Table 1.
Primers used for RT-PCR.
Table 2.
Primers used for pyrosequencing.
Figure 1.
Immunofluorescence staining for EMT markers.
Suspension cells (NCI-H69) are strongly positive for E-cadherin (red) (a) and Zona Occludens (red) (b), whereas adherent NCI-H69V cells are negative for E-cadherin (d). Majority of NCI-H69V cells were negative for Zona Occludens, but some single cells showed slight Zona Occludens expression (asterisk, e). Suspension cells (NCI-H69) are negative (c), whereas adherent NCI-H69V are positive (f) 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.
Figure 2.
Morphologic differences within SCLC cell lines.
NCI-H69 SCLC cell lines are growing in floating clusters (a) with a small subpopulation of adherently-growing cells that were selected by disposing floating cells over multiple passages (b) resembling the established subline NCI-H69V (c). NCI-N592 and NCI-H82 in typical floating clusters (d, f) and selected adherent cells (e, g). Adherent subpopulations reveal a spindle shape, filopodia and microtentacles. Cells were imaged by Zeiss Axioplan microscope and AxioCam Digital and analyzed by Zeiss LSM Image Browser. Bar represents 200 µm.
Figure 3.
Expression and methylation of EMT and neuroendocrine markers in different SCLC cell lines.
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 (a). 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 (a). 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 (a). 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). The expression change in adherent NCI-N592 toward a mesenchymal-like phenotype is more significant on the protein level than the mRNA level (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 (c). DNA methylation analysis (d) 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).
Figure 4.
Increased migration and invasion in adherent NCI-H69V sublines.
Adherently-growing NCI-H69 cells demonstrate significantly higher migration (a) and invasion (b) towards 10% FCS containing medium compared to the suspension cell line (*** p = .0001). Migration Index was defined as number of migrated cells divided by number of migrated cells in the untreated group (no FCS) after 48 hours. Invasion Index was defined as number of counted cells in five view fields divided by the number of migrated cells in the untreated group (no FCS) after 48 hours.
Figure 5.
Extracellular proteolytic activity comparing NCI-H69 and NCI-H69V sublines.
Live-cell proteolysis assay: suspension NCI-H69 and adherent NCI-H69V cells were incubated for 48 hours on matrigel containing 30 ng/µl DQ-collagen IV. (a) Degradation of collagen is determined by green fluorescence that surrounds positive cells. Bar represents 30 µm. (b) Diagram shows mean values of three independent experiments with standard deviation.
Figure 6.
Secretion and activation of MMPs by NCI-H69 and NCI-H69V sublines.
Secreted MMPs were analyzed in NCI-H69 suspension and NCI-H69V cells by antibody arrays (a). Mapping of spots (b), antibody arrays were then quantified by ImageJ. The bars represent means of two independent experiments (c), white bar: NCI-H69/gray bar: NCI-H69V/black bar: medium control. Activation of MMP-2 and MMP-9 were determined by gelatin zymography (d) and quantified by ImageJ (e). The diagram shows mean values of three independent experiments with standard deviation (e).
Figure 7.
Higher chemoresistance of H69V cells compared to NCI-H69.
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 (a, b). Mean values from the three independent experiments of vital, dead and apoptotic cells are displayed as percentage of untreated controls (c). Cell viability of NCI-H69 and NCI-H69V after Etoposide treatment expressed as mean values and standard error of three independent experiments (d).