Anti-epithelial Cell Adhesion Molecule Research Articles

Differential expression of MOC-31, Hep Par 1, and N-cadherin in primary carcinoma and metastatic adenocarcinoma in the liver

Background Immunohistochemistry plays a crucial role in the diagnosis of hepatocellular carcinoma (HCC) and in its distinction from other primary and metastatic neoplasms. In this study, we examined the expression of MOC-31 (Anti-epithelial cell adhesion molecule monoclonal antibody, clone number-31), hepatocyte paraffin 1 (Hep Par 1), and N-cadherin in primary carcinoma and metastatic adenocarcinoma (AC) in the liver. Aim The aim of this study was to evaluate the usefulness of MOC-31, Hep Par 1, and N-cadherin in the differential diagnosis of primary carcinoma and metastatic AC in the liver. Materials and methods The present study included 56 specimens from cases of primary and metastatic liver tumors, including 20 primary HCCs in the liver, five intrahepatic cholangiocarcinomas, and 31 metastatic ACs in the liver. They were studied to evaluate MOC-31, Hep Par 1, and N-cadherin expression using immunohistochemistry. Results The sensitivity of MOC-31 for AC in the studied group was 97.2%, whereas its specificity was 90%. The sensitivity of Hep Par 1 for HCC was 75%, whereas its specificity was 100%. The sensitivity of N-cadherin for primary liver carcinoma was 72%, whereas its specificity was 83.9%. Using the combination of the three antibodies, a final diagnosis could be established in 52 of 56 (92.9%) cases of studied group. In conclusion, a panel of these three antibodies can be helpful in the distinction between primary carcinoma and metastatic AC in the liver.

Integrated EpCAM-independent subtraction enrichment and iFISH strategies to detect and classify disseminated and circulating tumors cells.

Application of tumor cell surface adhesion molecule EpCAM-dependent antibody capture, and intracellular cytokeratins (CKs)-dependent immunostaining strategies to detect disseminated or circulating tumor cells (DTCs or CTCs), is limited by highly heterogeneous and dynamic expression or absence of EpCAM and/or CKs in CTCs and DTCs, particularly in their capturing and identifying CTCs/DTCs shed from diverse types of solid tumor, thus being biased and restricted to the only both EpCAM and CK positive cancer cells. Moreover, heterogeneity of chromosome and tumor biomarker of CTCs/DTCs cannot be co-examined by conventional CK/EpCAM-dependent techniques. Accordingly, a novel integrated cellular and molecular approach of EpCAM-independent subtraction enrichment (SE) and immunostaining-FISH (iFISH®) has recently been successfully developed. SE-iFISH® is able to effectively enrich, comprehensively identify and characterize both large and small size non-hematopoietic heteroploid CTCs, DTCs and circulating tumor microemboli in various biofluid specimens of either cancer patients or patient-derived-xenograft mice. Obtained tumor cells, free of anti-EpCAM perturbing and hypotonic damage, are eligible for primary tumor cell culture as well as a series of downstream analyses. Highly heterogeneous CTCs and DTCs could be classified into subtypes by in situ phenotyping protein expression of various tumor biomarkers and karyotyping of chromosome aneuploidy performed by iFISH®. Each CTC subtype may correlate with distinct clinical significance in terms of tumor metastasis, relapse, therapeutic drug sensitivity or resistance, respectively.

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.

Preparation of Homogeneous Nanostructures in 5 Minutes for Cancer Cells Capture

Grafting aptamers on nanostructured substrates has shown ultrasensitivity in isolation of circulating tumor cells (CTCs). Here, we report that over 80 cm2of homogenous nanostructured surface on glass substrates can be prepared in 5 min after one-step dry etching. The surface area was doubled; the average diameter of nanostructures is approximately 374 nm, which is more close to the nanostructures of natural extracellular matrix. Antiepithelial cell adhesion molecule aptamers grafted nanostructured glass substrates captured over 76% of PC3 cells compared to 30% of planar substrates. Bispecific aptamers cofunctionalized nanostructured substrates, however, fail to capture cancer cells probably due to the formation of heterodimers. This limitation reveals that multispecific aptamers, when applied to cell isolation, must be analyzed to exclude any potential formation of heterodimers due to complementary sequence matching.

Abstract 4298: Comparison of dual labeling strategies for NIRF/PET hybrid imaging

Abstract Objectives: Positron Emission Tomography (PET) plays a pivotal role in the surgical care of cancer patients pre- and post-operatively, however, no functional imaging approaches are currently available for real-time surgical guidance. Recently, antibodies dual-labeled with radioactive and near-infrared fluorescent (NIRF) labels have emerged as potential agents to extend whole-body nuclear imaging capabilities into the operating room, but structural limitations in conventional labeling reagents necessitate a random, two-step labeling process which can reduce bioactivity and batch reproducibility. Here, we evaluate the utility of a site-specific multimodal chelator (MMC) that combines PET/NIRF labeling into a single moiety for enhanced production of immunoconjugates for hybrid imaging. Methods: An anti-epithelial cell adhesion molecule (EpCAM)-specific antibody, mAb7, was treated with varying amounts of the reducing agent dithiothreitol (DTT) to partially reduce the interchain disulfides and permit conjugation with MMC-maleimide. The number of MMC moieties per mAb were quantified by isotopic dilution using non-radioactive Cu and immunoreactivity was assessed by ELISA. 64Cu labeling was performed and samples were purified by size exclusion prior to HPLC analysis. Stability of the immunoconjugates was evaluated by HPLC. Multimodal PET/NIRF imaging was performed in an orthotopic prostate tumor model to assess in vivo targeting. Results: mAbs treated with increasing amounts of DTT had higher conjugation ratios, with values ranging from 1-8 MMC moieties per mAb. ELISAs showed no differences in binding potency for the MMC-immunoconjugates. Radiochemical yields were high for all samples (>80%) and increased as a function of conjugation ratio. Interestingly, initial 64Cu labeling of the immunoconjugate with a MMC/mAb ratio of 4 had a corresponding HPLC trace showing >95% radiochemical purity, however, labeling 8 weeks later revealed formation of a second, prominent radioactive peak. Conversely, the HPLC profile of the immunoconjugate with a MMC/mAb ratio of 2 was consistent throughout the study, suggesting a possible correlation between the extent of partial reduction and protein stability. This observation was supported by in vivo imaging findings as lesions were better visualized in mice receiving 64Cu-MMC-mAb7 with a MMC/mAb ratio of 2. Conclusions: The results indicate that site-specific conjugation of MMC is effective for dual labeling antibodies. However, partial reduction conditions must be optimized in order to obtain a multimodal immunoconjugate with suitable imaging properties and stability. Citation Format: Sukhen C. Ghosh, Barrett R. Harvey, Holly Robinson, Kenneth L. Pinkston, Nathaniel Wilganowski, Banghe Zhu, Eva M. Sevick-Muraca, Ali Azhdarinia. Comparison of dual labeling strategies for NIRF/PET hybrid imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4298. doi:10.1158/1538-7445.AM2014-4298

Abstract 4830: Prognostic value of circulating tumor cells with podoplanin expression in locally advanced and metastatic head and neck squamous cell carcinoma

Abstract Podoplanin (PDPN) is a biomarker for poor prognosis in head and neck squamous cell carcinoma (HNSCC). A prospective study was designed to investigate the prognostic value of circulating tumor cells (CTCs) with PDPN expression (PDPN+-CTCs) in HNSCC patients. Sixty-one healthy donors and fifty-three HNSCC patients with locally advanced and metastatic diseases were enrolled in this study. CTCs were enriched from the peripheral blood by the negative selection method PowerMag that has been validated in our previous study. Enumeration of CTCs was performed by co-immunofluorescence staining using the anti-epithelial cell adhesion molecule (EpCAM) and anti-PDPN antibodies. The CTCs (EpCAM+) and PDPN+-CTCs (EpCAM+PDPN+) counts between the patients and healthy volunteers were statistically different (p < 0.0001). When assessed the progression-free survival (PFS) and overall survival (OS) of the patients, we found that the median PFS for the patient group was 8.6 (95% CI: 4.781-12.419) months and the median OS has not reached 50% after the follow-up duration of 9.1 (5.2-17.1) months. Analysis of the CTCs and PDPN+-CTCs counts revealed that neither of them can be considered as a predictive factor for PFS and OS. Notably, the ratio for the PDPN+-CTCs over total CTCs has progonic value in the assessment of PFS and OS. Based on the ROC curve, the prediction of 6-month death could be revealed at the cutoff value of 20% (AUC: 0.718, sensitivity: 94.7%, specificity: 51.7%, p = 0.011). The patients with PDPN+/EpCAM+-CTCs ratio >20% were associated with poor PFS (hazard ratio (HR): 4.666, 95% CI: 1.373-15.856, p = 0.016) and OS (HR: 12.400, 95% CI: 1.643-93.570, p = 0.015). Kaplan-Meier survival curve further confirmed that the PDPN+/EpCAM+-CTCs ratio had significant impact on PFS (p = 0.006) and OS (p = 0.008). In conclusion, CTCs enumeration clearly differentiates HNSCC patients from healthy individuals, while the PDPN+/EpCAM+-CTC ratio is more important than the absolute CTCs count in determining the degree of malignancy and serves as an independent prognostic factor for PFS and OS. Citation Format: Ching-Ping Tseng, Hung-Chih Lin, Chia-Hsun Hsieh, Hung-Ming Wang, Ju-Chien Cheng. Prognostic value of circulating tumor cells with podoplanin expression in locally advanced and metastatic head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4830. doi:10.1158/1538-7445.AM2014-4830

Abstract 4817: Circulating tumor cells enriched by the negative selection system PowerMag is a useful predictor for treatment response of metastatic colorectal cancer patients

Abstract Enumeration of circulating tumor cells (CTCs) provides prognostic and predictive value to guide treatment options. This study aims to (1) evaluate the merits of the PowerMag system, an immunomagnetic separation technique that we recently developed for negative selection of CTCs, in the prediction of treatment response; (2) perform analysis to investigate whether there is a correlation between the imaging response and the changes of CTCs counts before and during chemotherapy for the patients with metastatic colorectal cancer (mCRC). A total of 47 mCRC patients were enrolled in this prospective single-center study. CTCs were isolated from the peripheral blood of these patients by PowerMag at baseline and 2 weeks after starting first dose of chemotherapy. CTCs enumeration was then performed by immunofluorescence staining using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Tumor response at 12 weeks after chemotherapy of the whole cohort was monitored by computed tomography (CT) scans following RECIST criteria 1.0. Our data revealed that PowerMag is a cost-effective negative selection-based CTCs isolation platform with a CTCs detection rate of 89.4% (42/47). The CTCs counts could successfully differ mCRC patients from healthy donors (P = 0.0015). The higher baseline CTCs number was associated with initially metastatic status rather than recurrent settings (P = 0.042). In all assessable patients, a high correlation rate of 86.7% was found between changes of CTCs (decline or elevation) and CT responses (progressive disease, and partial response/stable disease) with a P value of 0.005 (Fisher exact test). These data indicate that the change of CTCs counts before and during treatment is in accord with the image response evaluation at 12 weeks after initiation of treatment. In conclusion, PowerMag is a cost-effective method for negative selection of CTCs from cancer patients. Moreover, comparison of CTCs counts before and during treatment is a useful predictor for disease progression of mCRC patients that warrants further investigation in large-scale validation trial. Citation Format: Jason Chia-Hsun Hsieh, Hung-Chih Lin, Hung-Chih Hsu, John Wen-Cheng Chang, Ching-Ping Tseng. Circulating tumor cells enriched by the negative selection system PowerMag is a useful predictor for treatment response of metastatic colorectal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2014-4817

Abstract 2097: Distinct levels of circulating tumor cell counts for the papillary thyroid cancer patients with distant metastases and in disease-free status

Abstract Distant metastasis of papillary thyroid cancer (PTC) is a multistep and complex process that frequently complicates the disease with poor prognosis. The presence of circulating tumor cells (CTCs) in the peripheral blood has been reported in several cancer types to be highly related to the development of distant metastasis. Nevertheless, the CTCs status during the progression of PTC is rarely investigated. This study aimed to define and compare the CTCs counts among the healthy control individuals (Group I, n = 14), the PTC patients in disease-free status (Group II, n = 9), and the patients with distant metastasis (Group III, n = 8). The patients in group III had metastases to the lung and mediastinum (n = 4), bones (n = 3), and lung and kidney (n = 1), respectively. CTCs were enriched from the peripheral blood by the negative selection method PowerMag that has been validated in our previous study. CTCs enumeration was performed by immunofluorescence staining using the anti-epithelial cell adhesion molecule (EpCAM) and anti-thyroid stimulating hormone receptor (TSHR) antibodies. The EpCAM+ and TSHR+ CTCs counts were 2.97 ± 2.03 and 9.97 ± 9.22 cells/mL for group I, 10.43 ± 9.03 and 19.17 ± 9.77 cells/mL for group II, and 100.06 ± 73.34 and 141.60 ± 82.08 cells/mL for group III individuals, respectively (p < 0.01). These data indicate that (1) some of the TSHR+-CTCs are not EpCAM+ and this type of cell population can be overlooked if CTCs were enriched by the method based on positive selection of EpCAM+ cells; (2) the PTC patients in disease-free status has higher CTCs counts than the healthy control individuals and, hence, continuous CTCs enumeration is recommended for the patient in the stage of disease-free; (3) the PTC patients with distant metastases have higher CTCs counts when compares to the healthy control subjects and the patients with disease-free status. In conclusion, CTCs enumeration is thereby warranted in the course of diagnosis and treatment of thyroid cancer. Citation Format: Hung-Chih Lin, Ju-Chien Cheng, Ching-Ping Tseng, Jen-Der Lin. Distinct levels of circulating tumor cell counts for the papillary thyroid cancer patients with distant metastases and in disease-free status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2097. doi:10.1158/1538-7445.AM2014-2097

Application of an improved magnetic immunosorbent in an Ephesia chip designed for circulating tumor cell capture

In this study, we describe a particular step in developing a microfluidic device for capture and detection of circulating tumor cells-specifically the preparation of an immunosorbent for implementation into the separation chip. We highlight some of the most important specifics connected with superparamegnetic microspheres for microfluidic purposes. Factors such as nonspecific adsorption on microfluidic channels, interactions with model cell lines, and tendency to aggregation were investigated. Poly(glycidyl methacrylate) microspheres with carboxyl groups were employed for this purpose. To address the aforementioned challenges, the microspheres were coated with hydrazide-PEG-hydrazide, and subsequently anti-epithelial cell adhesion molecule (EpCAM) antibody was immobilized. The prepared anti-EpCAM immunosorbent was pretested using model cell lines with differing EpCAM density (MCF7, SKBR3, A549, and Raji) in a batchwise arrangement. Finally, the entire system was implemented and studied in an Ephesia chip and an evaluation was performed by the MCF7 cell line.

Microfluidic immunodetection of cancer cells via site-specific microcontact printing of antibodies on nanoporous surface

We demonstrate an efficient method for cancer cell capture via cell line-specific protein deposition on nanoporous surface in microfluidic channels. Specifically, anti-epithelial cell adhesion molecules (EpCAM) were microcontact printed onto nanoporous silica substrate with optimal pore size of 4nm, porosity of 52.4±0.2%, and thin film thickness of 130±0.5nm. SkBr3, Colo205, and MDA-MB-435 cancer cells suspended in buffer solution were captured on the stamped nanoporous silica substrate. The method demonstrated significantly higher numbers of captured EpCAM-positive cancer cells within anti-EpCAM stamped areas. For site-selective cell capture, grooved microfluidic channels were designed to investigate effects of local confinement due to the laminar flows. Both theoretical modeling and experiments show that the integration of the microfluidic channels greatly enhances cell capture. Patterning of anti-EpCAM in areas of downward flow (optimal regions for cell capture), generated by grooves of the microchannel, enables higher capture numbers than that of stamped areas of upward flow (non-optimal). Fluorescence microscopy images were acquired for captured SkBr3 and Colo205 cells using anti-EpCAM on the nanoporous silica substrate. It was shown that higher numbers of cells were captured across all EpCAM-positive cell lines in optimal areas versus non-optimal areas. Spatial control and large scale patterning of proteins enable novel designs and productions of cost effective, high throughput, and integrated detection and analysis systems. Site-selective detection provides the capability of defining optimal locations for cell capture based on various channel geometries and flow profile. The demonstrated method shows great potential for point-of-care cancer diagnostic tools to quantify the progression and status of the disease.

A bispecific EpCAM/CD133-targeted toxin is effective against carcinoma

The discovery of chemoresistant cancer stem cells (CSCs) in carcinomas has created the need for therapies that specifically target these subpopulations of cells. Here, we characterized a bispecific targeted toxin that is composed of two antibody fragments and a catalytic protein toxin allowing it to bind two CSC markers on the same cell killing this resistant subpopulation. CD133 is a well-known CSC marker and has been successfully targeted and caused regression of head and neck squamous cell carcinoma (HNSCC) in vivo. To enable it to bind a broader range of CSCs, an anti-epithelial cell adhesion molecule (EpCAM) scFv was added to create dEpCAMCD133KDEL, a deimmunized bispecific targeted toxin on a single amino acid chain. This bispecific potently inhibited protein translation and proliferation in vitro in three different types of carcinoma. Furthermore, in a CSC spheroid model dEpCAMCD133KDEL eliminated Mary-X spheroids, an inflammatory breast carcinoma. Finally, this bispecific also caused tumor regression in an in vivo model of HNSCC. This represents the first bispecific CSC-targeted toxin and warrants further development as a possible therapy for carcinoma.

Detection of circulating tumour cells with the CellSearch system in patients with advanced-stage head and neck cancer: preliminary results

To assess the feasibility and clinical value of using the CellSearch system to detect circulating tumour cells in patients with advanced-stage head and neck squamous cell carcinoma. Circulating tumour cells were isolated and counted via positive selection utilising magnetically labelled anti-epithelial cell adhesion molecule and immunocytochemical staining for cytokeratin. The correlation between circulating tumour cell presence and clinical features was evaluated in nine patients newly diagnosed with advanced-stage (stage III or IV) head and neck squamous cell carcinoma. Circulating tumour cells were detected in three of the nine patients (33 per cent). Circulating tumour cell positivity was more prevalent in node stage 2 to 3 patients (3 of 5, 60 per cent) than node stage 0 to 1 patients (0 of 4, 0 per cent). Recurrent or progressive disease was observed in only one of the six patients (17 per cent) without circulating tumour cells, compared with two of the three patients (67 per cent) with circulating tumour cells. In this preliminary study, circulating tumour cells were successfully isolated in patients with advanced-stage head and neck squamous cell carcinoma, using the CellSearch system. Further investigation is needed to evaluate the prognostic significance of circulating tumour cells.

Single-walled carbon nanotubes as specific targeting and Raman spectroscopic agents for detection and discrimination of single human breast cancer cells

Raman active nano-complex agents based on single-walled carbon nanotubes (SWCNTs) are prepared and used for the swift and specific detection of breast cancer cells. SWCNTs are functionalized to bond covalently with the anti-epithelial cell adhesion molecule (anti-EpCAM) antibody, which is specific to the highly expressed EpCAM antigen on the surface of breast cancer cells (MCF-7), but not on normal cells. The Raman nano-complexes demonstrate excellent ability to detect in vitro single breast cancer cells (MCF-7) and discriminate between them and normal fibroblast cells during the first 30 min of the targeting process. Raman linearity scanning is collected from a monolayer cell mixture, including both cancer cells and normal cells incubated with anti-EpCAM-SWCNTs, using a 633-nm laser excitation. The results shows that the Raman signal collected from targeted MCF-7 cells is extremely high, whereas there is little signal from the normal cells.

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.

Abstract 1443: Improved molecular characterization of circulating tumor cells.

Abstract Circulating tumor cells (CTCs) of epithelial origin are present in the blood of carcinoma patients at very low frequency (< 10 / ml blood). In order to characterize CTCs, they need to be enriched from large volumes of blood (7.5 mLs). The CELLSEARCH® Circulating Tumor Cell (Epithelial) assay uses magnetic particles conjugated to anti-epithelial cell adhesion molecule (EpCAM) to selectively capture CTCs. After enrichment, the sample contains CTCs and a number (1000 - 5000) of white blood cells (WBCs). The presence of WBCs in the CTC enriched sample may interfere with the molecular analysis of CTC biomarkers that may be co-expressed by WBCs. We have improved the molecular characterization of CTCs by selectively depleting WBCs following the enrichment of CTCs. Normal donor blood was spiked with tumor cell lines and the CTCs enriched using the CELLSEARCH® Profile Kit. Contaminating WBCs were labeled with antibody to the leukocyte marker CD45 conjugated to fluorescein isothiocyanate (FITC). Samples were then incubated for 1 hour in microtiter plates coated with anti-FITC antibody. The WBCs bound to the anti-FITC on the microtiter plates and the unbound cells, containing the CTCs, were removed with the supernatant fluids. The unbound cells were counted and analyzed by flow cytometry and molecular methods. Flow cytometric results show greater than 85% of the CTCs were recovered in the supernatant fluids, while greater than 80% of WBCs had been depleted; demonstrating a substantial improvement in CTC purity. For molecular analysis, normal donor blood was spiked with a range (0, 10, 50, or 150) of Vertebral-Cancer of the Prostate (VCaP) cells and enriched as above using the CELLSEARCH® Profile Kit. Enriched cells from parallel samples were either lysed immediately for RNA isolation (non-depleted sample) or subjected to the CD45 depletion method (depleted samples) and then lysed for RNA isolation. To measure efficacy of depletion, RNA was also isolated from the cells that adhered to the microtiter plate during depletion (adherent fraction). RT-PCR results of the WBC specific markers PTPRC (CD45) and BST1 in WBC depleted samples show differences in cycle threshold (Δ Ct) values of 2.5 and 2.4 PCR cycles, respectively, compared to non-depleted samples. These measurements correspond to ∼80% reduction in WBC specific RNA load and are consistent with the flow cytometric observations. VCaP cell specific markers TMPRSS2:ERG and AR show Δ Ct values of only 0.04 and 0.02 cycles (or ∼ 3% reduction in VCaP-specific RNA load). This demonstrates that the cell population following WBC removal is significantly depleted of WBC with minimal loss of cancer cells. The method thus has a potential to improve detection of tumor-specific markers in clinical samples enriched for the presence of circulating tumor cells. Citation Format: Chandra Rao, Brad Foulk, Karl Nielsen, Bhavesh Vaidya, Jaymala Patel, Dana Gaffney, Denis Smirnov, Jayaprakash Karkera, Mark Connelly. Improved molecular characterization of circulating tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1443. doi:10.1158/1538-7445.AM2013-1443

Rare cell capture technology for the diagnosis of leptomeningeal metastasis in solid tumors

To evaluate the utility of rare cell capture technology (RCCT) in the diagnosis of leptomeningeal metastasis (LM) from solid tumors through identification of circulating tumor cells (CTCs) in the CSF. In this pilot study, CSF samples from 60 patients were analyzed. The main patient cohort consisted of 51 patients with solid tumors undergoing lumbar puncture for clinical suspicion of LM. Those patients underwent initial MRI evaluation and had CSF analyzed through conventional cytology and for the presence of CTCs using RCCT, based on immunomagnetic platform enrichment utilizing anti-epithelial cell adhesion molecule antibody-covered magnetic nanoparticles. An additional 9 patients with CSF pleocytosis but without solid tumors were separately analyzed to ensure accurate differentiation between CTCs and leukocytes. Among the 51 patients with solid tumors, 15 patients fulfilled criteria for LM. CSF CTCs were found in 16 patients (median 20.7 CTCs/mL, range 0.13 to >150), achieving a sensitivity of 100% as compared with 66.7% for conventional cytology and 73.3% for MRI. One patient had a false-positive CSF CTC result (specificity = 97.2%); however, that patient eventually met LM criteria 6 months after the tap. CSF CTCs were not found in any of the additional 9 patients with CSF pleocytosis. RCCT is an accurate, novel method for the detection of LM in solid tumors, potentially providing earlier diagnostic confirmation and sparing patients from repeat lumbar punctures.

Isolation of Rare Tumor Cells from Blood Cells with Buoyant Immuno-Microbubbles

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.

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.

Regression of liver metastases after treatment with intraperitoneal catumaxomab for malignant ascites due to breast cancer

Malignant ascites is quite rare in breast cancer and is mainly associated with a lobular histology. To date, no studies have evaluated locoregional therapy for malignant ascites in breast cancer. The anti-epithelial cell adhesion molecule (EpCAM), trifunctional antibody catumaxomab was approved in the European Union for the intraperitoneal (i.p.) treatment of malignant ascites in patients with EpCAM-positive carcinomas where standard therapy is not available or no longer feasible. We report the case of a 69-year-old female with pretreated breast cancer who received i.p. catumaxomab for the treatment of malignant ascites and showed a regression of liver metastases. The patient originally underwent a left mastectomy and ipsilateral axillary lymph node dissection for an invasive ductal carcinoma in 1995. Following several lines of treatment, she was enrolled in February 2010 in a phase IIIb study (CASIMAS) investigating the safety of a 3-h i.p. catumaxomab infusion. In addition to a local benefit, as shown by an improvement in malignant ascites and a prolongation of the paracentesis-free interval with i.p. catumaxomab, a computed tomography scan, performed some weeks after catumaxomab administration, showed a regression of liver metastases. In addition to a locoregional effect on EpCAM-positive disease, i.p. catumaxomab may also show systemic effects. The use of i.p. catumaxomab for the treatment of malignant ascites due to breast cancer should be explored further in appropriate clinical studies and its possible systemic effects should also be further investigated.

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes.

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via “wick-in-needle” technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.