Image analysis and processing was performed using AxioVision SE64 Rel


Image analysis and processing was performed using AxioVision SE64 Rel. epithelial cell Pyrithioxin dihydrochloride markers, including EpCAM. A highly relevant proportion of mesenchymal CTCs cannot therefore be isolated using techniques that are based on the Pyrithioxin dihydrochloride capture of cells expressing EpCAM. Herein, we provide evidence that a monoclonal antibody (mAb) directed against a membrane-bound form of Hsp70 (mHsp70)cmHsp70.1can be used for the isolation of viable CTCs from peripheral blood of tumor patients of different entities in a more quantitative manner. In contrast to EpCAM, the expression of mHsp70 remains stably upregulated on migratory, mesenchymal CTCs, metastases and cells that have been triggered to undergo EMT. Therefore, we Pyrithioxin dihydrochloride propose that approaches for isolating CTCs based on the capture of cells that express mHsp70 using the cmHsp70.1 mAb are superior to those based on EpCAM expression. separation of CTCs from peripheral blood are based on the capturing of cells using antibodies directed against cell surface expressed EpCAM (CD326) (22C26). The CellSearch? system (27)the FDA-approved gold standardcombines a magnetic separation technique based on EpCAM antibody-coated particles with subsequent cytokeratin (CK) staining and a microscopic analysis of the isolated cells (22). Another limitation of most CTC isolation techniques is the relatively small blood sample volume (7.5 ml) which is used and the low numbers of CTCs that can be derived therefrom. To overcome these disadvantages of CTC isolation, Rabbit Polyclonal to CBF beta GILUPI GmbH (Potsdam, Germany) has developed an EpCAM antibody-coated CellCollector? system which involves the direct insertion of a stainless steel wire, functionalized with gold and a hydrogel coating that incorporates anti-EpCAM antibodies, into the blood stream via a standard venous cannula in the cubital veins for 30 min. During this period, CTCs can be captured from the entire peripheral blood compartment (several liters of blood) of a cancer patient. Subsequently, the captured viable cells can be stained whilst attached to the wire and analyzed by fluorescence microscopy (28) or expanded for further analysis. The number of CTCs captured by the CellCollector? system before and after therapy has been shown to be associated with prognosis and therapeutic responsiveness (11). All the techniques described Pyrithioxin dihydrochloride above rely on the cell surface expression of EpCAM and the lack of the leukocyte marker CD45 by CTCs. However, many studies have shown that the transition of the adherent epithelial cells to the migratory mesenchymal state which enables the motility and invasiveness of CTCs and their dissemination to distant sites is associated with a loss in the expression of classical epithelial cell markers, including EpCAM (29). Yu et al. demonstrated that benign and non-invasive tumor cells exclusively express epithelial antigens, whereas a subpopulation of Pyrithioxin dihydrochloride invasive breast cancer cells express both epithelial and mesenchymal markers (30). Epithelial-to-mesenchymal transition (EMT) correlates with an increased migratory and metastatic potential of CTCs, invasiveness, poor overall survival and drug resistance (29, 30). It is therefore apparent that systems for isolating CTCs that rely only on the expression of epithelial markers by target cells are limited in their ability to detect CTCs arising after EMT. The search for universal tumor markers has revealed that the major stress-inducible heat shock protein 70 (Hsp70) is frequently expressed on the plasma membrane of primary tumor cells and distant metastases (31). This membrane Hsp70 (mHsp70) positivity has been identified on a large variety of different primary tumor types such as breast, lung, head and neck, colorectal, pancreas, brain and hematological malignancies, but not on corresponding normal cells and tissues (32, 33). A comparison of the cell surface density of Hsp70 has also revealed higher intensities of mHsp70 on metastases compared to corresponding primary tumors in mouse and human models (33C36). This finding provides a first indication that the expression of mHsp70 might not be downregulated by EMT and that it could therefore serve as a useful target for the isolation of CTCs in the circulation that have undergone EMT. Given that our group has developed.