KCa3.1 channel inhibition leads to an ICAM-1 dependent increase of cell-cell adhesion between A549 lung cancer and HMEC-1 endothelial cells

Figures

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  Figure 1. Illustration of adhesion force measurements using single cell force spectroscopy (SCFS) (A) Single A549 cells are picked with a WGA-coated AFM-cantilever and brought into contact with HMEC-1 endothelial cells. A constant force (1 nN) is applied to maintain the contact between both cells for a period of 2 s. The force needed to separate the two cells (adhesion force [nN]) is measured during retraction of the cantilever. (B) Representative example of a force-distance curve. The characteristic phases of the experiment are indicated by numbers. 1. Approach of the A549 cell to an endothelial cell. No cell-cell contact has been formed. 2. The NSCLC cell is “pressed” onto the endothelial cell, positive force is applied. 3. Retraction of the cantilever so that the A549 cell is lifted off of the endothelial cell. 4. Detachment of the A549 cell from the endothelial cell. The turning point of the curve represents the maximal adhesion force [nN], and the shaded area under the curve is a measure of the detachment work [fJ].

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  Figure 2. Inhibition of KCa3.1 channels increases the adhesion force between A549 NSCLC and HMEC-1 endothelial cells The adhesion force is measured using single cell force spectroscopy. (A) Typical force-distance curves recorded under control conditions (vehicle DMSO; left) and in the presence of the KCa3.1 channel blocker senicapoc (right). Summary of the adhesion force measurements between A549 and HMEC-1 cells. Data are obtained from N = 9 independent experiments. Each experiment has been performed such that any given A549 cell, attached to the AFM-cantilever, has come into contact with 20 different endothelial cells. *denotes p < 0.05. (B) Adhesion force measurements were performed under control conditions or in the presence of senicapoc. (C) A549 cells were transfected with siRNA against KCa3.1 channels and used for experiments 48 h after transfection. (D) HMEC-1 endothelial cells were transfected with siRNA against KCa3.1. 48 hours after transfection, the cells were incubated for 24 hours with 10 ng/ml TNF-α.

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  Figure 3. Downregulation of KCa3.1 increases ICAM-1 expression in HMEC-1 endothelial and A549 NSCLC cells (A) Western blots of whole cell lysates from HMEC-1 and A549 cells transfected with control siRNA (scramble) or siRNA targeting KCa3.1 channels (siKCa3.1). The blot is representative for three replicates. (B) ICAM-1 staining of non-permeablized HMEC-1 endothelial cells with an anti-ICAM-1 antibody recognizing an extracellular epitope. (C) Quantification of ICAM-1 fluorescence intensity [arbitrary units a.u.]; n = 30 cells from N = 3 experiments. *denotes p < 0.05. (D) ICAM-1 expression in membrane fractions of HMEC-1 endothelial cells obtained from biotinylation assays. We blotted the fraction of biotinylated membrane proteins pulled down with streptavidin beads (biotin) and proteins from the remaining cell lysates (lysate) of HMEC-1 cells, treated with and without TNF-α. Actin blots validate the surface biotinylation because no actin is detected in the fraction of biotinylated membrane proteins. The blots are representative for four experiments.

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  Figure 4. Inhibiton of ICAM-1 prevents KCa3.1 channel-dependent strengthening of adhesion (A) and (B) Adhesion measurements using single cell force spectroscopy. The experimental procedures are shown in Figure 1. Prior to the measurements, A549 cells were incubated for 30 min with 10 μg/ml ICAM-1 blocking antibody or an IgG control antibody. (A) Measurements of the maximum adhesion force [nN]. (B) Detachment work [fJ] that is needed to seperate A549 cells from the endothelial cells. N = 7 independent experiments, in which A549 cells were brought into contact with 20 different HMEC-1 endothelial cells each. *p < 0.05. (C and D) Cell-cell adhesion of A549 to HMEC-1 endothelial cells. (C) A549 cells pre-incubated either with 10 μg/ml ICAM-1 blocking antibody or with an IgG control antibody (8 μg/ml) were seeded for 2.5 hours on a confluent endothelial layer. After washing and fixation, adherent A549 cells were quantified via microscopy. N = 6 independent experiments, and 10 different image sections per experiment were analysed. *denotes p < 0.05. (D) Representative images of adherent A549 cells on a confluent HMEC-1 endothelial layer (white arrows indicate A549 cells).

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  Figure 5. The KCa3.1 channel blocker senicapoc inhibits invasion of A549 cells through an endothelial cell layer (A) Invasion assay. HMEC-1 endothelial cells are seeded on a “sandwiched” collagen matrix consisting of a thick cushion of collagen I superimposed by a thin reconstituted “basememt membrane” containing collagen IV. After endothelial cells have grown to confluency, they are incubated with TNF-α (10 ng/ml) for 24 hours. The following day, A549 cells are added and transendothelial migration is monitored for 13.5 h using live-cell imaging. (B) Series of images of an A549 cell transmigrating through the endothelial cell layer. White arrows indicate the transmigrating A549 cell. (C–F) Analysis of the transmigration experiments. The time course of the experiments is plotted in (C) and (E), the summary of the experiments in (D) and (F). N = 6 experiments each. (C) and (D) Invasion of A549 cells in the presence of DMSO (control conditions) or 30 μmol/l senicapoc. *denotes p < 0.05. (E) and (F) Invasion of A549 cells transfected with siRNA against KCa3.1 channels.

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  Figure 6. Transendothelial resistance measurements (TER) with HMEC-1 and HUVEC cells showing no impact of senicapoc on endothelial barrier function HMEC-1 and HUVEC cells are seeded in an impedance chamber equipped with a glass surface coated with 0.5% crosslinked gelatin. When confluent, cells are treated with or without 100 ng/ml TNF-α. After 24 hours of incubation with/without TNF-α, 30 μmol/l of senicapoc or DMSO are added. (A) Time courses of TER-measurements of HMEC-1 and HUVEC cells. The time point “0” reproduces the addition of TNF-α. The vertical broken line indicates the addition of senicapoc or DMSO. (B) Representative images of immunofluorescence stainings of HMEC-1 and HUVEC cells, treated with senicapoc or DMSO. Cells were stained for VE-cadherin.