Spatio-temporal patterns of pancreatic cancer cells expressing CD44 isoforms on supported membranes displaying hyaluronic acid oligomers arrays.

In this paper, we designed a quantitative model of biological membranes by the deposition of planar lipid membranes on solid substrates (called supported membranes), and immobilized biotinylated oligomers of hyaluronic acid (oligo-HA, 6-8 disaccharide units in length) to the membrane surface via neutravidin cross-linkers. By controlling the self-assembly of biotinylated lipid anchors, the mean distance between the oligo-HA molecules on the membrane could be controlled to nm accuracy. The adhesion and motility of pancreatic adenocarcinoma cells expressing different splice variants of the HA-binding cell surface receptor CD44 on these surfaces were investigated quantitatively. The combination of label-free, time-lapse imaging of living cells and statistical analysis suggests that the static morphology (global shape and cytoskeleton remodeling) of cells, their stochastic morphological dynamics, and the probability of directed motion reflect the metastatic behaviour of the cancer cells

Autonomous inhibition of apoptosis correlates with responsiveness of colon carcinoma cell lines to ciglitazone.

Colorectal cancer is a leading cause of mortality worldwide. Resistance to therapy is common and often results in patients succumbing to the disease. The mechanisms of resistance are poorly understood. Cells basically have two possibilities to survive a treatment with potentially apoptosis-inducing substances. They can make use of their existing proteins to counteract the induced reactions or quickly upregulate protective factors to evade the apoptotic signal. To identify protein patterns involved in resistance to apoptosis, we studied two colorectal adenocarcinoma cell lines with different growth responses to low-molar concentrations of the thiazolidinedione Ciglitazone: HT29 cells underwent apoptosis, whereas SW480 cells increased cell number. Fluorescence detection and autoradiography scans of 2D-PAGE gels were performed in both cell lines to assess protein synthesis and turnover, respectively. To verify the data we performed shotgun analysis using the same treatment procedure as in 2D-experiments. Biological functions of the identified proteins were mainly associated with apoptosis regulation, chaperoning, intrinsic inflammation, and DNA repair. The present study suggests that different growth response of two colorectal carcinoma cell lines after treatment with Ciglitazone results from cell-specific protein synthesis and differences in protein regulation

Shape descriptors of adhesion area.

<p>Comparison of projected area per adhered cell, elongation, and circularity of 1AS cells on supported membranes functionalized with poly-HA and oligo-HA at <<i>d</i>> ∼ 5.5 nm and <i>t</i>  =  2 h. Standard deviation is given for mean values of nine cells for each condition.</p

Time evolution of cell morphology.

<p>(A) A snap shot of an ASpSV14 cell on an oligo-HA-functionalized membrane (<<i>d</i>> ∼ 5.5 nm, <i>t</i>  =  9 h) captured by RICM. The peripheral edge of the cell was determined by the contrast in pixel intensity. (B) The amplitude of the fluctuation amplitude plotted as a function of <i>θ</i>. is the mean radial distance over <i>θ</i>  =  0–360°. (C) The amplitude map as a function of angle <i>θ</i> over time (<i>t</i>  =  140–1000 min). (D) The autocorrelation corresponding to the amplitude map in panel (C).</p

Cell shape fluctuation amplitude and autocorrelation map at early stage.

<p>Representative amplitude maps of (A) 1AS and (B) ASpSV14 cells plotted as function of <i>θ</i> recorded during the early stage of cell adhesion (<i>t</i>  =  0–200 min). The corresponding autocorrelation functions for 1AS and ASpSV14 are presented in panel (C) and (D), respectively.</p

The impact of the average distance between oligo-HA molecules on adhesion of 1AS cells.

<p>The fraction of 1AS cells adhered to the indicated supported membranes plotted as function of time. The three membranes studied were pure DOPC membranes (black line), and membranes displaying oligo-HA at <<i>d</i>> ∼ 11 nm (green line) and <<i>d</i>> ∼ 5.5 nm (blue line).</p

Cell adhesion on poly-HA and oligo-HA substrates.

<p>Micro-interferometry images of 1AS cells on supported membranes displaying (A) poly-HA and (B) oligo-HA at an average distance of <<i>d</i>> ∼ 5.5 nm at <i>t</i>  =  2 h. Scale bar: 8 µm.</p

Binding of cancer cells on oligo-HA tethered supported membranes.

<p>RICM images (left) and confocal fluorescence images (middle and right) of (A) 1AS cells (B) AS-K162R166 cells (C) ASpSV14 cells, and (D) AS-R44 cells incubated on supported membranes displaying oligo-HA at <<i>d</i>> ∼ 5.5 nm for 4 h. After fixation, DNA and actin were stained with DAPI and Alexa 488 phalloidin.</p

Cell viability and mitochondrial membrane potential after treatment with thiazolidinediones.

<p>Cell viability was assessed by neutral red uptake in SW480 and HT29 cells treated with increasing concentrations of (A) Ciglitazone or (B) Troglitazone for 6 and 24 hours. *p<0.05, values of HT29 cells differ from those of SW480 cells. Mitochondrial membrane potential (Δψ_m) was assessed by JC-1 FACS analysis in (C) HT29 and (D) SW480 cells treated with increasing concentrations of Ciglitazone for 24 and 48 hours. *p<0.05, values at 48 hours differ from those at 24 hours. #p<0.05, values at indicated concentrations differ from baseline.</p

Cell responsiveness of colorectal adenocarcinoma cell lines after treatment with Ciglitazone.

<p>2D-PAGE gels were performed in (A) HT29 and (B) SW480 cells after treatment with 5 µM Ciglitazone. (1) Fluorescence scans of untreated cells, (2) fluorescence scans of cells treated with Ciglitazone, (3) autoradiography scans of untreated cells, (4) autoradiography scans of cells treated with Ciglitazone. Cell cycle distribution of (C) HT29 and (D) SW480 cells treated with increasing concentrations of Ciglitazone.</p