Circulating tumor cells in metastatic lung cancer enriched by EpCAM expression and physical characteristics

Introduction: Circulating tumor cells (CTC) measured with the CellSearch system in patients with metastatic carcinomas are associated with poor survival. The frequency of CTC detected by the CellSearch system in non-small cell lung cancer (NSCLC) patients is relatively low, raising the question whether some CTC are not detected by the CellSearch system. To investigate this, additional antibodies were added to broaden the coverage of cytokeratins and leukocytes. Additionally, a device was designed that collects the sample material of the individual samples that are discarded by CellSearch. This collected waste is filtered for CTC isolation based on physical characteristics and the CTC are stained with a cocktail of antibodies. Methods: A device was designed that uses optical sensing to detect the presence of blood in the waste tube of the CellTracks Autoprep. It collects the waste of individual samples in a 50 mL conical tube. After collection the blood is passed with 100 mbar pressure through a 8x8 mm2 microfabricated silicon microsieve containing 300,000 pores of 5 µm in diameter (VyCAP, Deventer, The Netherlands). The performance was tested using four pre-stained cell lines: Colo320 (size 11 µm, ∼1,616 EpCAM antigens), SW480 (size 11 µm, ∼63,233 EpCAM antigens), T24 (size 16 µm, ∼2,167 EpCAM antigens) and SKBR3 (size 16 µm, ∼445,000 EpCAM antigens). Cells are spiked in 7.5 mL of blood collected in CellSave tubes from healthy volunteers. Spiked blood samples from healthy donors and patients with NCSLC and small cell lung cancer (SCLC) (enrollment is ongoing) were processed on the CellSearch and filtration system between 24 and 96 hours of collection. The cells on the microsieves were stained with a nucleic acid dye, antibodies recognizing leukocytes and all cytokeratins. Additional antibodies were added to the CellSearch test to cover all cytokeratins and broaden the coverage of leukocytes. Results: The recovery percentage of the CellSearch system for the different cell lines used was: 2% of COLO320, 91% of SW480, 2% of T24 and 87% of SKBR3. Additional recovery on the microsieves after filtration of the CellSearch waste was: 18% of COLO320, 6% of SW480, 59% of T24 and 2% of SKBR3. The combined recovery accounts for 20% of COLO320, 97% of SW480, 61% of T24 cells and 89% of SKBR3. In patients with NSCLC and SCLC either no CTC were detected at all, or in various proportions in the CellSearch cartridge, on the microsieves after filtration of the CellSearch waste or with the additional antibodies that were added. Conclusions: We combined the CellSearch system with a device for collecting and filtering the CellSearch waste. On cell lines this demonstrated that a low EpCAM expression results in the presence of CTC in the waste that would not be detected by the CellSearch system. In both NSCLC and SCLC additional CTC can be detected but it still remains to be determined whether the CTC not detected by the original CellSearch approach are also of clinical relevance

Improving the CellSearch® system

Introduction: The CellSearch® CTC test enumerates tumor cells present in 7.5 ml blood of cancer patients. improvements, extensions and different utilities of the cellsearch system are discussed in this paper. Areas covered: This paper describes work performed with the CellSearch system, which go beyond the normal scope of the test. All results from searches with the search term ‘CellSearch’ from Web of Science and PubMed were categorized and discussed. Expert commentary: The CellSearch Circulating Tumor Cell test captures and identifies tumor cells in blood that are associated with poor clinical outcome. How to best use CTC in clinical practice is being explored in many clinical trials. The ability to extract information from the CTC to guide therapy will expand the potential clinical utility of CTC

Big city life in the Second Commonwealth of Poland in Polish interwar feature films. An Introduction

Polish feature films from the interwar period are to a great extent a reflection of the reality of that time. This can be seen mainly in the presentation of particular architectural objects and fashion from that time. The films are also a source of knowledge about the problems of the time: the impoverishment of the society, economic crisis, prostitution, etc. These pictures reflect reality, yet they are subject to numerous transformations, as the result of the conventions of the chosen genre.Polski film fabularny omawianego okresu jest w znaczącym stopniu odbiciem ówczesnej rzeczywistości. Objawia się to głównie w dziedzinie urbanistyki, czyli przedstawiania na ekranie określonych obiektów architektonicznych, oraz w dziedzinie panującej wówczas mody. Jest ponadto źródłem wiedzy na temat typowych dla tego okresu problemów społecznych (pauperyzacja społeczeństwa, kryzys gospodarczy, prostytucja etc.). Obrazy te posiadają z jednej strony znamiona prawdy ekranowej, z drugiej – podane są licznym przekształceniom wynikającym z przyjętej konwencji gatunkowej

Capture of Tumor Cells on Anti-EpCAM-Functionalized Poly(acrylic acid)-Coated Surfaces

The presence of tumor cells in blood is predictive of short survival in several cancers and their isolation and characterization can guide toward the use of more effective treatments. These circulating tumor cells (CTC) are, however, extremely rare and require a technology that is sufficiently sensitive and specific to identify CTC against a background of billions of blood cells. Immuno-capture of cells expressing the epithelial cell adhesion molecule (EpCAM) are frequently used to enrich CTC from blood. The choice of bio conjugation strategy and antibody clone is crucial for adequate cell capture but is poorly understood. In this study, we determined the binding affinity constants and epitope binding of the EpCAM antibodies VU1D-9, HO-3, EpAb3-5, and MJ-37 by surface plasmon resonance imaging (SPRi). Glass surfaces were coated using a poly(acrylic acid) based coating and functionalized with anti-EpCAM antibodies. Binding of cells from the breast carcinoma cell line (SKBR-3) to the functionalized surfaces were compared. Although EpAb3-5 displayed the highest binding affinity HO-3 captured the highest amount of cells. Hence we report differences in the performance of the different antibodies and more importantly that the choice of antibody to capture CTC should be based on multiple assays