Greatly isolated heterogeneous circulating tumor cells using hybrid engineered cell membrane-camouflaged magnetic nanoparticles.

BACKGROUND: Circulating tumor cells (CTCs) are considered as a useful biomarker for early cancer diagnosis, which play a crucial role in metastatic process. Unfortunately, the tumor heterogeneity and extremely rare occurrence rate of CTCs among billions of interfering leukocytes seriously hamper the sensitivity and purity of CTCs isolation. METHODS: To address these, we firstly used microfluidic chips to detect the broad-spectrum of triple target combination biomarkers in CTCs of 10 types of cancer patients, including EpCAM, EGFR and Her2. Then, we constructed hybrid engineered cell membrane-camouflaged magnetic nanoparticles (HE-CM-MNs) for efficient capture of heterogeneous CTCs with high-purity, which was enabled by inheriting the recognition ability of HE-CM for various CTCs and reducing homologous cell interaction with leukocytes. Compared with single E-CM-MNs, HE-CM-MNs showed a significant improvement in the capture efficiency for a cell mixture, with an efficiency of 90%. And the capture efficiency of HE-CM-MNs toward 12 subpopulations of tumor cells was ranged from 70 to 85%. Furthermore, by using HE-CM-MNs, we successfully isolated heterogeneous CTCs with high purity from clinical blood samples. Finally, the captured CTCs by HE-CM-MNs could be used for gene mutation analysis. CONCLUSIONS: This study demonstrated the promising potential of HE-CM-MNs for heterogeneous CTCs detection and downstream analysis.

Intelligent Cell Profiling and Precision Release: Multimolecular Marker-Activated Transmembrane DNA Computing Nanosystem.

Accurate classification of tumor cells is of importance for cancer diagnosis and further therapy. In this study, we develop multimolecular marker-activated transmembrane DNA computing systems (MTD). Employing the cell membrane as a native gate, the MTD system enables direct signal output following simple spatial events of "transmembrane" and "in-cell target encounter", bypassing the need of multistep signal conversion. The MTD system comprises two intelligent nanorobots capable of independently sensing three molecular markers (MUC1, EpCAM, and miR-21), resulting in comprehensive analysis. Our AND-AND logic-gated system (MTDAND-AND) demonstrates exceptional specificity, allowing targeted release of drug-DNA specifically in MCF-7 cells. Furthermore, the transformed OR-AND logic-gated system (MTDOR-AND) exhibits broader adaptability, facilitating the release of drug-DNA in three positive cancer cell lines (MCF-7, HeLa, and HepG2). Importantly, MTDAND-AND and MTDOR-AND, while possessing distinct personalized therapeutic potential, share the ability of outputting three imaging signals without any intermediate conversion steps. This feature ensures precise classification cross diverse cells (MCF-7, HeLa, HepG2, and MCF-10A), even in mixed populations. This study provides a straightforward yet effective solution to augment the versatility and precision of DNA computing systems, advancing their potential applications in biomedical diagnostic and therapeutic research.

Single-cell SERS imaging of dual cell membrane receptors expression influenced by extracellular matrix stiffness.

Membrane receptors perform a diverse range of cellular functions, accounting for more than half of all drug targets. The mechanical microenvironment regulates cell behaviors and phenotype. However, conventional analysis methods of membrane receptors often ignore the effects of the extracellular matrix stiffness, failing to reveal the heterogeneity of cell membrane receptors expression. Herein, we developed an in-situ surface-enhanced Raman scattering (SERS) imaging method to visualize single-cell membrane receptors on substrates with different stiffness. Two SERS substrates, Au@4-mercaptobenzonitrile@Ag@Sgc8c and Au@4-pethynylaniline@Ag@SYL3c, were employed to specifically target protein tyrosine kinase-7 (PTK7) and epithelial cell adhesion molecule (EpCAM), respectively. The polyacrylamide (PA) gels with tunable stiffness (2.5-25 kPa) were constructed to mimic extracellular matrix. The simultaneous SERS imaging of dual membrane receptors on single cancer cells on substrates with different stiffness was achieved. Our findings reveal decreased expression of PTK7 and EpCAM on cells cultured on stiffer substrates and higher migration ability of the cells. The results elucidate the heterogeneity of membrane receptors expression of cells cultured on the substrates with different stiffness. This single-cell analysis method offers an in-situ platform for investigating the impacts of extracellular matrix stiffness on the expression of membrane receptors, providing insights into the role of cell membrane receptors in cancer metastasis.

Single-cell transcriptomics reveal metastatic CLDN4+ cancer cells underlying the recurrence of malignant pleural effusion in patients with advanced non-small-cell lung cancer.

BACKGROUND: Recurrent malignant pleural effusion (MPE) resulting from non-small-cell lung cancer (NSCLC) is easily refractory to conventional therapeutics and lacks predictive markers. The cellular or genetic signatures of recurrent MPE still remain largely uncertain. METHODS: 16 NSCLC patients with pleural effusions were recruited, followed by corresponding treatments based on primary tumours. Non-recurrent or recurrent MPE was determined after 3-6 weeks of treatments. The status of MPE was verified by computer tomography (CT) and cytopathology, and the baseline pleural fluids were collected for single-cell RNA sequencing (scRNA-seq). Samples were then integrated and profiled. Cellular communications and trajectories were inferred by bioinformatic algorithms. Comparative analysis was conducted and the results were further validated by quantitative polymerase chain reaction (qPCR) in a larger MPE cohort from the authors' centre (n = 64). RESULTS: The scRNA-seq revealed that 33 590 cells were annotated as 7 major cell types and further characterized into 14 cell clusters precisely. The cell cluster C1, classified as Epithelial Cell Adhesion Molecule (EpCAM)+ metastatic cancer cell and correlated with activation of tight junction and adherence junction, was significantly enriched in the recurrent MPE group, in which Claudin-4 (CLDN4) was identified. The subset cell cluster C3 of C1, which was enriched in recurrent MPE and demonstrated a phenotype of ameboidal-type cell migration, also showed a markedly higher expression of CLDN4. Meanwhile, the expression of CLDN4 was positively correlated with E74 Like ETS Transcription Factor 3 (ELF3), EpCAM and Tumour Associated Calcium Signal Transducer 2 (TACSTD2), independent of driver-gene status. CLDN4 was also found to be associated with the expression of Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A) and Vascular Endothelial Growth Factor A (VEGFA), and the cell cluster C1 was the major mediator in cellular communication of VEGFA signalling. In the extensive MPE cohort, a notably increased expression of CLDN4 in cells from pleural effusion among patients diagnosed with recurrent MPE was observed, compared with the non-recurrent group, which was also associated with a trend towards worse overall survival (OS). CONCLUSIONS: CLDN4 could be considered as a predictive marker of recurrent MPE among patients with advanced NSCLC. Further validation for its clinical value in cohorts with larger sample size and in-depth mechanism studies on its biological function are warranted. TRIAL REGISTRATION: Not applicable.

Curcumin-loaded liposomes modulating the synergistic role of EpCAM and estrogen receptor alpha in lung cancer management.

Lung cancer (LC) remains a leading cause of cancer-related mortality worldwide, necessitating the exploration of innovative therapeutic strategies. This study delves into the in vitro potential of liposomal therapeutics utilizing Curcumin-loaded PlexoZome® (CUR-PLXZ) in targeting EpCAM/TROP1 and Estrogen Receptor Alpha (ERα) signalling pathways for LC management. The prevalence of LC, particularly non-small cell lung cancer (NSCLC), underscores the urgent need for effective treatments. Biomarkers like EpCAM/TROP1 and ERα/NR3A1 play crucial roles in guiding targeted therapies and influencing prognosis. EpCAM plays a key role in cell-cell adhesion and signalling along with ERα which is a nuclear receptor that binds estrogen and regulates gene expression in response to hormonal signals. In LC, both often get overexpressed and are associated with tumour progression, metastasis, and poor prognosis. Curcumin, a phytochemical with diverse therapeutic properties, holds promise in targeting these pathways. However, its limited solubility and bioavailability necessitate advanced formulations like CUR-PLXZ. Our study investigates the biological significance of these biomarkers in the A549 cell line and explores the therapeutic potential of CUR-PLXZ, which modulates the expression of these two markers. An in vitro analysis of the A549 human lung adenocarcinoma cell line identified that CUR-PLXZ at a dose of 5 µM effectively inhibited the expression of EpCAM and ERα. This finding paves the way for targeted intervention strategies in LC management.

Utilizing Magnetic Levitation to Detect Lung Cancer-Associated Exosomes.

Extracellular vesicles, especially exosomes, have attracted attention in the last few decades as novel cancer biomarkers. Exosomal membrane proteins provide easy-to-reach targets and can be utilized as information sources of their parent cells. In this study, a MagLev-based, highly sensitive, and versatile biosensor platform for detecting minor differences in the density of suspended objects is proposed for exosome detection. The developed platform utilizes antibody-functionalized microspheres to capture exosomal membrane proteins (ExoMPs) EpCAM, CD81, and CD151 as markers for cancerous exosomes, exosomes, and non-small cell lung cancer (NSCLC)-derived exosomes, respectively. Initially, the platform was utilized for protein detection and quantification by targeting solubilized ExoMPs, and a dynamic range of 1-100 nM, with LoD values of 1.324, 0.638, and 0.722 nM for EpCAM, CD81, and CD151, were observed, respectively. Then, the sensor platform was tested using exosome isolates derived from NSCLC cell line A549 and MRC5 healthy lung fibroblast cell line. It was shown that the sensor platform is able to detect and differentiate exosomal biomarkers derived from cancerous and non-cancerous cell lines. Overall, this innovative, simple, and rapid method shows great potential for the early diagnosis of lung cancer through exosomal biomarker detection.

Diagnostic Performance of Epithelial Cell Adhesion Molecule for Early Detection of Hepatocellular Carcinoma Among HCV High-Risk Patients.

OBJECTIVE: Hepatocellular carcinoma (HCC) is often diagnosed at advanced stage where hopeless for effective therapies. Identification of more reliable biomarkers for early detection of HCC is urgently needed. Circulating tumor cells (CTCs) represent a unique liquid biopsy carrying comprehensive biological information of the primary tumor. Herein, we sought to develop a novel score based on the combination of the most significant CTCs biomarkers with routine laboratory tests for accurate detection of HCC. MATERIAL & METHODS: Epithelial cell adhesion molecule (EpCAM), α-fetoprotein, albumin, and platelets count were assayed in HCC patients (98), liver cirrhosis patients (77). Areas under receiving operating curve (AUCs) were calculated and used for construction on novel score. RESULTS: A novel score named EpCAM-HCC = AFP (U/L) × 0.11 - Albumin (g/dl) × 1.5 + EpCAM % × 2.9 - Platelets count (×109)/L× 0.75 - 93. EpCAM-HCC score produce AUC of 1 for differentiate patients with HCC from those with liver cirrhosis with sensitivity and specificity of a cut-off 1.7 (i.e., less than 1.7 the case is considered cirrhotic, whereas above 1.7 it is considered HCC. CONCLUSION: EpCAM-HCC score could replace AFP during screening of HCV patients and early detection of HCC.

A novel conditional active biologic anti-EpCAM x anti-CD3 bispecific antibody with synergistic tumor selectivity for cancer immunotherapy.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that plays several roles in cancer biology. EpCAM is an attractive therapeutic target because of its expression in most solid tumors. However, targeting EpCAM has been challenging because it is also highly expressed in normal epithelial tissues. Initial attempts to develop EpCAM-specific T-cell engagers were unsuccessful due to severe cytokine release effects, as well as serious on-target, off-tumor drug-related toxicities. We developed novel, conditionally active biological (CAB) bispecific antibodies that bind to both EpCAM and CD3 in an acidic tumor microenvironment. In healthy tissues, binding to EpCAM and CD3 is greatly reduced by a novel, dual CAB selection, where each binding domain is independently blocked by the presence of physiological chemicals known as Protein-associated Chemical Switches (PaCS). The CAB anti-EpCAM T-cell engagers displayed the anticipated bispecific binding properties and mediated the potent lysis of EpCAM-positive cancer cell lines through the recruitment of T cells in the tumor microenvironment. Xenograft studies showed that the efficacy of CAB bispecific antibodies is similar to that of a non-CAB anti-EpCAM bispecific antibody, but they have markedly reduced toxicity in non-human primates, indicating an unprecedentedly widened therapeutic index of over 100-fold. These preclinical results indicate that the dual CAB bispecific antibody is potentially both a powerful and safe therapeutic platform and a promising T cell-engaging treatment for patients with EpCAM-expressing tumors.

Development of a novel conditionally active EpCAM-specific T-cell engager with enhanced safety and tolerability for treatment of solid tumors.

Clinical features and impact of p53 status on sporadic mismatch repair deficiency and Lynch syndrome in uterine cancer.

The clinical features of sporadic mismatch repair deficiency (MMRd) and Lynch syndrome (LS) in Japanese patients with endometrial cancer (EC) were examined by evaluating the prevalence and prognostic factors of LS and sporadic MMRd in patients with EC. Targeted sequencing of five LS susceptibility genes (MLH1, MSH2, MSH6, PMS2, and EPCAM) was carried out in 443 patients with EC who were pathologically diagnosed with EC at the National Cancer Center Hospital between 2011 and 2018. Pathogenic variants in these genes were detected in 16 patients (3.7%). Immunohistochemistry for MMR proteins was undertaken in 337 of the 433 (77.9%) EC patients, and 91 patients (27.0%) showed absent expression of at least one MMR protein. The 13 cases of LS with MMR protein loss (93.8%) showed a favorable prognosis with a 5-year overall survival (OS) rate of 100%, although there was no statistically significant difference between this group and the sporadic MMRd group (p = 0.27). In the MMRd without LS group, the 5-year OS rate was significantly worse in seven patients with an aberrant p53 expression pattern than in those with p53 WT (53.6% vs. 93.9%, log-rank test; p = 0.0016). These results suggest that p53 abnormalities and pathogenic germline variants in MMR genes could be potential biomarkers for the molecular classification of EC with MMRd.

Interplay in galectin expression predicts patient outcomes in a spatially restricted manner in PDAC.

BACKGROUND: Galectins (Gal's) are a family of carbohydrate-binding proteins that are known to support the tumour microenvironment through their immunosuppressive activity and ability to promote metastasis. As such they are attractive therapeutic targets, but little is known about the cellular expression pattern of galectins within the tumour and its neighbouring stromal microenvironment. Here we investigated the cellular expression pattern of Gals within pancreatic ductal adenocarcinoma (PDAC). METHODS: Galectin gene and protein expression were analysed by scRNAseq (n=4) and immunofluorescence imaging (n=19) in fibroblasts and epithelial cells of pancreatic biopsies from PDAC patients. Galectin surface expression was also assessed on tumour adjacent normal fibroblasts and cancer associated primary fibroblasts from PDAC biopsies using flow cytometry. RESULTS: scRNAseq revealed higher Gal-1 expression in fibroblasts and higher Gal-3 and -4 expression in epithelial cells. Both podoplanin (PDPN+, stromal/fibroblast) cells and EpCAM+ epithelial cells expressed Gal-1 protein, with highest expression seen in the stromal compartment. By contrast, significantly more Gal-3 and -4 protein was expressed in ductal cells expressing either EpCAM or PDPN, when compared to the stroma. Ductal Gal-4 cellular expression negatively correlated with ductal Gal-1, but not Gal-3 expression. Higher ductal cellular expression of Gal-1 correlated with smaller tumour size and better patient survival. CONCLUSIONS: In summary, the intricate interplay and cell-specific expression patterns of galectins within the PDAC tissue, particularly the inverse correlation between Gal-1 and Gal-4 in ducts and its significant association with patient survival, highlights the complex molecular landscape underlying PDAC and provides valuable insights for future therapeutic interventions.

Detection of effusion tumor cells under different storage and processing conditions.

BACKGROUND: Circulating tumor cells (CTCs) shed into blood provide prognostic and/or predictive information. Previously, the authors established an assay to detect carcinoma cells from pleural fluid, termed effusion tumor cells (ETCs), by employing an immunofluorescence-based CTC-identification platform (RareCyte) on air-dried unstained ThinPrep (TP) slides. To facilitate clinical integration, they evaluated different slide processing and storage conditions, hypothesizing that alternative comparable conditions for ETC detection exist. METHODS: The authors enumerated ETCs on RareCyte, using morphology and mean fluorescence intensity (MFI) cutoffs of >100 arbitrary units (a.u.) for epithelial cellular adhesion molecule (EpCAM) and <100 a.u. for CD45. They analyzed malignant pleural fluid from three patients under seven processing and/or staining conditions, three patients after short-term storage under three conditions, and seven samples following long-term storage at -80°C. MFI values of 4',6-diamidino-2-phenylindol, cytokeratin, CD45, and EpCAM were compared. RESULTS: ETCs were detected in all conditions. Among the different processing conditions tested, the ethanol-fixed, unstained TP was most similar to the previously established air-dried, unstained TP protocol. All smears and Pap-stained TPs had significantly different marker MFIs from the established condition. After short-term storage, the established condition showed comparable results, but ethanol-fixed and Pap-stained slides showed significant differences. ETCs were detectable after long-term storage at -80°C in comparable numbers to freshly prepared slides, but most marker MFIs were significantly different. CONCLUSIONS: It is possible to detect ETCs under different processing and storage conditions, lending promise to the application of this method in broader settings. Because of decreased immunofluorescence-signature distinctions between cells, morphology may need to play a larger role.

Continuous genetic monitoring of transient mesenchymal gene activities in distal tubule and collecting duct epithelial cells during renal fibrosis.

Epithelial cells (ECs) have been proposed to contribute to myofibroblasts or fibroblasts through epithelial-mesenchymal transition (EMT) during renal fibrosis. However, since EMT may occur dynamically, transiently, and reversibly during kidney fibrosis, conventional lineage tracing based on Cre-loxP recombination in renal ECs could hardly capture the transient EMT activity, yielding inconsistent results. Moreover, previous EMT research has primarily focused on renal proximal tubule ECs, with few reports of distal tubules and collecting ducts. Here, we generated dual recombinases-mediated genetic lineage tracing systems for continuous monitoring of transient mesenchymal gene expression in E-cadherin+ and EpCAM+ ECs of distal tubules and collecting ducts during renal fibrosis. Activation of key EMT-inducing transcription factor (EMT-TF) Zeb1 and mesenchymal markers αSMA, vimentin, and N-cadherin, were investigated following unilateral ureteral obstruction (UUO). Our data revealed that E-cadherin+ and EpCAM+ ECs did not transdifferentiate into myofibroblasts, nor transiently expressed these mesenchymal genes during renal fibrosis. In contrast, in vitro a large amount of cultured renal ECs upregulated mesenchymal genes in response to TGF-ß, a major inducer of EMT.

TLR4, IgA and EpCAM Expression in Colorectal Cancer and Their Possible Association with Microbiota as a Pathogenic Factor; An Immunohistochemical and Genetic Study.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC) is thought to be related to immune response against gut microbiota. TLR4, IgA, and EpCAM have a role in intestinal local immune response and their altered expression related to both IBD and CRC. Lipopolysaccharide (LPS) is the main activator of TLR4. The objective of this study is to evaluate the possible role of intestinal microbiota in the pathogenesis of IBD and CRC through expression of TLR4, IgA and EpCAM. METHODS: One hundred five cases were divided into (Group 1/ Control: 10 sections of normal colonic mucosa, Group 2/CRC: 51 cases, Group 3/IBD: 44 cases). Immunohistochemistry for TLR4, IgA, and EpCAM was done. LPS was assessed in all groups. TLR4 gene and protein expression were assessed in colorectal cancer cell line by RT-PCR and immunocytochemistry. RESULTS: There was a significant correlation between TLR4 and tumor grade (P value 0.003 and 0.01 respectively). A significant correlation was found between IgA expression and T stage (P value 0.02) and between EpCAM expression and histologic type (P value 0.02). In comparison of CRC patients to controls; there was a statistically significant different expression of TLR4 positivity, IgA positivity and EpCAM (P value <0.001, 0.004, <0.001 respectively). Patients with CRC were compared to colitis patients and there was a statistically significant different expression of IgA positivity and EpCAM expression (P value <0.001). There was significant higher expression of TLR4 in CRC cell line than the fibroblast by both PCR and immunocytochemistry (P-value: 0.003 and 0.024 respectively). LPS level in CRC patients was significantly higher than the control and IBD groups (P values <0.001 and <0.001 respectively). CONCLUSION: TLR4, IgA, EpCAM expression in both CRC and IBD might be related to the pathogenic role of microbiota and could represent potential prevention modalities and therapeutic targets.

Durvalumab with or without tremelimumab plus chemotherapy in HRR non-mutated, platinum-resistant ovarian cancer (KGOG 3045): A phase II umbrella trial.

AIM: We investigated the efficacy and safety of durvalumab (D) with or without tremelimumab (T) in addition to single-agent chemotherapy (CT) in patients with platinum-resistant recurrent ovarian cancer (PROC) lacking homologous recombination repair (HRR) gene mutations. PATIENTS AND METHODS: KGOG 3045 was an open-label, investigator-initiated phase II umbrella trial. Patients with PROC without HRR gene mutations who had received ≥2 prior lines of therapy were enrolled. Patients with high PD-L1 expression (TPS ≥25%) were assigned to arm A (D + CT), whereas those with low PD-L1 expression were assigned to arm B (D + T75 + CT). After completing arm B recruitment, patients were sequentially assigned to arms C (D + T300 + CT) and D (D + CT). RESULTS: Overall, 58 patients were enrolled (5, 18, 17, and 18 patients in arms A, B, C, and D, respectively). The objective response rates were 20.0, 33.3, 29.4, and 22.2%, respectively. Grade 3-4 treatment-related adverse events were observed in 20.0, 66.7, 47.1, and 66.7 of patients, respectively, but were effectively managed. Multivariable analysis demonstrated that adding T to D + CT improved progression-free survival (adjusted HR, 0.435; 95% CI, 0.229-0.824; P = 0.011). Favorable response to chemoimmunotherapy was associated with MUC16 mutation (P = 0.0214), high EPCAM expression (P = 0.020), high matrix remodeling gene signature score (P = 0.017), and low FOXP3 expression (P = 0.047). Patients showing favorable responses to D + T + CT exhibited significantly higher EPCAM expression levels (P = 0.008) and matrix remodeling gene signature scores (P = 0.031) than those receiving D + CT. CONCLUSIONS: Dual immunotherapy with chemotherapy showed acceptable response rates and tolerable safety in HRR non-mutated PROC, warranting continued clinical investigation.

Immune evasion in lung metastasis of leiomyosarcoma: upregulation of EPCAM inhibits CD8+ T cell infiltration.

BACKGROUND: Leiomyosarcomas are among the most common histological types of soft tissue sarcoma (STS), with no effective treatment available for advanced patients. Lung metastasis, the most common site of distant metastasis, is the primary prognostic factor. We analysed the immune environment targeting lung metastasis of STS to explore new targets for immunotherapy. METHODS: We analysed the immune environment of primary and lung metastases in 38 patients with STS using immunohistochemistry. Next, we performed gene expression analyses on primary and lung metastatic tissues from six patients with leiomyosarcoma. Using human leiomyosarcoma cell lines, the effects of the identified genes on immune cells were assessed in vitro. RESULTS: Immunohistochemistry showed a significant decrease in CD8+ cells in the lung metastases of leiomyosarcoma. Among the genes upregulated in lung metastases, epithelial cellular adhesion molecule (EPCAM) showed the strongest negative correlation with the number of CD8+ cells. Transwell assay results showed that the migration of CD8+ T cells was significantly increased in the conditioned media obtained after inhibition or knock down of EPCAM. CONCLUSIONS: EPCAM was upregulated in lung metastases of leiomyosarcoma, suggesting inhibition of CD8+ T cell migration. Our findings suggest that EPCAM could serve as a potential novel therapeutic target for leiomyosarcoma.

c-MET-positive circulating tumor cells and cell-free DNA as independent prognostic factors in hormone receptor-positive/HER2-negative metastatic breast cancer.

BACKGROUND: Endocrine therapy resistance in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) is a significant clinical challenge that poses several unmet needs in the management of the disease. This study aimed to investigate the prognostic value of c-MET-positive circulating tumor cells (cMET+ CTCs), ESR1/PIK3CA mutations, and cell-free DNA (cfDNA) concentrations in patients with hormone receptor-positive (HR+) metastatic breast cancer (mBC). METHODS: Ninety-seven patients with HR+ mBC were prospectively enrolled during standard treatment at Samsung Medical Center. CTCs were isolated from blood using GenoCTC® and EpCAM or c-MET CTC isolation kits. PIK3CA and ESR1 hotspot mutations were analyzed using droplet digital PCR. CfDNA concentrations were calculated using internal control copies from the ESR1 mutation test. Immunocytochemistry was performed to compare c-MET overexpression between primary and metastatic sites. RESULTS: The proportion of c-MET overexpression was significantly higher in metastatic sites than in primary sites (p = 0.00002). Survival analysis showed that c-MET+ CTC, cfDNA concentration, and ESR1 mutations were significantly associated with poor prognosis (p = 0.0026, 0.0021, and 0.0064, respectively) in HR+/HER2- mBC. By contrast, EpCAM-positive CTC (EpCAM+ CTC) and PIK3CA mutations were not associated with progression-free survival (PFS) in HR+/HER2- mBC. Multivariate analyses revealed that c-MET+ CTCs and cfDNA concentration were independent predictors of PFS in HR+/HER2- mBC. CONCLUSIONS: Monitoring c-MET+ CTC, rather than assessing c-MET expression in the primary BC site, could provide valuable information for predicting disease progression, as c-MET expression can change during treatment. The c-MET+ CTC count and cfDNA concentration could provide complementary information on disease progression in HR+ /HER2- mBC, highlighting the importance of integrated liquid biopsy.

Mesothelial cells with mesenchymal features enhance peritoneal dissemination by forming a protumorigenic microenvironment.

Malignant ascites accompanied by peritoneal dissemination contain various factors and cell populations as well as cancer cells; however, how the tumor microenvironment is shaped in ascites remains unclear. Single-cell proteomic profiling and a comprehensive proteomic analysis are conducted to comprehensively characterize malignant ascites. Here, we find defects in immune effectors along with immunosuppressive cell accumulation in ascites of patients with gastric cancer (GC) and identify five distinct subpopulations of CD45(-)/EpCAM(-) cells. Mesothelial cells with mesenchymal features in CD45(-)/EpCAM(-) cells are the predominant source of chemokines involved in immunosuppressive myeloid cell (IMC) recruitment. Moreover, mesothelial-mesenchymal transition (MMT)-induced mesothelial cells strongly express extracellular matrix (ECM)-related genes, including tenascin-C (TNC), enhancing metastatic colonization. These findings highlight the definite roles of the mesenchymal cell population in the development of a protumorigenic microenvironment to promote peritoneal dissemination.

Tmprss2 maintains epithelial barrier integrity and transepithelial sodium transport.

The mouse cortical collecting duct cell line presents a tight epithelium with regulated ion and water transport. The epithelial sodium channel (ENaC) is localized in the apical membrane and constitutes the rate-limiting step for sodium entry, thereby enabling transepithelial transport of sodium ions. The membrane-bound serine protease Tmprss2 is co-expressed with the alpha subunit of ENaC. αENaC gene expression followed the Tmprss2 expression, and the absence of Tmprss2 resulted not only in down-regulation of αENaC gene and protein expression but also in abolished transepithelial sodium transport. In addition, RNA-sequencing analyses unveiled drastic down-regulation of the membrane-bound protease CAP3/St14, the epithelial adhesion molecule EpCAM, and the tight junction proteins claudin-7 and claudin-3 as also confirmed by immunohistochemistry. In summary, our data clearly demonstrate a dual role of Tmprss2 in maintaining not only ENaC-mediated transepithelial but also EpCAM/claudin-7-mediated paracellular barrier; the tight epithelium of the mouse renal mCCD cells becomes leaky. Our working model proposes that Tmprss2 acts via CAP3/St14 on EpCAM/claudin-7 tight junction complexes and through regulating transcription of αENaC on ENaC-mediated sodium transport.

Notch2 with retinoic acid license IL-23 expression by intestinal EpCAM+ DCIR2+ cDC2s in mice.

Despite the importance of IL-23 in mucosal host defense and disease pathogenesis, the mechanisms regulating the development of IL-23-producing mononuclear phagocytes remain poorly understood. Here, we employed an Il23aVenus reporter strain to investigate the developmental identity and functional regulation of IL-23-producing cells. We showed that flagellin stimulation or Citrobacter rodentium infection led to robust induction of IL-23-producing EpCAM+ DCIR2+ CD103- cDC2s, termed cDCIL23, which was confined to gut-associated lymphoid tissues, including the mesenteric lymph nodes, cryptopatches, and isolated lymphoid follicles. Furthermore, we demonstrated that Notch2 signaling was crucial for the development of EpCAM+ DCIR2+ cDC2s, and the combination of Notch2 signaling with retinoic acid signaling controlled their terminal differentiation into cDCIL23, supporting a two-step model for the development of gut cDCIL23. Our findings provide fundamental insights into the developmental pathways and cellular dynamics of IL-23-producing cDC2s at steady state and during pathogen infection.