Influence of CLL18 on chemoresistance.

<p>Cells were cultured in presence or absence of -TGFbeta or CCL18 in the concentrations indicated for 72 h. After the culture period, medium was changed and the cells were incubated with cisplatin for additional 72 hours at concentrations ranging from 0.4 to 25 µg/ml. Cell survival was measured by MTT assay. The horizontal line indicates the LD₅₀, vertical lines indicate the LD₅₀ for the respective pre-incubation condition (n = 4).</p

Mean CCL18 levels of all T stages was significantly increased compared with controls (p<0.0002).

<p>In addition, there was a significant difference between patient groups with lowest versus the two highest T-stages.</p

CCL18 induces chemotaxis in A549 lung cancer cells.

<p>Significances were calculated using paired comparison (Wilcoxon Signed Rank Test), control: untreated cells (n = 4).</p

Immunofluorescence staining for EMT markers.

<p>Suspension cells (NCI-H69) are strongly positive for E-cadherin (red) (<b>a</b>) and Zona Occludens (red) (<b>b</b>), whereas adherent NCI-H69V cells are negative for E-cadherin (<b>d</b>). Majority of NCI-H69V cells were negative for Zona Occludens, but some single cells showed slight Zona Occludens expression (asterisk, <b>e</b>). Suspension cells (NCI-H69) are negative (<b>c</b>), whereas adherent NCI-H69V are positive (<b>f</b>) for Vimentin (red). Ki-67 was co-stained (green). Cells were imaged by Zeiss Axioplan microscope and AxioCam Digital and analyzed by ZeissAxioVision software. Bar represents 20 µm.</p

Expression and methylation of EMT and neuroendocrine markers in different SCLC cell lines.

<p>EMT markers and transcription factors are expressed differently within the SCLC lines (NCI-H69, NCI-H82, and NCI-N592). NCI-H69 cells did not express mesenchymal markers or inductors in suspension cultures, evident on the mRNA level by RT-PCR (<b>a</b>). Zeb1, Snail/Snai1, Slug/Snai2, and FSP are expressed in the adherent subline (NCI-H69V), but E-cadherin is only expressed in suspension cells. Mesenchymal markers Fibronectin and Vimentin are upregulated in the adherent sublines (<b>a</b>). The mRNA expression in NCI-H82 and NCI-N592 compared with its adherent subcultures generally revealed a similar tendency toward a mesenchymal phenotype in the adherent cells (<b>a</b>). Western blot analysis displays decreased protein levels in the epithelial markers E-cadherin and Zona occludens 1, and expression of mesenchymal markers Vimentin and ZEB1 in NCI-H69V. EMT inductors Snail/Snai1, Slug/Snai2 are expressed in NCI-H69V (<b>b</b>). The expression change in adherent NCI-N592 toward a mesenchymal-like phenotype is more significant on the protein level than the mRNA level (<b>b</b>). The neuroendocrine markers NSE and L-Dopa are expressed in the parental cell line NCI-H69, but are not detectable in the adherent subline NCI-H69V (<b>c</b>). DNA methylation analysis (<b>d</b>) of the EMT hallmark gene Vimentin demonstrates strong methylation in NCI-H69 and significant hypomethylation in NCI-H69V, E-cadherin methylation is significantly higher in H69V. As control, cultured lung fibroblasts isolated from three patients for Vimentin methylation levels and from two patients for E-cadherin methylation levels without fibrotic diseases served as normal control. The bar shows the mean of three independent measurements. The differences between the mean methylation levels of the analyzed amplicon was tested using Student's unpaired t-test (* p value from 0.01 to 0.05, ** p value from 0.01 to 0.001 and *** p value less than 0.001).</p

Higher chemoresistance of H69V cells compared to NCI-H69.

<p>NCI-H69V treated with 200 µM Etoposide showed significantly increased chemoresistance in comparison to NCI-H69 cells. After 48 h treatment with Etoposide, cells were stained with Propidium iodide and DiOC6 to detect vital, dead and apoptotic cells via flow cytometry. Dot plots of one representative experiment repeated three times (<b>a, b</b>). Mean values from the three independent experiments of vital, dead and apoptotic cells are displayed as percentage of untreated controls (<b>c</b>). Cell viability of NCI-H69 and NCI-H69V after Etoposide treatment expressed as mean values and standard error of three independent experiments (<b>d</b>).</p

Secretion and activation of MMPs by NCI-H69 and NCI-H69V sublines.

<p>Secreted MMPs were analyzed in NCI-H69 suspension and NCI-H69V cells by antibody arrays (<b>a</b>). Mapping of spots (<b>b</b>), antibody arrays were then quantified by ImageJ. The bars represent means of two independent experiments (<b>c</b>), white bar: NCI-H69/gray bar: NCI-H69V/black bar: medium control. Activation of MMP-2 and MMP-9 were determined by gelatin zymography (<b>d</b>) and quantified by ImageJ (<b>e</b>). The diagram shows mean values of three independent experiments with standard deviation (<b>e</b>).</p