Anti-epithelial Cell Adhesion Molecule Antibody Research Articles

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.

Efficient isolation, culture, purification, and stem cell expression profiles of primary tumor cells derived from uterine cervical squamous cell carcinoma.

Since not too many human uterus cervical squamous cell carcinoma (CSCC) cell lines in existence, efficient isolation, culture, and purification protocols for primary CSCC cells were optimized as a tool for the study of uterus CSCC. The protocols for partial multiple enzymatic digestion and explant cell culture were combined and then the resulting mixed cell component cultures were purified by magnetic-activated cell sorting. Colony-forming assay was utilized for detection of cell carcinogenesis potential, and immunofluorescence was used to detect protein expression of CSCC. Finally, flow cytometry (FCM) was performed to analyze cancer stem cells (CSCs) phenotypic markers as well as programmed cell death ligand 1(PD-L 1). Freshly isolated cells containing tumor cells and cancer-associated fibroblasts (CAFs) efficiently proliferate to 85% confluence on a 6cm petri dish in 5-7days. Anti-epithelial cell adhesion molecule antibody (EpCAM) microbeads were used to successfully separate a homogeneous subpopulation of epithelial tumor cells. Both EpCAM+ and EpCAM- cell subpopulations were able to be passaged more than 30 times. Proportions of tumor cell populations expressed CSCs markers such as CD133, CD24, aldehyde dehydrogenase 1 (ALDH1), and CD44. The vimentin+ & EpCAM- population, defined with CAFs, could express CD146 mesenchymal stem cells marker. Meanwhile, PD-L 1 was identified in most subpopulation of CD44+ cells at low passage numbers. Efficient isolation, culture, and purification protocols for primary CSCC cells were successfully built. Additionally, the profiling of CSCs cell markers might provide promising therapeutic targets and clinic strategies.

Self-Propelling Targeted Magneto-Nanobots for Deep Tumor Penetration and pH-Responsive Intracellular Drug Delivery

Self-propelling magnetic nanorobots capable of intrinsic-navigation in biological fluids with enhanced pharmacokinetics and deeper tissue penetration implicates promising strategy in targeted cancer therapy. Here, multi-component magnetic nanobot designed by chemically conjugating magnetic Fe3O4 nanoparticles (NPs), anti-epithelial cell adhesion molecule antibody (anti-EpCAM mAb) to multi-walled carbon nanotubes (CNT) loaded with an anticancer drug, doxorubicin hydrochloride (DOX) is reported. Autonomous propulsion of the nanobots and their external magnetic guidance is enabled by enriching Fe3O4 NPs with dual catalytic-magnetic functionality. The nanobots propel at high velocities even in complex biological fluids. In addition, the nanobots preferably release DOX in the intracellular lysosomal compartment of human colorectal carcinoma (HCT116) cells by the opening of Fe3O4 NP gate. Further, nanobot reduce ex vivo HCT116 tumor spheroids more efficiently than free DOX. The multicomponent nanobot’s design represents a more pronounced method in targeting tumors with self-assisted anticancer drug delivery for ‘far-reaching’ sites in treating cancers.

Size-matching hierarchical micropillar arrays for detecting circulating tumor cells in breast cancer patients' whole blood.

Circulating tumor cells (CTCs) are important markers for cancer diagnosis and treatment, but it is still a challenge to recognize and isolate CTCs because they are very rare in the blood. To selectively recognize CTCs and improve the capture efficiency, micro/nanostructured substrates have been fabricated for this application; however the size of CTCs is often ignored in designing and engineering micro/nanostructured substrates. Herein, a spiky polymer micropillar array is fabricated for capturing CTCs with high efficiency. The surface of the micropillar is cactus-like, and is composed of nanospikes. This hierarchical polymer array is designed according to the size of CTCs, which allows for more interactions of the CTCs with the array by setting the size of gaps among the micropillars to match with the CTCs. This polymer array is created by molding on an ordered silicon array, and then it is coated with an antiepithelial cell adhesion molecule antibody (anti-EpCAM). After co-culture with MCF-7 cells for 45 min, the capture efficiency of this array for CTCs is up to 91% ± 2%. Moreover, the anti-EpCAM modified polymer micropillar arrays present an excellent capacity to isolate CTCs from the whole blood samples of breast cancer patients. This study may provide a new concept for capturing target cells by designing and engineering micro/nanostructured substrates according to the size of target cells.

Antibody Based EpCAM Targeted Therapy of Cancer, Review and Update.

Todays, after four decades from the discovery of monoclonal antibodies by Kohler and Milstein in 1975, a dozen of antibodies are used in cancer targeted therapy with different strategies. The success of these antibodies depends on the specificity of antigens expressed on the cancer cells. Epithelial Cell Adhesion Molecule (EpCAM), a homophilic cell-cell adhesion glycoprotein is a well- known tumor antigen, which expresses on epithelial tumors and circulating tumor cells as well as cancer stem cells. The EpCAM signaling pathway is associated with proliferation, differentiation and adhesion of epithelial cancer cells. Here we review EpCAM structure, expression profile and its signaling pathway in cancer cells. In addition, we focused on structure, mechanism of action and success of anti EpCAM antibodies which have been used in different clinical trials. Based on literatures, Edrecolomab showed limited efficacy in the phase III studies. The wholly human monoclonal antibody Adecatumumab is dose- and target-dependent in metastatic breast cancer patients expressing EpCAM. The chimeric antibody, Catumaxomab, has been approved for the treatment of malignant ascites; however, this Mab showed considerable results in intrapleural administration in cancer patients. Anti EpCAM toxin conjugated antibodies like, Oportuzumab Monatox (scFv antibody and Pseudomonas exotoxin A (ETA)), Citatuzumab Bogatox (Fab fragment with bouganin toxin) and immono-conjugate antibody Tucotuzumab (monoclonal antibody with IL2), have shown acceptable results in different clinical trials. Almost, all of the antibodies were well-tolerated; however, still more clinical trials are needed for the approval of antibodies for the treatment of specific tumors.

Biomimetic Immuno-Magnetosomes for High-Performance Enrichment of Circulating Tumor Cells.

A novel biomimetic immuno-magnetosome (IMS) is developed by coating a leukocyte membrane (decorated with anti-epithelial cell-adhesion molecule antibody) on a magnetic nanocluster. In addition to the good stability and magnetic controllability, the IMS also exhibits satisfactory binding avidity to circulating tumor cells but stealth property to leukocytes. As a result, rare tumor cells can be effectively enriched with undetectable leukocyte background.

Degradable Zinc-Phosphate-Based Hierarchical Nanosubstrates for Capture and Release of Circulating Tumor Cells.

Circulating tumor cells (CTCs) play a significant role in cancer diagnosis and personalized therapy, and it is still a significant challenge to efficiently capture and gently release CTCs from clinical samples for downstream manipulation and molecular analysis. Many CTC devices incorporating various nanostructures have been developed for CTC isolation with sufficient capture efficiency, however, fabricating such nanostructured substrates often requires elaborate design and complicated procedures. Here we fabricate a degradable zinc-phosphate-based hierarchical nanosubstrate (HZnPNS), and we demonstrate its excellent CTC-capture performance along with effective cell-release capability for downstream molecular analysis. This transparent hierarchical architecture prepared by a low-temperature hydrothermal method, enables substantially enhanced capture efficiency and convenient imaging. Biocompatible sodium citrate could rapidly dissolve the architecture at room temperature, allowing that 88 ± 4% of captured cells are gently released with a high viability of 92 ± 1%. Furthermore, antiepithelial cell adhesion molecule antibody functionalized HZnPNS (anti-EpCAM/HZnPNS) was successfully applied to isolate CTCs from whole blood samples of cancer patients, as well as release CTCs for global DNA methylation analysis, indicating it will serve as a simple and reliable alternative platform for CTC detection.

Abstract B130: Correlation of in vitro cytotoxicity and catabolism of antibody-drug conjugates (ADCs) in target cancer cells

Abstract A sensitive and convenient ELISA method has been developed to quantify catabolites generated upon treatment of target cancer cells with an antibody-drug conjugate (ADC). The levels of catabolites generated by different ADCs in target cells were correlated with their in vitro cytotoxic potencies. The ELISA method, exemplified for maytansinoid ADCs, measures catabolite levels as low as 0.03 pmol, requiring only 1 million cells, and is suitable as a high-throughput assay to compare processing efficiency of multiple conjugates with different antibodies or linkers. An anti-epidermal growth factor receptor antibody conjugate, anti-EGFR-CX-DM1, using our maytansinoid payload and a new, triglycyl (CX) peptide linker was highly active in vitro and in vivo against xenografts of the HSC-2 cell line. In contrast, an anti-EGFR-SMCC-DM1 conjugate using the non-cleavable SMCC linker had little activity. The CX-DM1 conjugate generated 1.7-fold greater amount of total maytansinoid catabolite compared to SMCC-DM1 (3.5 and 2.0 pmol per million cells in one day, respectively), with an 11-fold higher level of catabolite in the media for CX-DM1 (2.8 and 0.25 pmol per million cells, respectively), suggesting trapping of SMCC-DM1 catabolite in the lysosomes of this cell line. The carboxylic acid catabolite of CX-DM1 is presumably effluxed as an uncharged molecule out of acidic lysosomes into the cytoplasm, where it binds to and inhibits microtubules, and is also effluxed out of cells into the media. In contrast, the SMCC-DM1 conjugate generates a charged Lys-SMCC-DM1 catabolite that is not effectively transported out of the lysosome. In another cell line, HCC827, where both anti-EGFR CX-DM1 and SMCC-DM1 conjugates were equally cytotoxic, high levels of total catabolites were generated (2.7 and 2.0 pmol/million cells, respectively) and relatively high levels detected in the media (1.7 and 0.9 pmol/million cells, respectively), suggesting efficient processing and catabolite transport for both CX-DM1 and SMCC-DM1 conjugates in this cell line. The activity and processing of maytansinoid conjugates of an anti-epithelial cell adhesion molecule antibody (anti-EpCAM) with a hindered-disulfide linker (SPDB-DM4) and a charged sulfonate-bearing hindered-disulfide linker (sulfo-SPDB-DM4) were compared in a multi-drug resistant (MDR) and a non-MDR cell line. The sulfo-SPDB-DM4 conjugate was cytotoxic against the MDR cell line, HCT-15, while the SPDB-DM4 conjugate was inactive. A greater amount of total catabolite was generated for sulfo-SPDB-DM4 than for SPDB-DM4 (0.7 and 0.4 pmol/million cells, respectively), with lower MDR-mediated efflux for sulfo-SPDB-DM4 compared to SPDB-DM4 (intracellular amounts of 0.5 vs 0.1 pmol/million cells, respectively). In a non-MDR cell line, COLO 205, both sulfo-SPDB-DM4 and SPDB-DM4 conjugates were cytotoxic and generated high levels of total catabolites (2.6 and 2.0 pmol/million cells, respectively) and similar levels in the media (0.6 and 0.8 pmol/million cells, respectively), suggesting efficient catabolism and catabolite transport for both conjugates in this cell line. The ELISA method is a convenient and sensitive tool to measure the intra- and extracellular levels of maytansinoid catabolites generated by processing of ADCs. In several cell lines and ADCs, the method gave good correlation with in vitro cytotoxicity. Citation Format: Rajeeva Singh, Paulin Salomon, Yulius Setiady, Jose Ponte. Correlation of in vitro cytotoxicity and catabolism of antibody-drug conjugates (ADCs) in target cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B130.

Polymeric microfluidic devices exhibiting sufficient capture of cancer cell line for isolation of circulating tumor cells

Here, we developed polymeric microfluidic devices for the isolation of circulating tumor cells. The devices, with more than 30,000 microposts in the channel, were produced successfully by a UV light-curing process lasting 3min. The device surface was coated with anti-epithelial cell adhesion molecule antibody by just contacting the antibody solution, and a flow system including the device was established to send a cell suspension through it. We carried out flow tests for evaluation of the device's ability to capture tumor cells using an esophageal cancer cell line, KYSE220, dispersed in phosphate-buffered saline or mononuclear cell separation from whole blood. After the suspension flowed through the chip, many cells were seen to be captured on the microposts coated with the antibody, whereas there were few cells in the device without the antibody. Owing to the transparency of the device, we could observe the intact and the stained cells captured on the microposts by transmitted light microscopy and phase contrast microscopy, in addition to fluorescent microscopy, which required fluorescence labeling. Cell capture efficiencies (i.e., recovery rates of the flowing cancer cells by capture with the microfluidic device) were measured. The resulting values were 0.88 and 0.95 for cell suspension in phosphate-buffered saline, and 0.85 for the suspension in the mononuclear cell separation, suggesting the sufficiency of this device for the isolation of circulating tumor cells. Therefore, our device may be useful for research and treatments that rely on investigation of circulating tumor cells in the blood of cancer patients.

Selective cell capture and analysis using shallow antibody-coated microchannels

Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath et al., Nature 450, 1235-1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples.

Expected clinical applications of circulating tumor cells in breast cancer

Tumor cell invasion and intravascular filtration lead to the presence of circulating tumor cells (CTCs) in peripheral blood. CTCs have, thus, been counted in patients with cancer to analyze metastatic mechanisms or in the hope of developing clinical applications for diagnosis and therapy; various CTC-related studies have been performed. However, the clinical significance of CTCs remains to be established because of the extremely small number of CTCs in peripheral blood as compared with the number of blood cells. Technical problems (e.g. reproducibility and reliability) in the detection of CTCs also remain to be solved. The use of flow cytometric analysis, which can be performed with tumor-cell markers such as anti-epithelial cell adhesion molecule antibodies and anti-cytokeratin antibodies and non-tumor-cell markers such as anti-CD45 antibodies has enhanced specificity for the detection of tumor cells. The CellSearch System(®) can detect 1 CTC in 7.5 mL of peripheral blood, with high reproducibility. Its detection rate and accuracy for CTCs have been confirmed. In the United States, clinical trials have used this system to detect CTCs in patients with metastatic breast cancer, metastatic colorectal cancer, and metastatic prostate cancer, and CTCs have been confirmed to be a useful prognostic factor. This system was also suggested to be useful for monitoring treatment response in patients with metastatic breast cancer and was approved by the United States Food and Drug Administration in 2004. Measuring CTC counts can facilitate the early prediction of treatment response and thereby avoid unnecessary therapy. CTCs may also be a useful biomarker for molecular targeted agents, enabling the identification of patients most likely to respond to a given treatment and facilitating treatment selection. However, the widespread use of CTC monitoring as a routine examination requires a further improvement in measurement sensitivity, the establishment of criteria for quantitative and qualitative evaluations, and additional clear-cut evidence supporting the clinical significance of CTCs. We expect that CTCs will be established to be a new diagnostic and therapeutic index for breast cancer.

Phase II Study of the Human Anti-Epithelial Cell Adhesion Molecule Antibody Adecatumumab in Prostate Cancer Patients with Increasing Serum Levels of Prostate-Specific Antigen after Radical Prostatectomy

Background: Rising serum levels of prostate-specific antigen (PSA) after radical prostatectomy are indicative of recurrent prostate cancer. This double-blind, placebo-controlled phase II study evaluated the anti-tumour activity of the anti-epithelial cell adhesion molecule (EpCAM) antibody adecatumumab in delaying biochemical disease progression. Patients and Methods: Prostate cancer patients with increasing serum PSA levels following radical prostatectomy were randomized to low- (2 mg/kg) or high-dose adecatumumab (6 mg/kg) or placebo. The primary efficacy endpoint was the mean change from baseline in total serum PSA at week 24. Secondary endpoints included PSA response rate, prolongation of serum PSA doubling time and time to biochemical disease progression. Results: The primary and secondary endpoints of the study were not met in the predefined analyses. In a retrospective analysis of patients with baseline PSA ≤1 ng/ml and a high EpCAM expression, both the mean increase in PSA from baseline to week 24 and the PSA doubling time at week 15 were significantly improved in the high-dose adecatumumab group compared with the placebo group. Most frequent treatment-related clinical adverse events were gastrointestinal (diarrhoea and nausea) or general events (chills), showing a dose dependency but no grade 3/4 intensity in any patient. Conclusion: In men with rising PSA levels after radical prostatectomy and no evidence of clinical relapse, adecatumumab delayed disease progression in a subgroup of patients with baseline PSA levels ≤1 ng/ml and high EpCAM-expressing tumours.