Epithelial Cell Adhesion Molecule Antibody Research Articles

Magnetically-activated, nanostructured cellulose for efficient capture of circulating tumor cells from the blood sample of head and neck cancer patients

In this report, the relative efficiency of cellulose nanocrystals (CNCs) and nanofibers (CNFs) to capture circulating tumor cells (CTCs) from the blood sample of head and neck cancer (HNC) patients was evaluated. Detection and enumeration of CTCs are critical for monitoring cancer progression. Both types of nanostructured cellulose were chemically modified with Epithelial Cell Adhesion Molecule (EpCAM) antibody and iron oxide nanoparticles. The EpCAM antibody facilitated the engagement of CTCs, promoting entrapment within the cellulose cage structure. Iron oxide nanoparticles, on the other hand, rendered the cages activatable via the use of a magnet for the capture and separation of entrapped CTCs. The efficiency of the network structures is shown in head and neck cancer (HNC) patients' blood samples. It was observed that the degree of chemical functionalization of hydroxyl groups located within the CNCs or CNFs with anti-EpCAM determined the efficiency of the system's interaction with CTCs. Further, our result indicated that inflexible scaffolds of nanocrystals interacted more efficiently with CTCs than that of the fibrous CNF scaffolds. Network structures derived from CNCs demonstrated comparable CTC capturing efficiency to commercial standard, OncoDiscover®. The output of the work will provide the chemical design principles of cellulosic materials intended for constructing affordable platforms for monitoring cancer progression in ‘real time’.

Effective Circulating Tumor Cell Isolation Using Epithelial and Mesenchymal Markers in Prostate and Pancreatic Cancer Patients

Circulating tumor cells (CTCs) display antigenic heterogeneity between epithelial and mesenchymal phenotypes. However, most current CTC isolation methods rely on EpCAM (epithelial cell adhesion molecule) antibodies. This study introduces a more efficient CTC isolation technique utilizing both EpCAM and vimentin (mesenchymal cell marker) antibodies, alongside a lateral magnetophoretic microseparator. The effectiveness of this approach was assessed by isolating CTCs from prostate (n = 17) and pancreatic (n = 5) cancer patients using EpCAM alone, vimentin alone, and both antibodies together. Prostate cancer patients showed an average of 13.29, 11.13, and 27.95 CTCs/mL isolated using EpCAM alone, vimentin alone, and both antibodies, respectively. For pancreatic cancer patients, the averages were 1.50, 3.44, and 10.82 CTCs/mL with EpCAM alone, vimentin alone, and both antibodies, respectively. Combining antibodies more than doubled CTC isolation compared to single antibodies. Interestingly, EpCAM antibodies were more effective for localized prostate cancer, while vimentin antibodies excelled in metastatic prostate cancer isolation. Moreover, vimentin antibodies outperformed EpCAM antibodies for all pancreatic cancer patients. These results highlight that using both epithelial and mesenchymal antibodies with the lateral magnetophoretic microseparator significantly enhances CTC isolation efficiency, and that antibody choice may vary depending on cancer type and stage.

Abstract 2131: Immunoaffinity isolation of EpCAM expressing exosomes utilizing high throughput microfluidic chip with IEDDA chemistry (EpCAM-TCOOncoBean Chip)

Abstract The overall objective of this study is to investigate the microfluidic isolation of tumor derived exosome (TDEs) using the OncoBean Chip, functionalized with the unique inverse electron demand Diels-Alder (IEDDA) for downstream analysis of patient TDEs for therapeutic applications. This chemistry allows for capture and subsequent release of exosomes for immunomodulation and functional studies. We developed an approach for targeting TDEs in patient’s blood plasma by using the epithelial cell adhesion molecule (EpCAM) antibody, a highly expressed biomarker associated with non-small cell lung cancer.The OncoBean Chip is a high throughput radial-flow microfluidic device designed for the specific isolation of TDEs. The bean shaped micro-nodes allow for a varying shear profile to better capture nanosized particles by positioning them towards the middle curvature. We optimized the device using exosomes extracted from cell culture media by ultracentrifugation. First, the surface of the device was salinized with (3-Aminopropyl) triethoxysilane, as an anchor, following plasma activation of the surface. Then, bonded with 3,3'-dithiobis(sulfosuccinimidyl propionate) as a crosslinker, to facilitate the release. Finally, trans-cyclooctene (TCO) is attached as the exposed agent for capture of TDEs. The exosome sample is functionalized with an EpCAM antibody (Ab) conjugated to a tetrazine (Tz) molecule via incubation. The anti-EpCAM-Tz conjugate bonds to surface EpCAM on the TDEs creating a TDE-EpCAM-Tz complex for isolation. The TDEs are processed through the functionalized OncoBean Chip for Ab specific isolation. We tested and validated the EpCAM specific isolation and confirmed TDEs were forming complexes with the Ab-Tz conjugates and not arbitrarily sticking to the nodes. This was done by flowing a sample of TDE-Tz through a functionalized device where little to no capture was expected. Then we evaluated the isolation of EpCAM specific TDEs by using H1650 and A549 exosomes and comparing the capture with CD63, a common exosome marker. Limited capture is expected in the A549 group due to low EpCAM expression for the Ab-Tz conjugate to bind too.Additionally, EV isolation was confirmed and quantified by NTA which reveals capture efficiency up to 90% for the H1650 TDEs. Capture efficiency of the no Ab control was found to be at 30% and below while the A549 control, was found to be at about 20% and below. Hence, our OncoBean Chip with IEDDA chemistry has successfully demonstrated efficient and specific isolation of EpCAM and CD63 expressing TDEs from two different cell lines. Data from Western Blot analysis and scanning electron microscopy further confirm the physical characteristics and protein expression of the TDEs established by the International Society of Extracellular Vesicles (ISEV). Citation Format: Nna-Emeka Onukwugha, Sunitha Nagrath, Henry McEacheron. Immunoaffinity isolation of EpCAM expressing exosomes utilizing high throughput microfluidic chip with IEDDA chemistry (EpCAM-TCOOncoBean Chip) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2131.

Surface-Initiated Synthesis of Cell-Specific Glycopolymers Using Live Mammalian Cells as Templates.

Molecular recognition is an important process in life activities where specificity is the key. However, the method to gain specificity are often complex and time-consuming. Herein, a novel, versatile, and effective way is developed to obtain cell-specific glycosurfaces by surface-initiated Cu-mediated reversible deactivation radical polymerization(Cu-RDRP)in an open to air fashion. Mammalian cells are used for the first time as live templates to realize cell-sugar monomer-aptation-polymerization which can produce cell-specific glycosurfaces. Both epithelial cell adhesion molecule (EpCAM) positive cells L929 and EpCAM negative cells Hela as models are used to acquire two cell-specific glycosurfaces, which can distinguish template-cells from others. The strategy is effective and convenient without the need of fixative pretreatment of cells. It is found that the specific capture does not rely on EpCAM antibodies, and the specificity is related to the composition and chain sequence of the glycopolymer brushes rather than surface morphology. In addition, these glycosurfaces keep the ability to identify the target cells after ten regenerative treatments, which provides another advantage for practical applications.

EpCAM expression negatively regulates E-cadherin function in colorectal carcinomas.

This study aimed to characterise epithelial cell adhesion molecule (EpCAM) expression patterns in colorectal carcinomas (CRC) from Nigerian patients, its association with E-cadherin and tumour characteristics, to forecast patient selection for anti-EpCAM therapy among whom no data existed previously. Tissue microarray blocks of formalin-fixed and paraffin-embedded CRC tissues, with their non-cancer margins of resection, were sectioned and stained with EpCAM and E-cadherin primary antibodies. Scoring for antibody staining was done semiquantitatively by combining staining proportion and intensity. The outcome was correlated with patient age, gender and tumour histological parameters with p ≤ 0.05 regarded as statistically significant. Sixty-three carcinoma tissues had staining status for the two markers and were included in this study. Of these, 36 (57.1%) showed positive EpCAM expression (immunoscore ≥3) out of which 83% (30/36 positive cases) were overexpressed (combined immunoscore ≥4) while 12 (19%) tissues were positive for E-cadherin. Non-tumour margins of resection tissues showed less EpCAM positivity in 24% (6/25) of histospots. The difference in staining between tumour and non-tumour margin tissues with EpCAM was significant (p < 0.001). Also, EpCAM overexpression was significantly associated with reduced E-cadherin (p < 0.035) expression in tumour cells. Tumour extent within the gut wall was equal (50% each) for early and late pT stages among EpCAM overexpressing tumours but two-thirds (8/12) of cases expressing E-cadherin had later pT stage paradoxically, while distant metastasis was negligible among tumours bearing both markers. Also, tumours overexpressing EpCAM had significant association with tumour-associated lymphocytes (p < 0.02 each). CRC in this study preferentially overexpress EpCAM over E-cadherin whose strong cell-cell contact inhibitory role is weakened even when expressed, resulting in further local tumour spread. This, and the observed immune response, supports targeted therapy among eligible patients.

Multicoloured flow cytometry to detect metastatic carcinoma in lymph node by epithelial cell adhesion molecule.

The metastatic carcinoma in the lymph node may be missed in routine fine needle aspiration cytology (FNAC). There are limited studies on the role of epithelial cell adhesion molecule (EpCAM) in detecting metastatic carcinoma in the FNAC of lymph nodes by flow cytometry (FCM). To evaluate the role of EpCAM in flow cytometry detecting metastatic carcinoma in the fine needle aspiration cytology (FNAC) of the lymph node. In this prospective study, successive 42 cases of lymph nodes were subjected to FNAC followed by flow cytometry to detect the EpCAM positive cell population. The sample was used for cytology and FCM (CD45, CD14, and EpCAM antibodies tagged with fluorochromes). The percentage of EpCAM positive cell population in each case was calculated and compared in the metastatic carcinomas and reactive lymphoid hyperplasia (RLH) cases. There were 29 cases of metastatic carcinoma and 13 non-neoplastic cases (12 RLH and one granulomatous inflammation). The average percentages of EpCAM in metastatic carcinoma and reactive lymphoid cells were 11.37 and 1.24, respectively. The independent sample t-test showed a significant difference (0.001) in the percentage of EpCAM in the two groups. The cut of value of 3% EpCAM in FCM showed 97% sensitivity and 92% specificity to detect metastatic carcinoma in FNAC of the lymph node. The percentage of EpCAM in FCM may be helpful in detecting metastatic carcinoma in the lymph node. The FCM is a rapid and quantitative test with high sensitivity and specificity.

Abstract 5568: Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12

Abstract Introduction: The application of chimeric antigen receptor (CAR) T cells in solid tumors has met many challenges, arising from the paucity of effective yet safe targets and T cell dysfunction within the immunosuppressive milieu. Clinical trials over the years have reported severe on-target off-tumor toxicities associated with CAR T cell therapies targeting tumor-associated antigens (TAAs). Here, we propose a strategy to target TAAs effectively and safely: 1) improve tumor-targeting specificity by affinity-tuning; 2) armor T cells with antigen-dependent expression of interleukin-12 (IL-12) to compensate loss of CAR T cell activity caused by affinity-tuning. In this system, IL-12 expression is tightly regulated by the nuclear factor of the activated T cells promoter (NFAT) to limit IL-12 to the environ of activated affinity-tuned CAR T cells within the tumor. We demonstrate here the feasibility of this strategy using epithelial cell adhesion molecule (EpCAM) as a model TAA. Methods: Affinity variants were generated by alanine scanning mutagenesis in the complementary-determining region 3 (CDR3) of an EpCAM antibody (UBS54). The affinity of CAR variants was determined by flow cytometry-based saturation binding assay. On-target off-tumor cytotoxicity of CAR T cells was examined against human primary normal epithelial cells. In vivo anti-tumor efficacy was evaluated in mouse models of gastric cancer with cell line-derived (SNU-638 and MKN-45) and patient-derived xenografts. Results: Substitution of alanine into the UBS54 CDR3 led to identification of a tyrosine (6th residue in heavy chain CDR3) as a hot spot for affinity tuning. Among several more subtle amino acid substitutions for Tyr-6, we found that CAR molecule with valine substituent (Y6V) possesses 10 µM affinity toward EpCAM, rendering CAR T cells to be selective to EpCAM-high tumors while being not reactive to primary normal epithelial cells in vitro. In gastric cancer mouse models, compared to UBS54 CAR T cells that mediated rapid tumor remission, Y6V CAR T cells produced mainly partial responses. To revitalize affinity-tuned CAR T cells, we further engineered CAR T cells to release IL-12 under NFAT. We found that both the activity of the NFAT promoter and the level of IL-12 release were tightly regulated by the antigen density of targets when inducible IL-12 is combined with 10 µM affinity Y6V CAR; however, such dependence is lost when 1 µM affinity UBS54 was examined. In mouse models of gastric cancer, inducible lL-12 armored Y6V CAR T cells produced enhanced anti-tumor responses without elevating systemic exposure to IL-12, evidenced by low levels of IL-12 in mouse sera (below 100 pg/ml). Conclusions: Combination of affinity-tuned CAR with inducible expression of IL-12 is a promising strategy to develop CAR T cell therapy against TAAs with the potential for reduced on-target off-tumor toxicity and tumor resistance. Citation Format: Yanping Yang, Huan Yang, Yago Alcaina, Jaclyn E. McCloskey, Janusz Puc, Alyssa Birt, Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5568.

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.

Circulating Tumor Cell Detection in Lung Cancer Animal Model.

BackgroundMetastasis and recurrence of primary cancer are the main causes of cancer mortality. Disseminated tumor cells refer to cancer cells that cause metastasis from primary cancer to other organs. Several recent studies have suggested that circulating tumor cells (CTCs) are associated with the clinical stage, cancer recurrence, cancer metastasis, and prognosis. There are several methods of isolating CTCs from whole blood; in particular, using a membrane filtration system is advantageous due to its cost-effectiveness and availability in clinical settings. In this study, an animal model of lung cancer was established in nude mice using the human large cell lung cancer cell line H460.MethodsSix-week-old nude mice were used. The H460 lung cancer cell line was injected subcutaneously into the nude mice. Blood samples were obtained from the orbital area before cell line injection, 2 weeks after injection, and 2 weeks after tumor excision. Blood samples were filtered using a polycarbonate 12-well Transwell membrane (Corning Inc., Corning, NY, USA). An indirect immunofluorescence assay was performed with the epithelial cell adhesion molecule antibody. The number of stained cells was counted using fluorescence microscopy.ResultsThe average size of the tumor masses was 35.83 mm. The stained cells were counted before inoculation, 2 weeks after inoculation, and 2 weeks after tumor excision. Cancer cells generally increased after inoculation and decreased after tumor resection.ConclusionThe CTC detection method using the commercial polycarbonate 12-well Transwell (Corning Inc.) membrane is advantageous in terms of cost-effectiveness and convenience.

In situ monitoring of circulating tumor cell adhered on three-dimensional graphene/ZnO macroporous structure by resistance change and electrochemical impedance spectroscopy

A three-dimensional (3D) graphene macroporous foam substrate with ZnO nanorod array was developed for specific and non-invasive detection of circulating tumor cells (CTCs). With the help of the epithelial cell adhesion molecule antibody (anti-EpCAM) and the array structure, the EpCAM-positive CTCs can adhere on the detection substrate (rGO-ZnO-antiEpCAM foam). Moreover, the resistance and the electrochemical impedance spectroscopy (EIS) of the rGO-ZnO-antiEpCAM foam was changing during the CTCs adhering process with high sensitivity. Compared to the EpCAM-negative cancer cell, the resistance of the substrate is rising with the amount of the EpCAM-positive CTCs. The possible reason is that the negative charges of CTCs affected the conductivity of the p-type reduced graphene oxide (rGO), and when CTCs were adhering to the rGO-ZnO-antiEpCAM foam, the resistance of the rGO-ZnO-antiEpCAM foam increased. In addition, the EIS measurement also shows the regular changes during the adhering process. From the equivalent circuit modeling of EIS, the charge transfer resistance (Rct) on the surface of the rGO-ZnO-antiEpCAM foam electrode had a linear growth. This may be due to the fixation of negatively charged Breast cancer cells (MCF-7) on the substrate, the charge transfer process was hindered, which caused the semicircle domain of the EIS curve to expand significantly with time. These results reveal that CTCs can not only be specifically adhered on the rGO-ZnO-antiEpCAM foam, but also the electrical microenvironment of the detection substrate was changed during CTCs adhering process. The construction of the detection substrate, the measurement and analysis methods of the electrical signals are of great significance in CTCs acquisition and sensing, which will provide an effective strategy for the real-time monitoring of CTCs at the clinic.

Specific recognition and photothermal release of circulating tumor cells using near-infrared light-responsive 2D MXene nanosheets@hydrogel membranes

2D materials with attractive optical properties are promising for individualized cancer immunotherapy. Isolation, capture, and release of circulating tumor cells (CTCs) are of great significance for promoting the process of early diagnosis of cancers. MXene nanosheets incorporated gelatin hydrogel offers the possibility of achieving near-infrared (NIR) light response to initiate the photothermal effect. Herein, the design and preparation of Ti3C2Tx MXene nanosheets-embedded thermoresponsive gelatin hydrogel membrane with NIR light-responsive for the specific capture and release of CTCs were reported. The membrane was fabricated by casting Ti3C2Tx-embedded gelatin onto a substrate and then modified with epithelial-cell adhesion-molecule antibody (anti-EpCAM) for the specific recognition and separation of CTCs from whole blood. The captured cells can be released without damage with dual-mode containing temperature-responsive release (gelatin deconstructed at 37 °C) and photothermal site-release (Ti3C2Tx induced by NIR light). Furthermore, we were able to achieve an average efficient release rate of 89 % of captured cells with stable cell viability of 87 % via the NIR light irradiation. This work may provide the promising potential for retrieval of single cells in clinical diagnosis.

Using a combination of gangliosides and cell surface vimentin as surface biomarkers for isolating osteosarcoma cells in microfluidic devices

BackgroundOsteosarcoma (OS) is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. Liquid biopsies are an alternative to current diagnostic imaging modalities that can be used to monitor treatment efficacy and the development of metastases. This study addresses the use of novel biomarkers to detect circulating osteosarcoma cells. ProceduresFlow cytometry was used to evaluate the relative expression of epithelial cell adhesion molecule (EpCAM), ganglioside 2 and 3 (GD2/3), and cell surface vimentin (CSV) on a panel of OS cell lines. A microfluidic device was used to affirm the efficacy of GD2/3 and CSV to capture CTCs. Once captured, CTCs on the device are enumerated and the capture efficiency for each marker is measured. Patient samples were captured using the LFAM chip. ResultsWe report the evaluation of GD2, GD3, and CSV as markers for OS cell capture in cell lines and in patient samples. The results of our capture studies correlate with our flow cytometry data and have shown a low capture efficiency of OS cells using EpCAM antibodies, while showing a moderate capture efficiency of OS cells using the GD2, GD3, and CSV antibodies independently. The combination of biomarkers demonstrate a high capture efficiency of approximately 80%. This is further supported by the detection of 1–1.5 CTCs per mL of blood using GD2 + CSV in OS patient samples. ConclusionsThe combination of GD2 + CSV significantly increased the capture efficacy of OS cells. The detection of CTCs through routine blood sampling may be used clinically for earlier detection of metastases and monitoring the therapeutic effect of treatments in metastatic osteosarcomas.

Comparison of circulating tumor cell (CTC) detection rates with epithelial cell adhesion molecule (EpCAM) and cell surface vimentin (CSV) antibodies in different solid tumors: a retrospective study.

PurposeStatus of epithelial-mesenchymal transition (EMT) varies from tumors to tumors. Epithelial cell adhesion molecule (EpCAM) and cell surface vimentin (CSV) are the most common used targets for isolating epithelial and mesenchymal CTCs, respectively. This study aimed to identify a suitable CTC capturing antibody for CTC enrichment in each solid tumor by comparing CTC detection rates with EpCAM and CSV antibodies in different solid tumors.MethodsTreatment-naive patients with confirmed cancer diagnosis and healthy people who have performed CTC detection between April 2017 and May 2018 were included in this study. CTC detection was performed with CytoSorter® CTC system using either EpCAM or CSV antibody. In total, 853 CTC results from 690 cancer patients and 72 healthy people were collected for analysis. The performance of CTC capturing antibody was determined by the CTC detection rate.ResultsEpCAM has the highest CTC detection rate of 84.09% in CRC, followed by BCa (78.32%). CTC detection rates with EpCAM antibody are less than 40% in HCC (25%), PDAC (32.5%) and OC (33.33%). CSV has the highest CTC detection rate of 90% in sarcoma, followed by BC (85.71%), UC (84.62%), OC (83.33%) and BCa (81.82%). CTC detection rates with CSV antibody are over 60% in all 14 solid tumors. Except for CRC, CSV has better performances than EpCAM in most solid tumors regarding the CTC detection rates.ConclusionEpCAM can be used as a target to isolate CTCs in CRC, LC, GC, BCa, EC, HNSCC, CC and PCa, especially in CRC, while CSV can be used in most solid tumors for isolating CTCs.

Detection of Epithelial Cell Adhesion Molecule in Feline Normal and Tumor Cell Lines and Tissues With Selected Commercial Anti-human EpCAM Antibodies.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein expressed at intercellular junctions in epithelial cells. As an epithelial biomarker, it used for immunologic-based capture of epithelial-derived circulating tumor cells (CTCs) in human patients with different carcinomas. EpCAM expression has not been described in normal or neoplastic epithelial tissues in cats. Our goal was to find a commercial antibody that recognizes surface EpCAM expression for CTC detection. We tested two anti-human EpCAM antibodies, designated for use with flow cytometry, for detection of surface EpCAM expression on feline cell lines derived from normal mammary and renal epithelia and mammary and oropharyngeal squamous cell carcinomas in cats. Only one of the antibodies, a goat polyclonal antibody, labeled normal and neoplastic feline mammary epithelial cells and oropharyngeal squamous cell carcinoma cells; no labeling was observed for normal feline kidney epithelial cells. At low dilution, this antibody immunohistochemically stained the intercellular junctions of normal pancreatic, intestinal and mammary epithelium, as well as neoplastic mammary epithelium in feline tissues; however, oral mucosa, skin, and an oropharyngeal squamous cell carcinoma showed no positive immunostaining. The antibody only weakly bound feline squamous cell carcinoma cell lines under static adhesion. Our results indicate that EpCAM is expressed in specific epithelia in cats but is variably expressed in feline mammary tumors and oropharyngeal squamous cell carcinoma. A higher avidity cross-reactive or feline-specific antibody will be required to further investigate EpCAM expression in normal and neoplastic feline tissue or for detecting CTCs in the blood of tumor-bearing cats.

Highly sensitive electrochemical detection of cancer biomarker based on anti-EpCAM conjugated molybdenum disulfide grafted reduced graphene oxide nanohybrid

An ultrasensitive, electrochemical biosensor has been fabricated by utilizing molybdenum disulfide (MoS2) grafted reduced graphene oxide (MoS2@rGO) nanohybrid as a sensing platform. Biomolecular-assisted synthetic method was adopted to synthesize MoS2@rGO nanohybrid, where L-cys was used to reduce GO. The MoS2@rGO nanohybrid exhibits improved electrochemical performance when it has been electrophoretically deposited onto the indium tin oxide (ITO) coated glass substrate. Further, epithelialcell adhesion moleculeantibodies (anti-EpCAM) specific to cancer biomarker has been covalently immobilized on the MoS2@rGO/ITO electrodes for label-free detection of EpCAM. Electrochemical results confirm that anti-EpCAM/MoS2@rGO/ITO based biosensor can detect EpCAM in the concentration range of 0.001–20 ng mL−1 with a detection limit of 44.22 fg mL−1 (S/N = 3). The biosensor's excellent analytical performance has been attributed to the efficient immobilization of EpCAM antibodies on the MoS2@rGO surface, which results in high specificity for EpCAM antigen. The fabricated biosensor showed good selectivity, reproducibility, and stability. The successful detection of EpCAM antigen in spiked samples (human saliva, serum and urine) makes this platform an alternative method for early screening of cancer biomarker.

Application of multicolour flow cytometry in the detection of metastatic carcinoma in serous effusions: Special emphasis in atypical cytology.

We evaluated the role of simultaneous use of multiple antibodies in flow cytometry (FCM) to detect metastatic carcinomas in effusion samples. Cytological examination of 75 successive cases of effusion samples was performed. There were 48 peritoneal, 26 pleural and one pericardial fluid. Multi-coloured FCM examination was undertaken using a cocktail of CD45, CD14 and epithelial cell adhesion molecule (EpCAM), antibodies tagged with different fluorochromes. The percentage of EpCAM positivity was calculated in the CD45 and CD14 dual negative population by selective gating. The EpCAM value was correlated with the cytological findings, follow-up data and MOC-31 immunostaining. There were 20 benign, 35 malignant and 20 atypical cases diagnosed on cytomorphology. The primary sources of carcinomas were mainly from the ovary, followed by lung, gall bladder, intestine and other areas. Out of 20 cytologically benign cases, there were two malignant cases on the final follow-up, and EpCAM on FCM picked up all 18 benign cases and one malignant case. Out of 35 cytologically detected malignant cases, EpCAM picked up 32 malignant cases. The EpCAM detected 15/18 malignant and both benign cases out of 20 cytological atypical cases. EpCAM antibody by FCM showed 87% sensitivity, 100% specificity, 100% positive predictive value and 74% negative predictive value. This comprehensive study highlights the potential use of multi-coloured FCM along with cytological examination to diagnose metastatic carcinoma in effusion samples. Multi-coloured FCM is rapid and quantitative and is helpful in atypical cases.

N-doped carbon dots modified with the epithelial cell adhesion molecule antibody as an imaging agent for HepG2 cells using their ultra-sensitive response to Al3+

Carbon dots (CDs) are emerging as an ideal multifunctional materials due to their ease of preparation and excellent properties in medical imaging technology, environmental monitoring, chemical analysis and other fields. N-doped CDs modified with the epithelial cell adhesion molecule antibody (anti-EpCAM-NCDs) were synthesized in an ingenious and high-output approach. Due to the fluorescence enhancement effect of the introduced N atoms, the obtained anti-EpCAM-NCDs exhibited a strong green emission with an absolute quantum yield of up to 32.5%. Anti-EpCAM-NCDs have immunofluorescent properties and an active targeting function. The fluorescence effect and fluorescence quenching of anti-EpCAM-NCDs are used to image cells and detect Al3+, respectively. Experimental results show that this probe exhibited a wide linear response to Al3+ over a concentration range of 0−100 μM with a detection limit and quantification limit of 3 nM and 6 nM, respectively. Significantly, anti-EpCAM-NCDs, which have negligible cytotoxicity, excellent biocompatibility and high photostability, could be used for the intracellular imaging of HepG2 cells and the detection of Al3+ in environmental and biological samples. As an efficient multifunctional material, anti-EpCAM-NCDs hold great promise for a number of applications in biological systems.

Identification of Tumor Specific Peptide as EpCAM Ligand and Its Potential Diagnostic and Therapeutic Clinical Application.

Tumor targeting agents are being developed for early tumor detection and therapeutics. We previously identified the peptide SNFYMPL (SNF*) and demonstrated its specific binding to human esophageal specimens of high-grade dysplasia (HGD) and adenocarcinoma with imaging ex vivo. Here, we aim to identify the target for this peptide and investigate its potential applications in imaging and drug delivery. With SNF* conjugated affinity chromatography, mass spectrum, Western blot, enzyme-linked immunosorbent assay (ELISA), and molecular docking, we found that the epithelial cell adhesion molecule (EpCAM) was the potential target of SNF*. Next, we showed that FITC-labeled SNF* (SNF*-FITC) colocalized with EpCAM antibody on the surface of esophageal adenocarcinoma cells OE33, and SNF*-FITC binding patterns significantly changed after EpCAM knockdown or exogenous EpCAM transfection. With the data from TCGA, we demonstrated that EpCAM was overexpressed in 17 types of cancers. Using colon and gastric adenocarcinoma cells and tissues as examples, we found that SNF*-FITC bound in a pattern was colocalized with EpCAM antibody, and the SNF* binding did not upregulate the EpCAM downstream Wnt signals. Subsequently, we conjugated SNF* with our previously constructed poly(histidine)-PEG/DSPE copolymer micelles. SNF* labeling significantly improved the micelle binding with colon and gastric adenocarcinoma cells in vitro, and enhanced the antitumor effects and decreased the toxicities of the micelles in vivo. In conclusion, we identified and validated SNF* as a specific peptide for EpCAM. The future potential use of SNF* peptide in multiple tumor surveillance and tumor-targeted therapeutics was demonstrated.

Random and aligned electrospun PLGA nanofibers embedded in microfluidic chips for cancer cell isolation and integration with air foam technology for cell release

BackgroundCirculating tumor cells (CTCs) comprise the high metastatic potential population of cancer cells in the blood circulation of humans; they have become the established biomarkers for cancer diagnosis, individualized cancer therapy, and cancer development. Technologies for the isolation and recovery of CTCs can be powerful cancer diagnostic tools for liquid biopsies, allowing the identification of malignancies and guiding cancer treatments for precision medicine.MethodsWe have used an electrospinning process to prepare poly(lactic-co-glycolic acid) (PLGA) nanofibrous arrays in random or aligned orientations on glass slips. We then fabricated poly(methyl methacrylate) (PMMA)-based microfluidic chips embedding the PLGA nanofiber arrays and modified their surfaces through sequential coating with using biotin–(PEG)7–amine through EDC/NHS activation, streptavidin (SA), and biotinylated epithelial-cell adhesion-molecule antibody (biotin-anti-EpCAM) to achieve highly efficient CTC capture. When combined with an air foam technology that induced a high shear stress and, thereby, nondestructive release of the captured cells from the PLGA surfaces, the proposed device system operated with a high cell recovery rate.ResultsThe morphologies and average diameters of the electrospun PLGA nanofibers were characterized using scanning electron microscopy (SEM) and confocal Raman imaging. The surface chemistry of the PLGA nanofibers conjugated with the biotin–(PEG)7–amine was confirmed through time-of-flight secondary ion mass spectrometry (ToF–SIMS) imaging. The chip system was studied for the effects of the surface modification density of biotin–(PEG)7–amine, the flow rates, and the diameters of the PLGA nanofibers on the capture efficiency of EpCAM-positive HCT116 cells from the spiked liquid samples. To assess their CTC capture efficiencies in whole blood samples, the aligned and random PLGA nanofiber arrays were tested for their abilities to capture HCT116 cells, providing cancer cell capture efficiencies of 66 and 80%, respectively. With the continuous injection of air foam into the microfluidic devices, the cell release efficiency on the aligned PLGA fibers was 74% (recovery rate: 49%), while it was 90% (recovery rate: 73%) on the random PLGA fibers, from tests of 200 spiked cells in 2 mL of whole blood from healthy individuals. Our study suggests that integrated PMMA microfluidic chips embedding random PLGA nanofiber arrays may be suitable devices for the efficient capture and recovery of CTCs from whole blood samples.

Synthesis of saporin-antibody conjugates for targeting EpCAM positive tumour cells.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein involved in cell proliferation and differentiation. Ribosomal inactivating proteins derived from plants specifically target ribosomes and irreversibly inhibit protein synthesis. EpCAM antibody and saporin were conjugated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide chemistry. The mass of the conjugates were characterised using matrix-assisted laser desorption ionisation (MALDI). The saporin-EpCAM (SAP-EpAB) conjugates were tested in-vitro against MCF-7 (breast cancer cells), WERI-Rb1 (retinoblastoma) cells. The flow cytometry and fluorescence microscopy were performed to show the binding efficiency of SAP-EpAB conjugate. Whole transcriptome changes of sap-conjugate treated cells were studied using affymetrix microarrays. MALDI-TOF analysis and polyacrylamide gel electrophoresis confirmed the conjugation of SAP with EpCAM antibody. Flow cytometry and fluorescent microscopy analysis revealed the binding of SAP-EpAB conjugates to the MCF-7, WERI-Rb1 cells. Apoptosis assay by annexin-V has shown an increased apoptotic and necrotic population in conjugate treated cells. MTT assay confirmed the tumour cell death and had shown the IC50 value of 0.8 µg for conjugate in MCF-7 (breast cancer cells), and 1 µg for WERI-Rb1 (retinoblastoma) cells. The microarray analysis revealed downregulation of the tumourigenic genes and upregulation of pro-apoptotic genes leading to apoptosis of tumour cells.