Pharmacological targeting of netrin-1 inhibits EMT in cancer.

Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFß1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.

Early-onset tufting enteropathy in HAI-2-deficient mice is independent of matriptase-mediated cleavage of EpCAM.

Congenital tufting enteropathy (CTE) is a life-threatening intestinal disorder resulting from loss-of-function mutations in EPCAM and SPINT2. Mice deficient in Spint2, encoding the protease inhibitor HAI-2, develop CTE-like intestinal failure associated with a progressive loss of the EpCAM protein, which is caused by unchecked activity of the serine protease matriptase (ST14). Here, we show that loss of HAI-2 leads to increased proteolytic processing of EpCAM. Elimination of the reported matriptase cleavage site strongly suppressed proteolytic processing of EpCAM in vitro and in vivo. Unexpectedly, expression of cleavage-resistant EpCAM failed to prevent intestinal failure and postnatal lethality in Spint2-deficient mice. In addition, genetic inactivation of intestinal matriptase (St14) counteracted the effect of Spint2 deficiency in mice expressing cleavage-resistant EpCAM, indicating that matriptase does not drive intestinal dysfunction by excessive proteolysis of EpCAM. Interestingly, mice expressing cleavage-resistant EpCAM developed late-onset intestinal defects and exhibited a shortened lifespan even in the presence of HAI-2, suggesting that EpCAM cleavage is indispensable for EpCAM function. Our findings provide new insights into the role of EpCAM and the etiology of the enteropathies driven by Spint2 deficiency.

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.

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.

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.

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.

Multilineage murine stem cells generate complex organoids to model distal lung development and disease.

Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three-dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self-organization of FACS-sorted bronchioalveolar stem cells (BASCs) upon co-culture with lung-resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single-cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue-resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial-mesenchymal-myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro.

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.

Ultrasensitive and Wash-Free Detection of Tumor Extracellular Vesicles by Aptamer-Proximity-Ligation-Activated Rolling Circle Amplification Coupled to Single Particle ICP-MS.

Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 105-107 particles/mL, along with a detection limit of 1.1 × 104 particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.

Dual-Recognition-Mediated Autocatalytic Amplification Assay for the Subpopulations of PD-L1 Positive Extracellular Vesicle.

The PD-L1 protein on extracellular vesicles (EVs) is a promising biomarker for tumor immunotherapy. However, PD-L1+ EVs have various cell origins, so further analysis of the subpopulations is essential to help understand better their relationship with tumor immunotherapy. Different from the previous work which focus on the level of total PD-L1+ EVs expression, we, herein, report a dual-recognition mediated autocatalytic amplification (DRMAA) assay to detect the PD-L1 derived from tumors (EpCAM+), immune T cells (CD3+), and total (Lipids) EVs, respectively. The DRMAA assay employed proximity hybridization to construct a complete trigger sequence and then catalyzed the cross-hybridization of three hairpin probes, producing a three-way DNA junction (3-WJ) structure carrying the newly exposed trigger sequence. The 3-WJ complex subsequently initiated an autocatalytic amplification reaction and higher sensitivity than the traditional catalytic hairpin assembly assay was obtained. It was found that the EpCAM+ and PD-L1+ EVs were more effective than others in distinguishing lung cancer patients from healthy people. Surprisingly, the CD3+ and PD-L1+ EVs in lung cancer patients were also upregulated, indicating that immune cell-derived PD-L1+ EVs are also non-negligible marker in a tumor microenvironment. Our results suggested that the DRMAA assay would improve the study of subpopulations of PD-L1+ EVs to provide new insights for cancer immunotherapies.

Multiplex Sensing of Biomarkers on the Cancer Cell Surface by an Epithelial-Mesenchymal Transition (EMT) Sensing Panel Enables Precise Differentiating of Cancer Cells at Various EMT Stages.

Epithelial-mesenchymal transition (EMT) is a complex process that plays a critical role in tumor progression. In this study, we present an EMT sensing panel for the classification of cancer cells at different EMT stages. This sensing panel consists of three types of fluorescent probes based on boronic acid-functionalized carbon-nitride nanosheet (BCN) derivatives. The selective response toward different EMT-associated biomarkers, namely, EpCAM, N-cadherin, and sialic acid (SA), was achieved by conjugating the corresponding antibodies to each BCN derivative, whereas the rare-earth-doping ensures simultaneous sensing of the three biomarkers with fluorescent emission of the three probes at different wavelengths. Sensitive sensing of the three biomarkers was achieved at the protein level with LODs reaching 1.35 ng mL-1 for EpCAM, 1.62 ng mL-1 for N-cadherin, and 1.54 ng mL-1 for SA. The selective response of these biomarkers on the cell surface also facilitated sensitive detection of MCF-7 cells and MDA-MB-231 cells with LODs of 2 cells/mL and 2 cells/mL, respectively. Based on the simultaneous sensing of the three biomarkers on cancer cells that underwent different extents of EMT, precise discrimination and classification of cells at various EMT stages were also achieved with an accuracy of 93.3%. This EMT sensing panel provided a versatile tool for monitoring the EMT evolution process and has the potential to be used for the evaluation of the EMT-targeting therapy and metastasis prediction.

Epcam regulates intrahepatic bile duct reconstruction in zebrafish, providing a potential model for primary cholangitis model.

Epithelial cell adhesion molecules (EpCAMs) have been identified as surface markers of proliferating ductal cells, which are referred to as liver progenitor cells (LPCs), during liver regeneration and correspond to malignancies. These cells can differentiate into hepatocytes and biliary epithelial cells (BECs) in vitro. EpCAM-positive LPCs are involved in liver regeneration following severe liver injury; however, the in vivo function of EpCAMs in the regenerating liver remains unclear. In the present study, we used a zebrafish model of LPC-driven liver regeneration to elucidate the function of EpCAMs in the regenerating liver in vivo. Proliferating ductal cells were observed after severe hepatocyte loss in the zebrafish model. Analyses of the liver size as well as hepatocyte and BEC markers revealed successful conversion of LPCs to hepatocytes and BECs in epcam mutants. Notably, epcam mutants exhibited severe defects in intrahepatic duct maturation and bile acid secretion in regenerating hepatocytes, suggesting that epcam plays a critical role in intrahepatic duct reconstruction during LPC-driven liver regeneration. Our findings provide insights into human diseases involving non-parenchymal cells, such as primary biliary cholangitis, by highlighting the regulatory effect of epcam on intrahepatic duct reconstruction.

Development of Scaffold-Free Three-Dimensional Cholangiocyte Organoids to Study the Progression of Primary Sclerosing Cholangitis.

Organoids are novel in vitro models to study intercellular cross talk between the different types of cells in disease pathophysiology. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), scaffold-free multicellular three-dimensional cholangiocyte organoids (3D-CHOs) were developed using primary liver cells derived from normal subjects and patients with PSC. Human liver samples from healthy donors and patients with PSC were used to isolate primary cholangiocytes [epithelial cell adhesion molecule (EpCam)+/ cytokeratin-19+], liver endothelial cells (CD31+), and hepatic stellate cells (HSCs; CD31-/CD68-/desmin+/vitamin A+). 3D-CHOs were formed using cholangiocytes, HSCs, and liver endothelial cells, and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence, quantitative RT-PCR, and transmission electron microscopy. Transcription profiles for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, and SCTR), fibrosis (ACTA2, COL1A1, DESMIN, and TGFß1), angiogenesis (PECAM, VEGF, CDH5, and vWF), and inflammation (IL-6 and TNF-α) confirmed PSC phenotypes of 3D-CHOs. Because cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal immunofluorescence was used to demonstrate localization of the neurokinin-1 receptor within cytokeratin-19+ cholangiocytes and desmin+ HSCs. Moreover, 3D-CHOs from patients with PSC confirmed PSC phenotypes with up-regulated neurokinin-1 receptor, tachykinin precursor 1, and down-regulated membrane metalloendopeptidase. Scaffold-free multicellular 3D-CHOs showed superiority as an in vitro model in mimicking PSC in vivo phenotypes compared with two-dimensional cell culture, which can be used in PSC disease-related research.

Host protein EPCAM interacting with EtMIC8-EGF is essential for attachment and invasion of Eimeria tenella in chickens.

The five epidermal growth factor-like domains (EGF) of Eimeria tenella microneme protein 8 (EtMIC8) (EtMIC8-EGF) plays a vital role in host cell attachment and invasion. These processes require interactions between parasite proteins and receptors on the surface of host cells. In this study, five chicken membrane proteins potentially interacting with EtMIC8-EGF were identified using the GST pull-down assay and mass spectrometry analysis, and only chicken (Gallus gallus) epithelial cell adhesion molecule (EPCAM) could bind to EtMIC8-EGF. EPCAM-specific antibody and recombinant EPCAM protein (rEPCAM) inhibited the EtMIC8-EGF binding to host cells in a concentration-dependent manner. Furthermore, the rEPCAM protein showed a binding activity to sporozoites in vitro, and a significant reduction of E. tenella invasion in DF-1 cells was further observed after pre-incubation of sporozoites with rEPCAM. The specific anti-EPCAM antibody further significantly decreased weight loss, lesion score and oocyst output during E. tenella infection, displaying partial inhibition of E. tenella infection. These results indicate that chicken EPCAM is an important EtMIC8-interacting host protein involved in E. tenella-host cell adhesion and invasion. The findings will contribute to a better understanding of the role of adhesion-associated microneme proteins in E. tenella.

PARP-1, EpCAM, and FRα as potential targets for intraoperative detection and delineation of endometriosis: a quantitative tissue expression analysis.

BACKGROUND: Endometriosis is a gynecological disease characterized by the presence of endometrial tissue in abnormal locations, leading to severe symptoms, inflammation, pain, organ dysfunction, and infertility. Surgical removal of endometriosis lesions is crucial for improving pain and fertility outcomes, with the goal of complete lesion removal. This study aimed to analyze the location and expression patterns of poly (ADP-ribose) polymerase 1 (PARP-1), epithelial cell adhesion molecule (EpCAM), and folate receptor alpha (FRα) in endometriosis lesions and evaluate their potential for targeted imaging. METHODS: Gene expression analysis was performed using the Turku endometriosis database (EndometDB). By immunohistochemistry, we investigated the presence and distribution of PARP-1, EpCAM, and FRα in endometriosis foci and adjacent tissue. We also applied an ad hoc platform for the analysis of images to perform a quantitative immunolocalization analysis. Double immunofluorescence analysis was carried out for PARP-1 and EpCAM, as well as for PARP-1 and FRα, to explore the expression of these combined markers within endometriosis foci and their potential simultaneous utilization in surgical treatment. RESULTS: Gene expression analysis revealed that PARP-1, EpCAM, and FOLR1 (FRα gene) are more highly expressed in endometriotic lesions than in the peritoneum, which served as the control tissue. The results of the immunohistochemical study revealed a significant increase in the expression levels of all three biomarkers inside the endometriosis foci compared to the adjacent tissues. Additionally, the double immunofluorescence analysis consistently demonstrated the presence of PARP-1 in the nucleus and the expression of EpCAM and FRα in the cell membrane and cytoplasm. CONCLUSION: Overall, these three markers demonstrate significant potential for effective imaging of endometriosis. In particular, the results emphasize the importance of PARP-1 expression as a possible indicator for distinguishing endometriotic lesions from adjacent tissue. PARP-1, as a potential biomarker for endometriosis, offers promising avenues for further investigation in terms of both pathophysiology and diagnostic-therapeutic approaches.

Intracellular domain of epithelial cell adhesion molecule induces Wnt receptor transcription to promote colorectal cancer progression.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) has been widely studied as a tumor antigen due to its expression in varieties of solid tumors. Moreover, the glycoprotein contributes to critical cancer-associated cellular functionalities via its extracellular (EpEX) and intracellular (EpICD) domains. In colorectal cancer (CRC), EpCAM has been implicated in the Wnt signaling pathway, as EpICD and ß-Catenin are coordinately translocated to the nucleus. Once in the nucleus, EpICD transcriptionally regulates EpCAM target genes that; however, remains unclear whether Wnt signaling is modulated by EpICD activity. METHODS: Patient-derived organoids (PDOs), patient-derived xenografts (PDXs), and various CRC cell lines were used to study the roles of EpCAM and EpICD in Wnt receptor expression. Fluorescence and confocal microscopy were used to analyze tumors isolated from PDX and other xenograft models as well as CRC cell lines. EpCAM signaling was intervened with our humanized form of EpCAM neutralizing antibody, hEpAb2-6. Wnt receptor promoters under luciferase reporters were constructed to examine the effects of EpICD. Luciferase reporter assays were performed to evaluate promoter, γ-secretase and Wnt activity. Functional assays including in vivo tumor formation, organoid formation, spheroid and colony formation experiments were performed to study Wnt related phenomena. The therapeutic potential of EpCAM suppression by hEpAb2-6 was evaluated in xenograft and orthotopic models of human CRC. RESULTS: EpICD interacted with the promoters of Wnt receptors (FZD6 and LRP5/6) thus upregulated their transcriptional activity inducing Wnt signaling. Furthermore, activation of Wnt-pathway-associated kinases in the ß-Catenin destruction complex (GSK3ß and CK1) induced γ-secretase activity to augment EpICD shedding, establishing a positive-feedback loop. Our hEpAb2-6 antibody blocked EpICD-mediated upregulation of Wnt receptor expressions and conferred therapeutic benefits in both PDX and orthotopic models of human CRC. CONCLUSIONS: This study uncovers relevant functions of EpCAM where Wnt receptors are upregulated via the transcriptional co-factor activity of EpICD. The resultant enhancement of Wnt signaling induces γ-secretase activity further stimulating EpICD cleavage and its nuclear translocation. Our humanized anti-EpCAM antibody hEpAb2-6 blocks these mechanisms and may thereby provide therapeutic benefit in CRC.

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.

Chimeric antigen receptor-T cells targeting epithelial cell adhesion molecule antigens are effective in the treatment of colorectal cancer.

OBJECTIVE: To construct chimeric antigen receptor (CAR)-T cells targeting epithelial cell adhesion molecule (EpCAM) antigen (anti-EpCAM-CAR-T). METHODS: A third-generation CAR-T cell construct used a single-chain variable fragment derived from monoclonal antibody against human EpCAM. Peripheral blood mononuclear cells were extracted from volunteers. The proportion of cluster of differentiation 8 positive (CD8+) and CD4 + T cells was measured using flow cytometry. Western blot was used to detect the expression of EpCAM-CAR. The killing efficiency was detected using the MTT assay and transwell assay, and the secretion of killer cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected using the ELISA. The inhibitory effect of EpCAM-CAR-T on colorectal cancer in vivo was detected using xenografts. RESULTS: It was found that T cells expanded greatly, and the proportion of CD3+, CD8 + and CD4 + T cells was more than 60%. Furthermore, EpCAM-CAR-T cells had a higher tumour inhibition rate in the EpCAM expression positive group than in the negative group (P < 0.05). The secretion of killer cytokines TNF-α and IFN-γ in the EpCAM expression positive cell group was higher than that in the negative group (P < 0.05). In the experimental group treated with EpCAM-CAR-T cells, the survival rate of nude mice was higher (P < 0.05), and the tumour was smaller than that in the blank and control groups (P < 0.05). The secretion of serum killer cytokines TNF-α and IFN-γ in tumour-bearing nude mice in the experimental group treated with EpCAM-CAR-T cells was higher than that in the blank and control groups (P < 0.05). CONCLUSION: This study successfully constructed EpCAM-CAR cells and found that they can target and recognise EpCAM-positive tumour cells, secrete killer cytokines TNF-α and IFN-γ and better inhibit the growth and metastasis of colorectal cancer in vitro and in vivo than unmodified T cells.

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.

BerEP4-Negative Basal Cell Carcinoma: An Immunophenotype Not to Forget.

ABSTRACT: Basal cell carcinoma (BCC) is the most common cancer worldwide. Although not typically metastatic, BCC can be locally destructive. BerEP4 is an antibody against CD326, an epithelial cell adhesion molecule (EpCAM) that is expressed on epithelial progenitor cells and carcinomas. BerEP4 has been reported to have a 100% positive sensitivity in basal cell carcinomas, but a much lower sensitivity for a variety of other carcinomas, including clear cell renal cell carcinoma and metastatic renal cell carcinoma. A 74-year-old woman presented with a BerEP4-negative, but anti-renal cell antibody-positive BCC, and the stark clinical implications of misdiagnosis. This case stresses the importance of considering BerEP4-negative BCC, even when other abnormal features are present.