Anti-epithelial Cell Adhesion Molecule Research Articles

MoSe2-based magnetic nanosystem for clinical enrichment, retrieval and elimination of circulating tumor cells

The detection, analysis and clearing of circulating tumor cells (CTCs) from patients’ bloodstream at an early stage are important to diagnose the primary tumor progression and decrease cancer metastasis-related mortality. Herein, we developed a molybdenum diselenide nanosheet (MoSe2 NS) based magnetic nanosystem, which was surface functionalized with the anti-epithelial cell adhesion molecule (anti-EpCAM) antibody. CTCs were successfully isolated by the magnetic nanosystem from blood samples of clinical cancer patients. Highly efficient CTCs enrichment was achieved when the nanosystem was applied with a concentration down to 25 µg ml−1 in both static and biomimetic circulating conditions. The nanosystem was demonstrated biocompatible without obvious cytotoxicity, allowing in vitro culture of the captured cells for downstream analysis. Further, the magnetic nanosystem presented a reinforced photothermal effect due to the integration of several photothermal agents including MoSe2 NS, ferroferric oxide (Fe3O4) nanoparticles, and polydopamine (pDA), allowing fast in-situ elimination of the captured cells under NIR laser (808 nm) irradiation. This work represents a multimode system that enables highly efficient enrichment of rare CTCs from patients’ blood for either retrieval or clearing, with great promise for early cancer diagnosis and possible treatment screening.

Optimizing recombinant antibody fragment production: A comparison of artificial intelligence and statistical modeling.

Maximizing the recombinant protein yield necessitates optimizing the production medium. This can be done using a variety of methods, including the conventional "one-factor-at-a-time" approach and more recent statistical and mathematical methods such as artificial neural network (ANN), genetic algorithm, etc. Every approach has advantages and disadvantages of its own, yet even when a technique has flaws, it is nevertheless used to get the best results. Here, one categorical variable and four numerical parameters, including post-induction time, inducer concentration, post-induction temperature, and pre-induction cell density, were optimized using the 232 experimental assays of the central composite design. The direct and indirect effects of factors on the yield of anti-epithelial cell adhesion molecule extracellular domain fragment antibody were examined using statistical methods. The analysis of variance results indicate that the response surface methodology (RSM) model is effective in predicting the amount of produced single-chain fragment variable (p-value = 0.0001 and R2 = 0.905). For ANN modeling, the evaluation using normalized root mean square error (NRMSE) and R2 values shows a good fit (R2 = 0.942) and accurate predictions (NRMSE = 0.145). The analysis of error parameters and R2 of a dataset, which contained 30 data points randomly selected from the complete dataset, showed that the ANN model had a higher R2 value (0.968) compared to the RSM model (0.932). Furthermore, the ANN model demonstrated stronger predictive ability with a lower NRMSE (0.048vs. 0.064). Induction at the cell density of 0.7 and an isopropyl β-D-1-thiogalactopyranoside concentration of 0.6mM for 32 h at 30°C in BW25113 was the ideal culture condition leading to the protein yield of 259.51mg/L. Under the optimum conditions, the output values predicted by the ANN model (259.83mg/L) were more in line with the experimental data (259.51mg/L) than the RSM (276.13mg/L) expected value. This outcome demonstrated that the ANN model outperforms the RSM in terms of prediction accuracy.

An integrated microfluidic system for automatic detection of cholangiocarcinoma cells from bile.

Cholangiocarcinoma (CCA) is an aggressive cancer that originates from the epithelial cells lining the bile ducts. Due to its location deep within the body and nonspecific symptoms in the early stages, it is often diagnosed at the advanced stage, thus leading to worse prognosis. Circulating tumor cells within liquid biopsies (i.e. blood) have been considered as promising biomarkers for CCA diagnosis, though current methods for profiling them are not satisfactory in terms of sensitivity and specificity. Herein we developed a new cancer cell probing and immuno-tracking assay known as "CAPTURE", which was performed on an integrated microfluidic system (IMS) to automate CCA diagnosis from bile with a sample amount of only 1 mL. The assay utilized magnetic beads surface-coated with two affinity reagents, a nucleic acid aptamer (HN16) and a glycosaminoglycan (SCH 45-mix), for capturing cancer cells in bile; the "gold standard" anti-epithelial cell adhesion molecule was used as a comparison. In a single-blind test of 54 CCA-positive (+) and 102 CCA-negative (-) clinical samples, sensitivities and specificities of 96 and 80%, respectively, were documented with the CAPTURE assay on-bench. An IMS composed of a centrifugal module for sample pretreatment and a CAPTURE module for cell capture and staining was integrated with a new "vertical integration module" for detecting cancer cells from bile without human intervention. Furthermore, a novel micro-tier structure within the centrifugal module was designed to block passage of gallbladder stones with diameters >1 mm, thereby preventing their interference during the subsequent CAPTURE assay. Improved sensitivity and specificity (100 & 83%, respectively) by using three affinity reagents were achieved on the IMS when using 26 clinical bile samples, confirming its clinical bio-applicability for CCA diagnosis. This approach could be therefore used for early-stage CCA diagnostics, ideally enabling effective treatment, as well as reducing potential for relapse.

A multicomponent nanosystem for capturing circulating tumor cells from cancer patients with PD-L1 as an immunotherapy oncotarget.

Capturing circulating tumor cells (CTCs) from the peripheral blood of cancer patients, where they are disseminated among billions of other blood cells, is one of the most daunting challenge. We report OncoDiscover®, a multicomponent nano-system consisting of iron oxide (Fe3O4) nanoparticles (NPs), polyamidoamine generation 4 dendrimers (PAMAM-G4-NH2), graphene oxide (GO) sheets and an anti-epithelial cell adhesion molecule (anti-EpCAM) antibody (Fe-GSH-PAMAM-GO-EpCAM) for the selective and precise capture of CTCs. We further evaluated this system for therapeutically important oncotargets, exemplifying overexpression of the programmed death ligand 1 (PD-L1) as a functional assay on CTCs in cancer patients. We retrospectively evaluated 134 cancer patients for the presence of CTCs and 113 (84%) showed the presence of CTCs. About 62 (55%) of the CTC +ve patients showed CTCs with PD-L1 expression. The personalized cancer detection demonstrated by the OncoDiscover® CTC analysis tool is highly relevant for cancer monitoring and treatment outcomes.

Two Distinct Characteristics of Immune Microenvironment in Human Hepatocellular Carcinoma with Wnt/β-Catenin Mutations

Introduction: Immunotherapy is becoming a promising approach for unresectable-hepatocellular carcinoma (HCC); the anti-tumor response is affected by the tumor microenvironment (TME). Although Wnt/β-catenin mutations are reported to cause non-inflamed phenotype, their role on TME remains controversial. We aimed to clarify the heterogeneity of immunophenotype in HCC with Wnt/β-catenin mutations. Methods: This study includes 152 resected HCCs; mutations in the catenin beta-1, adenomatous polyposis coli, or AXIN1, or AXIN2 genes were defined as Wnt/β-catenin mutations. With hierarchical cluster analyses, TME was classified into inflamed or non-inflamed classes based on the gene expressions associated with T-cell activation. Expression profiles of molecules related to cell differentiation and biliary-stem cell markers were compared between the TME classes to investigate whether differences in tumor traits were associated with TME. Results: Forty of 152 (26.3%) HCCs carried the Wnt/β-catenin mutations. Of these, 33 were classified as non-inflamed (33/40, 82.5%) and 7 as inflamed (7/40, 17.5%). Non-inflamed class was characterized by low number of CD3+, CD4+, and CD8+ cells on immunostaining, and high mRNA expressions of AXIN2 and GLUL, which are involved in the canonical Wnt/β-catenin signaling and hepatocyte differentiation, respectively. Non-inflamed tumors showed higher enhancement on the hepatobiliary-phase of gadolinium-ethoxybenzyl-diethylenetriamine (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) compared to inflamed tumors. HCCs classified as inflamed class are revealed to have high numbers of CD3+, CD4+, and CD8+ tumor infiltrating lymphocytes on immunostaining. This class is associated with increased expression of anti-epithelial cell adhesion molecule and FOXM1 accompanied by upregulation of genes related to interferon-gamma signaling, dendritic cell migration, regulatory T cells, and myeloid-derived suppressor cell activation and recognized as low enhancement nodule on Gd-EOB-DTPA-enhanced MRI. Conclusion: Heterogeneity of tumor traits and TME was observed in HCC with Wnt/β-catenin mutation. The potential was indicated that tumor traits and TME are determined not only by the activation of the HNF4A but also by FOXM1, both of which are downstream transcription factor of the Wnt/β-catenin signaling pathway.

A novel method for detecting circulating tumor cells immunity based on micro-nano technique

This study was to develop a new method for detecting circulating tumor cells (CTCs) with high sensitivity and specificity, therefore to detect the colorectal cancer as early as possible for improving the detection rate of the disease. To this end, we prepared some micro-column structure microchips modified with graphite oxide-streptavidin (GO-SA) on the surface of microchips, further coupled with a broad-spectrum primary antibody (antibody1, Ab1), anti-epithelial cell adhesion molecule (anti-EpCAM) monoclonal antibody to capture CTCs. Besides, carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) were coupled with colorectal cancer related antibody as specific antibody 2 (Ab2) to prepare complex. The sandwich structure consisting of Ab1-CTCs-Ab2 was constructed by the microchip for capturing CTCs. And the electrochemical workstation was used to detect and verify its high sensitivity and specificity. Results showed that the combination of immunosensor and micro-nano technology has greatly improved the detection sensitivity and specificity of the immunosensor. And we also verified the feasibility of the immunosensor for clinical blood sample detection, and successfully recognitized detection and quantization of CTCs in peripheral blood of colorectal cancer patients by this immunosensor. In conclusion, the super sandwich immunosensor based on micro-nano technology provides a new way for the detection of CTCs, which has potential application value in clinical diagnosis and real-time monitoring of disease.

A novel whole blood purifier for efficient capture and separation of circulating tumor cells

Circulating tumor cells (CTCs) as important biomarkers for noninvasive clinical diagnosis and prognostic evaluation are significant in predicting the overall survival and progression-free survival of cancer patients. However, the current typical CTCs separation and enrichment techniques were limited to a single collection of small-volume blood samples, which was inadequate to comprehensively profile the distribution of CTCs in the systemic blood. In addition, those techniques cannot reduce metastasis of CTCs unless adjuvant chemotherapy. Herein, inspired by hemodialysis, we designed a whole blood purifier (WBP) composed of a functionalized special spiral-like glass tube modified by anti-epithelial cell adhesion molecule (anti-EpCAM). The WBP allowed real-time capture, enrichment and removal of CTCs from systemic blood circulation, and the purified blood was immediately returned to the body. Furthermore, the WBP did not cause any organic damages in vivo. This approach achieves the high accuracy of liquid biopsy technology and is expected to become an effective clinical adjuvant therapy for tumor metastasis.

Role of circulating tumour cells (CTCs) in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC).

Liquid biopsy is emerging as a non-invasive tool, providing a personalized snapshot of a primary and metastatic tumour. It aids in detecting early metastasis, recurrence or resistance to the disease. We aimed to assess the role of circulating tumour cells (CTCs) as a predictive biomarker in recurrent/metastatic head and neck cancer (head and neck squamous cell carcinoma (HNSCC)). Thirty-five patients receiving palliative chemotherapy underwent blood sampling [2 mL in Ethylenediaminetetraacetic acid (EDTA) vial] at baseline and at 3 months intervals. The CTCs were isolated and evaluated using anti-epithelial cell adhesion molecule antibody-based enrichment using the OncoDiscover platform. CTCs isolated from 80% of patients (n = 28) showed the sensitivity of cell detection at the baseline and 3 months intervals. The median CTC count was 1/1.5 mL of blood and the concordance with clinic-radiological outcomes was 51.4%. The median CTC count (1 (range:0-4) to 0 (range:0-1)) declined at 3 months in responders, while the non-responders had an increase in levels (0 (range :0-2) to 1 (range :0-3)). Although CTCs positively correlated with progression-free survival (PFS) and overall survival (OS), the association of CTCs did not show a significant difference with these parameters (PFS: 6 months versus 4 months; hazard ratio: 0.68; 95% confidence interval (CI): 0.29-1.58, p = 0.323; OS: 10 months versus 8 months; hazard ratio: 0.54; 95% (CI):0.18-1.57 p = 0.216) between CTC positive and CTC negative patients at 3 months. This study highlights the utility of CTC as a disease progression-monitoring tool in recurrent HNSCC patients. Our findings suggest the potential clinical utility of CTC and the need for exploration in upfront settings of the disease as well (NCT: CTRL/2020/02/023378).

Removal of Circulating Tumor Cells from Blood Samples of Cancer Patients Using Highly Magnetic Nanoparticles: A Translational Research Project.

The count of circulating tumor cells (CTCs) has been associated with a worse prognosis in different types of cancer. Perioperatively, CTCs detach due to mechanical forces. Diagnostic tools exist to detect and isolate CTCs, but no therapeutic technique is currently available to remove CTCs in vivo from unprocessed blood. The aim of this study was to design and test new magnetic nanoparticles to purify whole blood from CTCs. Novel magnetic carbon-coated cobalt (C/Co) nanoparticles conjugated with anti-epithelial cell adhesion molecule (EpCAM) antibodies were synthesized, and their antifouling and separation properties were determined. The newly developed C/Co nanoparticles showed excellent separation and antifouling properties. They efficiently removed tumor cells that were added to healthy subjects’ blood samples, through an anti-EpCAM antibody interaction. The nanoparticles did not interact with other blood components, such as lymphocytes or the coagulation system. In blood samples of carcinoma patients suffering from metastatic disease, on average, ≥68% of CTCs were removed. These nanoparticles could prompt the development of a blood purification technology, such as a dialysis-like device, to perioperatively remove CTCs from the blood of cancer patients in vivo and potentially improve their prognosis.

Polycarboxybetaine-Based Hydrogels for the Capture and Release of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are indicators for the detection, diagnosis, and monitoring of cancers and offer biological information for the development of personalized medicine. Techniques for the specific capture and non-destructive release of CTCs from millions of blood cells remain highly desirable. Here, we present a CTC capture-and-release system using a disulfide-containing poly(carboxybetaine methacrylate) (pCB) hydrogel. The non-fouling characteristic of pCB prevents unwanted, nonspecific cell binding, while the carboxyl functionality of pCB is used for the conjugation of anti-epithelial cell adhesion molecule (anti-EpCAM) antibodies for the capture of CTCs. The results demonstrated that the anti-EpCAM-conjugated pCB hydrogel captured HCT116 cells from blood, and the capture ratio reached 45%. Furthermore, the captured HCT116 cells were released within 30 min from the dissolution of the pCB hydrogel by adding cysteine, which breaks the disulfide bonds of the crosslinkers. The cells released were viable and able to grow. Our system has potential in the development of a device for CTC diagnosis.

A feasibility study of EMF(erlotinib+methotrexate+5-fluorouracil) regimen in recurrent head and neck squamous cell carcinoma (HNSCC) and role of circulating tumour cells(CTCs) in assessment of outcomes.

e18038 Background: Head and neck cancer is a huge burden in South East Asia with frequent relapse after curative therapy while the rest presenting in advanced unresectable stages. Financial constraints for targeted and immunotherapy make it inaccessible for the bulk of population. Thus, low-cost but efficacious regimen is highly implicated. We assessed if readily available triplet therapy of EMF, is superior in terms of extending life and maintaining quality of life along with evaluation of CTCs as a predictive biomarker in such patients. Methods: This was a single arm, phase II, investigator initiated interventional study, wherein 35 patients were enrolled. Platinum resistant/refractory patients of HNSCC were treated with combination of erlotinib 150mg daily, methotrexate 40 mg/m2 and 5-fluourouracil 500 mg/m2 (d1, d8) q28 days till progression or unacceptable toxicities. The primary endpoint was overall response rate(ORR) at 3 months; additional endpoints were disease control rate(DCR) at 3 months, overall survival (OS), progression free survival (PFS) safety and patient reported quality of life(QOL). The role of CTCs in gauging the responders and non-responders was monitored using anti Epithelial Cell Adhesion Molecule antibody based enrichment - OncoDiscover Drug Controller General of India (DCGI) approved platform. Results: The ORR and DCR at 3 months was 45.7% and 68.5%, respectively. The median PFS was 5 months (95%CI: 3.9-6 months) and median OS was 9 months (95%CI:7.4 -10.5 months). The 3 and 6 months PFS rates was 86 + 6% and 45 + 9%, respectively, while OS rates at 3 and 6 months were 91+ 5% and 68+ 8%, respectively. Rash, mucositis and fatigue were common adverse events occurring in 23 (65%), 14 (40%) and 9 (25.7%) respectively. The grade 3 events seen were rash in 5 (14.2%) and diarrhea in 2 (5.7%). Clinically significant improvement was seen in domains of role functioning, social functioning, fatigue, pain and global health status, swallowing, dryness of mouth and feeling ill. The mean CTC count at baseline was 0.90 + 1.1 /1.5ml of blood. Responders showed decline in levels from 1.19 + 0.25 to 0.33 + 0.48, while non-responders had increasing trend: 0.29 + 0.48 to 1+ 0.10 at 3 months (p = 0.010); with concordance rates with response being 52.9%. Additionally, CTC clearance at 3 months had numerically better PFS ̃ 6 months (95% CI: 4.72-7.72) and OS of 10 months (95% CI: 2.3-5.65) vs 4 months (95% CI: 2.3-5.65), p = 0.258 and 8 months (95% CI: 4.3-11.6), p = 0.203 in those with persistence of CTCs. Conclusions: The triplet regimen of EMF is a feasible, safe therapeutic option with favourable response rates and improved QOL in patients with platinum resistant/refractory HNSCC. CTCs have a promising futuristic role as a predictive biomarker and can be extrapolated in clinical upfront setting too. Clinical trial information: CTRI/2020/02/023378.

Isolation and label-free detection of circulating tumour cells by fluidic diffraction chips with a reflective laser beam system

A photonic crystal (PC) based line array of poly(methacrylic acid) (PMAA) brushes was grafted from a photoresist template using a trench array. The array was functionalised with anti-epithelial cell adhesion molecule antibodies (EpY). A laser beam was employed to analyse the two-dimensional (2D) and three-dimensional (3D) reflective signals of PCs at an incidence angle of 45°. The EpY-tailed PMAA PC possessed an optical feature with a characteristic diffraction effect along two laser input configurations including the SII configuration, in which the projection of the laser beam on the plane of the SPM chip was parallel to the strips, and the ST configuration, in which they were perpendicular. A fluidic diffraction chip based on the EpY-tailed PMAA PC with 1-μm resolution was fabricated to examine the ability to detect circulating tumour cells (CTCs) along the ST configuration. The CTCs attached on the EpY-tailed PMAA PC, resulting in the change in the diffraction intensity. Dependence of change degree of the diffraction intensity exhibited a linear range of concentration of CTC from 0 to 64 cells and a limit of detection of 5 cells in 3 mL. CTC detection using both fluidic diffraction chips and a commercial IsoFlux system was carried out in clinical trials, including three healthy donors and 12 patients at various stages of colorectal cancer for comparison. Our platform provides a simple label-free method with high accuracy for rapid CTC counting, which has great potential in clinical treatment applications.

Trispecific T-cell engagers for dual tumor-targeting of colorectal cancer

ABSTRACT Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR−EpCAM−) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity.

Multifunctional Cellular Targeting, Molecular Delivery, and Imaging by Integrated Mesoporous-Silica with Optical Nanocrescent Antenna: MONA.

Multifunctional nanoprobes have attracted significant attention in a wide range of disciplines such as nanomedicine, precision medicine, and cancer diagnosis and treatment. However, integrating multifunctional ability in a nanoscale structure to precisely target, image, and deliver with cellular spatial/temporal resolution is still challenging in cellulo applications. This is because the development of such high-precision resolution needs to be carried out without labeling, photobleaching, and structurally segregating live cells. In this study, we present an integrated nanostructure of a mesoporous-silica nanosphere with an optical nanocrescent antenna (MONA) for multifunctional cellular targeting, drug delivery, and molecular imaging with spatiotemporal resolution. MONA comprises a systematically constructed Au nanocrescent (AuNC) antenna as a nanosensor and optical switch on a mesoporous-silica nanosphere as a cargo to molecular delivery. MONA made of antiepithelial cell adhesion molecules (anti-EpCAM)-conjugated AuNC facilitates the specific targeting of breast cancer cells, resulting in a highly focused photothermal gradient that functions as a molecular emitter. This light-driven molecular, doxorubicin (DOX) delivery function allows rapid apoptosis of breast cancer cells. Since MONA permits the tracking of quantum biological electron-transfer processes, in addition to its role as an on-demand optical switch, it enables the monitoring of the dynamic behavior of cellular cytochrome c pivoting cell apoptosis in response to the DOX delivery. Owing to the integrated functions of molecular actuation and direct sensing at the precisely targeted spot afforded by MONA, we anticipate that this multifunctional optical nanoantenna structure will have an impact in the fields of nanomedicine, cancer theranostics, and basic life sciences.

Antibody-Peptide Epitope Conjugates for Personalized Cancer Therapy.

This study develops a high-throughput preclinical platform to identify patient-specific antibody-peptide epitope conjugates that target cancer cells and demonstrates the potential of this immunotherapy approach for treating ovarian carcinoma.

Esophageal Cancer-Derived Extracellular Vesicle miR-21-5p Contributes to EMT of ESCC Cells by Disorganizing Macrophage Polarization.

Simple SummaryMacrophage polarization-associated extracellular vesicles (EVs) play crucial roles in tumor progression. The role of miR-21-5p in EVs during esophageal squamous cell carcinoma (ESCC) development must be clarified. This study aimed to identify the relationship between ESCC cells and macrophages in different polarization states during the delivery of EVs-miR-21-5p. We found that M0 macrophages took up overexpressed EVs-miR-21-5p secreted by EC109 or EC9706 cells, which transformed them into M2 macrophages through the PTEN/AKT/STAT6 pathway. This, in turn, contributed to secretion of high levels of TGF-β1 by M2 macrophages and promoted esophageal cancer cell epithelial-mesenchymal transition via the TGF-β/Smad2 axis. These findings indicate that EVs-miR-21-5p may be a critical molecule for ESCC.The disorganized polarization of tumor-associated macrophages (TAMs) exerts a critical effect on tumor progression. MicroRNAs (miRNAs) in extracellular vesicles (EVs) secreted from cancer cells may contribute to this process. However, the relationship between TAMs and EVs-miRNAs-mediated regulation in esophageal squamous cell carcinoma (ESCC) remains unclear. In the present study, immunoaffinity magnetic beads combined with antiepithelial cell adhesion molecules (EpCAM) were used to isolate and identify EVs-miR-21-5p from the plasma of ESCC patients. An in vitro coculture system was designed to evaluate the effect of esophageal cancer cells with miR-21-5p overexpression on macrophage polarization. We found that phorbol myristate acetate-induced THP-1 macrophages took up EVs-miR-21-5p from EC109 or EC9706 cells and were transformed into M2 macrophages. This, in turn, contributed to the excessive migration and invasion of esophageal cancer cells. The mechanism underlying these changes may involve activation of M2 macrophages by upregulated ESCC-derived EVs-miR-21-5p through the PTEN/AKT/STAT6 pathway. This may result in esophageal cancer cell epithelial-mesenchymal transition (EMT) via TGF-β/Smad2 signaling. Our results indicate positive feedback between M2 macrophage polarization and EMT of esophageal cancer cells in the tumor microenvironment via shuttling of miR-21-5p in tumor-derived EVs.

Detection of Circulating Tumor Cells in Resectable Pancreatic Ductal Adenocarcinoma: A Prospective Evaluation as a Prognostic Marker.

Circulating tumor cells (CTCs) are useful biomarkers of many solid tumors, but are infrequently detected in early stage pancreatic ductal adenocarcinomas (PDACs). The first drainage of pancreatic venous blood flow come to portal vein and pass through the liver, and they finally go out for peripheral blood. We thought that comparing CTCs from portal vein and peripheral blood could enable us to understand the clinical meaning of CTCs from each different site in PDACs. Therefore, we aimed to determine 1) whether CTCs could be reliably identified in early stages (operable) of PDACs, 2) if there are any differences in the detected number of CTC in portal vein blood and peripheral blood, and 3) whether CTCs can be sensitive biomarkers for the prognosis of resectable PDAC patients. Newly diagnosed PDAC patients who underwent operation with curative intention between 2013 and 2015 were prospectively enrolled. Blood draws from portal and peripheral vein ran through the microfabricated porous filter, and anti-epithelial cell adhesion molecule (EpCAM) and anti-Plectin-1 antibodies were used for CTC identification. Baseline clinical characteristics, tumor characteristics, treatment, and clinical outcomes were assessed. The clinical stages of the 32 enrolled patients were as follows: IA/IB 1 (3.1%); IIA 9 (28.1%); IIB 17 (53.1%); III 5 (15.6%). Twenty-seven patients (84.4%) received R0 resection, while five patients (15.6%) received R1 resection. EpCAM+ CTCs were detected in 20 portal blood (62.5%) and 22 peripheral blood (68.8%). Plectin-1+ CTCs were identified in 14 portal blood (43.8%) and 16 peripheral blood (50%). Plectin-1-expressing CTCs were picked from CTC platform (microfabricated porous filter) and we could find out all KRAS mutation. Patients with detectable EpCAM+ CTC less than one in peripheral blood showed longer overall survival (OS) compared to patients with detectable CTCs more than one (35.5 months vs. 16.0 months). EpCAM and Plectin-1 successfully identified CTCs at the early stage of PDACs. Also, the number of CTCs could be a prognostic marker for survival in resectable PDACs.

Anti-epithelial cell adhesion molecule RNA aptamer-conjugated liposomal doxorubicin as an efficient targeted therapy in mice bearing colon carcinoma tumor model.

To overcome the lack of selectivity and nonspecific biodistribution of drugs in the body, targeted delivery of chemotherapeutic agents with aptamers is a very effective method. In this strategy, aptamers could be specifically identified and attach to targeted molecules on the cancerous cells and deliver the chemotherapeutic agents to desired tissue with minimal or no damage to the normal cells. In this study, we designed anti-epithelial cell adhesion molecule (EpCAM) RNA aptamer conjugated PEGylated liposomal doxorubicin (ER-lip) to investigate its in vitro and in vivo anticancer abilities. Data showed that EpCAM aptamer was able to enhance cell uptake and cytotoxic effects of Dox in C26 cell line. The biodistribution study indicated that ER-lip enhanced the tumor accumulation of Dox compared to Caelyx. Also, double staining of isolated tumor cells with anti-CD44-PE-cy5 and anti-EpCAM Cy-7 antibodies indicated that tumor cells expressed a high level of EpCAM+ CD44+ cells (p≤.001) compared to cultured C26 cell line. in vivo results showed that ER-lip promoted survival and reduced tumor growth rate in animal model compared to Caelyx. In conclusion, these results suggested that the ER-lip could be served as promising formulation for the treatment of cancers with the high expression of EpCAM.

Integrin α6 β4 on Circulating Tumor Cells of Metastatic Breast Cancer Patients.

Background:The detection of circulating tumor cells (CTCs) is prognostic during the disease in women with metastatic breast cancer. Integrins are key role players in nearly every step of cancer progression. In this study, we aimed to analyze integrin alpha6beta4 expression on CTCs isolated from blood samples of patients with advanced breast cancer.Materials and Methods:In this single-center study, peripheral blood samples from 23 breast cancer patients were obtained and analyzed for the presence of CTCs by EasySep™ Direct Human CTC Enrichment Kit combined with subsequent immunocytochemical staining of anti-cytokeratin and anti-epithelial cell adhesion molecules, and β4 integrin on CTCs. Data were correlated with clinicopathological parameters.Results:CTCs were detected in 100% of the patients. The ratio of integrin β4+ CTC was 61% ± 8% of total CTCs. No significant correlation between histopathological parameters and CTC detection was found.Conclusion:Our results demonstrated the importance of α6 β4 integrin expression on CTCs in distant metastasis.

Immunohistochemical study of hepatocyte paraffin 1 antibody, monoclonal antibody 31, and cytokeratin 7 in primary and secondary liver carcinomas

Background Primary hepatic malignant epithelial neoplasia includes several different types of tumors, among which includes cholangiocarcinoma (CC), the second most frequent cancer of the liver after hepatocellular carcinoma (HCC). However, the most common hepatic epithelial malignant neoplasms are metastatic adenocarcinomas (AC). Hepatocyte paraffin 1 antibody (Hep Par 1) is a monoclonal antibody that reacts with normal and neoplastic liver cell. Anti-epithelial cell adhesion molecule monoclonal antibody 31 (MOC-31) is an antibody directed against a cell surface glycoprotein. Cytokeratin 7 (CK7) is expressed in normal and neoplastic epithelia. Aim To study the usefulness of the immunohistochemistry of Hep Par 1, MOC-31, and CK7 in differentiating primary and secondary carcinomas in the liver. Materials and methods The present study included 45 specimens from cases of primary and metastatic liver tumors, including 15 primary HCCs in the liver, four intrahepatic CCs, and 26 metastatic ACs in the liver. They were studied to evaluate Hep Par 1, MOC-31, and CK7 expression. Results The sensitivity of Hep Par 1 for HCC in the study group was 73.3%, whereas its specificity was 100%. The sensitivity of MOC-31 for AC in the study group was 96.4%, whereas its specificity was 91%. The sensitivity of CK7 for CC in the study group was 100%, whereas its specificity was 54%. Conclusion Hep Par 1 has very sensitive and specific immunostaining for HCC, MOC-31 is a useful marker for AC with high sensitivity and specificity, and CK7 is highly sensitive for CC but its specificity is low. A combination of Hep Par 1 and MOC-31 is the most useful combination of two antibodies for the distinction between HCC and AC.