IL-1β/MCLK pathway

Emerging evidence has demonstrated the association between microplastics (MPs) with a diameter of <5 mm and the risk of intestinal diseases. However, the molecular mechanisms contributing to MP-induced intestinal barrier dysfunction have not been fully appreciated. In this study, C57BL/6 J mice were exposed to polystyrene microplastics (PS-MPs, 0.2, 1 or 5 μm) at 1 mg/kg body weight daily by oral gavage for 28 days. We found that PS-MPs exposure induced oxidative stress and inflammatory cell infiltration in mice colon, leading to an increased expression of pro-inflammatory cytokine. Moreover, there were an increase in intestinal permeability and decrease in mucus secretion, accompanied by downregulation of tight junction (TJ)-related zonula occluden-1 (ZO-1), occluding (OCLN) and claudin-1 (CLDN-1) in mice colon. Especially, 5 μm PS-MPs (PS5)-induced intestinal epithelial TJ barrier damage was more severe than 0.2 μm PS-MPs (PS0.2) and 1 μm PS-MPs (PS1). In vitro experiments indicated that PS5-induced oxidative stress upregulated the expression of nuclear factor kappa B (NF-κB), nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome, and myosin light chain kinase (MLCK). Meanwhile, pre-treatment with the antioxidant NAC, NLRP3 inhibitor MCC950 and MLCK inhibitor ML-7 considerably reduced PS5-triggered reactive oxygen species (ROS) production and inflammatory response, inhibited the activation of the NF-κB/NLRP3/MLCK pathway, and upregulated ZO-1, OCLN and CLDN-1 expression in Caco-2 cells. Taken together, our study demonstrated that PS-MPs cause intestinal barrier dysfunction through the ROS-dependent NF-κB/NLRP3/IL-1β/MLCK pathway.

Surface Proteome of Extracellular Vesicles and Correlation Analysis Reveal Breast Cancer Biomarkers

Breast cancer (BC) is the second most frequently diagnosed cancer and accounts for approximately 25% of new cancer cases in Canadian women. Using biomarkers as a less-invasive BC diagnostic method is currently under investigation but is not ready for practical application in clinical settings. During the last decade, extracellular vesicles (EVs) have emerged as a promising source of biomarkers because they contain cancer-derived proteins, RNAs, and metabolites. In this study, EV proteins from small EVs (sEVs) and medium EVs (mEVs) were isolated from BC MDA-MB-231 and MCF7 and non-cancerous breast epithelial MCF10A cell lines and then analyzed by two approaches: global proteomic analysis and enrichment of EV surface proteins by Sulfo-NHS-SS-Biotin labeling. From the first approach, proteomic profiling identified 2459 proteins, which were subjected to comparative analysis and correlation network analysis. Twelve potential biomarker proteins were identified based on cell line-specific expression and filtered by their predicted co-localization with known EV marker proteins, CD63, CD9, and CD81. This approach resulted in the identification of 11 proteins, four of which were further investigated by Western blot analysis. The presence of transmembrane serine protease matriptase (ST14), claudin-3 (CLDN3), and integrin alpha-7 (ITGA7) in each cell line was validated by Western blot, revealing that ST14 and CLDN3 may be further explored as potential EV biomarkers for BC. The surface labeling approach enriched proteins that were not identified using the first approach. Ten potential BC biomarkers (Glutathione S-transferase P1 (GSTP1), Elongation factor 2 (EEF2), DEAD/H box RNA helicase (DDX10), progesterone receptor (PGR), Ras-related C3 botulinum toxin substrate 2 (RAC2), Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), Aconitase 2 (ACO2), UTP20 small subunit processome component (UTP20), NEDD4 binding protein 2 (N4BP2), Programmed cell death 6 (PDCD6)) were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. In total, 846 surface proteins were identified from the second approach, of which 11 were already known as BC markers. This study supports the proposition that Evs are a rich source of known and novel biomarkers that may be used for non-invasive detection of BC. Furthermore, the presented datasets could be further explored for the identification of potential biomarkers in BC.

INFLAMMATION-TRIGGERED CLAUDIN-23 DOWNREGULATION IMPACTS INTESTINAL EPITHELIAL BARRIER FUNCTION

Abstract Intestinal epithelium maintains gut homeostasis by protecting the underlying tissue compartments from harmful contents. Although it is suggested that epithelial barrier function compromise contributes to the pathogenesis of inflammatory bowel disease (IBD), the underlying mechanisms are not well understood. Claudins (CLDN) are tight junctions (TJ) proteins that determine epithelial barrier properties during homeostasis and whose expression is altered in IBD, thereby contributing to a leaky barrier. We recently identified the expression of an atypical CLDN family member, CLDN23, in the regulation of homeostatic intestinal epithelial barrier function. Analysis of the human colonic crypt-luminal axis revealed increased CLDN23 expression in differentiated intestinal epithelial cells (IEC) facing the lumen compared to the proliferative crypt-base IECs. Complementary cell biologic approaches using IEC-specific Cldn23 knockout mice (Cldn23ERΔIEC) and human colon cell lines revealed that CLDN23 strengthens epithelial barrier function. Mechanistically, CLDN23 promoted redistribution of barrier-forming CLDN3 and CLDN4 from the lateral membrane to the TJ through cis- and trans-interactions, resulting in narrowed CLDN3 and CLDN4 pore diameters. Since inflammation compromises the epithelial barrier and contributes to disease pathogenesis in IBD, we first examined CLDN23 expression in colonic mucosal tissue from IBD individuals. Immunofluorescence and RNA scope labeling revealed that CLDN23 expression was significantly decreased in colonic epithelial cells of IBD individuals. Furthermore, reduced CLDN23 expression was also detected in primary colonic epithelial cells (colonoids) from mice treated with pro-inflammatory cytokines TNFα and IFNγ compared to untreated colonoids. Barrier function assays of control Cldn23f/f and Cldn23ERΔIEC mice-derived colonoids treated with TNFα and IFNγ identified decreased transepithelial electrical resistance in colonoids lacking CLDN23 and treated with these pro-inflammatory cytokines. These findings suggest a protective role for CLDN23 in regulating barrier function under homeostatic and inflammatory conditions.