Maja Günthert

Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: endothelial versus epithelial features.

SummaryECV 304 cells reported as originating from human umbilical vein endothelial cells by spontaneous transformation have been used as a model cell line for endothelia over the last decade. Recently, deoxyribonucleic acid fingerprinting revealed an identical genotype for ECV 304 and T24 cells (urinary bladder carcinoma cell line). In order to resolve the apparent discrepancy between the identical genotype and the fact that ECV304 cells phenotypically show important endothelial characteristics, a comparative study was performed. Immortalized porcine brain microvascular endothelial cells/C1–2, and Madin Darby canine kidney cells were included as typical endothelial and epithelial cells, respectively. Various methods, such as confocal laser scanning microscopy, Western blot, and protein activity tests, were used to study the cell lines. ECV304 and T24 cells differ in criteria, such as growth behavior, cytoarchitecture, tight junction arrangement, transmembrane electrical resistance, and activity of γ-glutamyltransferase. Several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells. Desmoglein and cytokeratin, both known as epithelial markers, were found in ECV304 as well as T24 cells. However, differences were found for the two cell lines with respect to the type of cytokeratin: in ECV304 cells mainly cytokeratin 18 (45 kDa) is found, whereas in T24 cells cytokeratin 8 (52 kDa) is predominant. As we could demonstrate, the ECV 304 cell line exposes many endothelial features which, in view of the scarcity of suitable endothelial cell lines, still make it an attractive in vitro model for endothelia.

Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides.

Human monoclonal antibodies are promising agents for the development of more selective anticancer therapeutics. However, the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass. Recent studies have shown that a peptide derived from the HIV TAT protein could improve the distribution of cytoplasmic reporter proteins when administered systemically as fusion proteins or cross-linked chimeras. In this article, we tested by quantitative biodistribtution analysis whether conjugation to TAT peptides could improve the tumor targeting properties of scFv(L19)-Cys: an engineered human antibody fragment specific for the ED-B domain of fibronectin, a marker located in the modified extracellular matrix surrounding tumor neovasculature. Our results show that TAT peptides, consisting either of L-amino acids or D-amino acids, can efficiently transduce target cells when conjugated to fluorophores and/or antibody fragments, suggesting a receptor-independent cell entry mechanism. However, conjugation of scFv(L19)-Cys to TAT peptides resulted in a severely reduced tumor targeting performance compared to the unconjugated antibody, as measured in murine F9 teratocarcinoma-bearing mice, after intravenous injection of the radiolabeled antibody preparations. Our results outline the usefulness of TAT peptides for the efficient in vitro transduction of cells with globular proteins. In particular, the use of TAT peptides composed of D-amino acids may significantly reduce proteolytic degradation. At the same time, the poor biodistribution properties of antibody-TAT conjugates cast doubts over the applicability of this methodology for the delivery of biopharmaceuticals in vivo.

MDCK cell cultures as an epithelial in vitro model : Cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy

AbstractPurpose. Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier. Methods. Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, α-tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration. Results. TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0−1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2−6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7−14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV (≥ 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer. Conclusions. Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitromodel to study drug transport and/or interactions with drugs and excipients.

Comparative analysis of the membrane proteome of closely related metastatic and nonmetastatic tumor cells.

The identification of proteins that are preferentially expressed on the membrane of metastatic tumor cells is of fundamental importance in cancer research. Here, we report the systematic comparison of the membrane proteome of two closely related murine teratocarcinoma cell lines (F9B9 and F9DR), of which only one (F9DR) is capable of forming liver metastases in vivo. The proteomic methodology used in this study featured the surface protein biotinylation on tumor cells followed by protein purification on streptavidin resin and relative quantification of corresponding tryptic peptides by mass spectrometric procedures. The study allowed the identification of 998 proteins and the determination of their relative abundance. Proteins previously known to be associated with metastatic spread were found to be either up-regulated (e.g., synaptojanin-2) or down-regulated (e.g., Ceacam1) in F9DR cells. A dramatic increase in abundance at the cell membrane was observed for a broad variety of proteins (e.g., high-mobility group protein B1), which were mainly thought to reside in intracellular compartments, a finding that was confirmed using confocal laser scanning microscopy and immunochemical analysis of cell cultures. Furthermore, we showed by microautoradiographic analysis that certain target proteins can readily be reached by intravenously administered radiolabeled antibodies. Finally, we showed that the most promising antigens for antibody-based pharmacodelivery approaches are strongly and selectively expressed on the surface of tumor cells in three different syngeneic mouse models of liver metastases. Taken together, our results indicate that the expression of intracellular proteins on the membrane of metastatic cells is a feature much more common than previously expected.

EFFECT OF THE MODULATION OF THE MEMBRANE LIPID COMPOSITION ON THE LOCALIZATION AND FUNCTION OF P-GLYCOPROTEIN IN MDR1-MDCK CELLS

SummaryMultidrug resistance (MDR) is a major obstacle in cancer therapy. It results from different mechanisms; among them is P-glycoprotein (P-gp)-mediated drug efflux out of cells. The mechanism of action remains elusive. The membrane lipid surrounding of P-gp, especially cholesterol, has been postulated to play an important role. To determine the effect of cholesterol depletion on P-gp, Madin Darby canine kidney (MDCK) cells, transfected with the mdr1 gene (MDR1-MDCK cells), were treated with methyl-β-cyclodextrin (MβCD). The localization and function of P-gp were analyzed using confocal laser scanning microscopy. Treatment with 100 mM MβCD did not affect viability but altered the structural appearance of the cells and abolished efflux of rhodamine 123, a P-gp substrate. The MβCD treatment released P-gp from intact cells into the supernatant and reduced the amount of P-gp in total membrane preparations. The P-gp was shifted from the raft fractions (1% Triton X-100, 4° C) to higher density fractions in MβCD-treated cells. The amount of cholesterol was significantly decreased in the raft fractions. Treatment of cells with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosylceramide synthase inhibitor, also led to a shift of P-gp to higher density fractions. These results show that removal of cholesterol modulates the membrane lipid composition, changes the localization of P-gp, and results in loss of P-gp function.

P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope

Madin Darby canine kidney (MDCK) cells transfected with the multidrug resistance mdr1 gene, MDR1-MDCK (Pastan et al., 1988, Proc. Natl. Acad. Sci. USA 85 4486-4470), were used in a combined approach to study expression, localisation and functionality of the P-glycoprotein (P-gp) membrane transporter in the same cell culture preparations. Cells were characterised with regard to their growth curve, transepithelial electrical resistance (TEER), and cytoarchitecture. Efflux of the P-gp substrate rhodamine123 (rho123) was monitored with confocal laser scanning microscopy (CLSM). The transfected cells grew in multilayers. After reaching confluence they exhibited a complete tight junction (TJ) network. P-gp was strongly expressed at the uppermost apical surface of the multilayer already after 4 days in culture. The lower cell layers were not clearly polarised. P-gp-mediated transport could be followed by efflux of the fluorescent rho123 from the cells into the apical extracellular space. Verapamil, a P-gp inhibitor, significantly decreased efflux. For MDCK parent cells the rho123 assay was negative up to about day 20, and only at later times (day 25) low P-gp activity was detected. These results clearly show that despite the fact that the transfected cells form irregular layers, they provide a good model for screening of P-gp substrates and inhibitors.

Formation of Multilayers in the Caco-2 Cell Culture Model: A Confocal Laser Scanning Microscopy Study

AbstractPurpose. To introduce confocal laser scanning microscopy (CLSM)combined with digital image restoration to characterise Caco-2 cellsunder different culture conditions, and thus to define additional validcriteria for the optimisation of culture models. Methods. Growth curves were established and transepithelial electricalresistance (TEER) measured for cells grown in EMEM or DMEMmedium on Cyclopore™ membranes. Cytoskeleton, cell nuclei and tightjunctions (TJ) were investigated by CLSM. Results. Cultures reached a plateau of ∼4.5 × 105 cells/cm2 after∼ 10 days. At the same time TEER reached 750 Ω cm2. An irregular,fairly complete network of TJ was present at confluence (∼2 d).Between 15 and 30 days a regular TJ network was established. Cellsformed mixed mono- and multilayers under most conditions with twoexceptions: flat monolayers were observed on polycarbonate filterswith EMEM and with the Biocoat™ intestinal epithelium differentiationenvironment system. In multilayers TJ were found in the upper aswell as in the lower cell layers although the regular vertical polaritywas disturbed. Conclusions. CLSM represents an important tool to investigate thecytoarchitecture of Caco-2 cells. 3D-analysis of confocal data givesimportant clues on the characteristics of cell layers and thus helps tovalidate optimisation strategies.

Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures.

The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.

Correlative 3D microscopy: CLSM and FIB/SEM tomography used to study cellular entry of vaccinia virus

The development of new anti-viral strategies requires detailed information on the replication cycle of viruses. In this study we focused on the early steps of viral interaction, the surface dynamics after binding to non-infected cells. We used the more abundant infectious form of the virus, the intracellular mature virus (IMV). IMVs have a dumbbell-shaped core, a single lipid bilayer, and a size of 360 nm in the longest dimension [1]. IMVs have been observed to bind to filopodia, actin-containing, finger-like cell protrusions. Live cell imaging showed that fluorescent virus particles associate with filopodia, and glide along them to the cell body [2]. They also induce the extrusion of large, transient membrane blebs, which upon retraction cause endocytic internalization of the virus [3].

Deconvolution Combined with Digital Colocalization Analysis to Study the Spatial Distribution of Tight and Adherens Junction Proteins

The relative spatial distribution of proteins was investigated with immunofluorescent methods by confocal laser scanning microscopy and digital image restoration. For confocal data sets recorded with a voxel dimension of 50 × 50 × 150 nm noise and blur can be decreased and the resolution in the z-axis increased by applying the maximum likelihood estimation algorithm of the Huygens software. This approach was successfully applied to the study of tight and adherens junctions in relation to the actin cytoskeleton in Madin Darby Canine Kidney cells. Colocalization analysis was done for pairs of probes using a histogram-based method. F-actin, occludin, zonula occludens 1, and E-cadherin were included in the study. Double-labeled preparations were used. The combination of deconvolution with the colocalization of confocal data sets offers a powerful tool to investigate the spatial arrangement of proteins.