Clinical and molecular characterization of the CRC cell lines.

<p>Clinical and molecular characterization of the CRC cell lines.</p

KEGG pathway analysis of genes distinguishing cells grown in 2D and lrECM culture conditions.

<p>Shown are all categories (pathways/hits) represented by three or more differently regulated genes when comparing 2D and lrECM 3D culture conditions.</p

The 3D microenvironment impairs the regulation of genes involved in proliferation.

<p>Total RNA was isolated from cells cultivated in 2D and 3D microenvironments. Quantitative RT-PCR was performed and differences in gene expression levels were calculated using the 2^−ΔΔCT method. The mean fold change in expression of the target gene in 2D or 3D culture conditions was calculated using 2^−ΔΔCT, where ΔΔCT = (CT _Target – C _GAPDH)_{culture condition} - (CT _Target - C _GAPDH)_2D. 2^−ΔΔCT-values of all CRC cell lines (n = 7) were pooled and are presented as a box plot. <b>A)</b> Indicates genes being downregulated in 3D microenvironments, whereas <b>B)</b> shows upregulated genes. Two-tailed <i>P</i>-values were calculated by the Mann-Whitney-U test (*** indicates a <i>P</i>-value <0.0001).</p

Differential gene expression in 2D and lrECM cultivated cells.

<p><b>A)</b> Hierarchical cluster analysis of 2D and lrECM 3D on-top cultivated CRC cells lines. A total of 23000 transcripts were clustered. Each cell line builds an independent cluster for itself. With the exception for LOVO and HT-29, within each cell line two separate clusters were observed, due to the cultivation method of the cells: 2D versus lrECM 3D on-top. <b>B)</b> Heatmap of 225 significantly differentially regulated genes between 2D and lrECM 3D on-top cultivated cells. (Mann-Whitney-U test, <i>P</i>_corr <0.05). <b>C)</b> Literature-based network analysis of significantly differentially regulated genes (Natural Language Processor Engine, GeneSpring GX 10.5). Known interactions of 14 from a total of 225 significantly differentially regulated genes are shown. A red arrow indicates the increased expression of each gene in lrECM cultivation conditions while a green arrow indicates a reduced gene expression.</p

Influence of culture conditions on EGFR signaling molecules and EGFR inhibition of CRC cells.

<p><b>A)</b> Schematic representation of the EGFR signalling pathway. <b>B)</b> Immunoblot analysis of AKT, phospho-AKT (S473), p44/42 MAPK and phospho p44/42 MAPK (Thr202/Tyr204). Equal amounts of total protein isolated from cells cultivated as 2D or lrECM 3D cultures were analyzed by SDS/PAGE/immunoblotting as indicated. β−actin served as loading control. SKBR3 served as a positive control for the expression of EGFR signaling molecules. <b>C)</b> Expression of the EGFR protein in 2D or lrECM (3D) cultivated CACO-2, DLD-1, HT-29, SW-480, LOVO, COLO 205 and COLO-206F cells. Cell lysate (40 µg protein per lane) was analyzed by SDS/PAGE/immunoblotting using monoclonal anti EGFR antibody. β-actin served as loading control. <b>D–G)</b> Treatment of 2D or lrECM cultivated CRC cells with AG1478. Two-tailed <i>P</i>-values were calculated by the paired t-test (** indicates a <i>P</i>-value <0.001; *** indicates a <i>P</i>-value <0.0001; ns = not significant). CACO-2 cells were treated for 48 h <b>(D)</b> or 96 h <b>(E)</b> with different concentrations of AG1478 as indicated. <b>F–G)</b> DLD-1 and HT-29 cells were incubated with the EGFR inhibitor AG1478 for 48 h.</p

Invasive capacity of CRC cell lines.

<p>Invasion was analyzed using the Boyden chamber based cell invasion assay. Transmigrated cells were stained with crystal violet and counted. Different invasive capacity is indicated by the number of+symbols (>1000 cells: ++++++; >500 cells: +++++; >100 cells: ++++; >50 cells: +++; >10 cells: ++; >5 cells: +). Grey shaded = “mass” type; white = “grape-like” type.</p

New Model for Gastroenteropancreatic Large-Cell Neuroendocrine Carcinoma: Establishment of Two Clinically Relevant Cell Lines

<div><p>Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) according to their proliferation index into G1- or G2-neuroendocrine tumors (NET) and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC). Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1) or lymph node metastases (NEC-DUE2) from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. <i>In vitro</i> and <i>in vivo</i> tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. <i>In vitro</i> and <i>in vivo</i> experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup.</p></div

Cytogenetic changes in large-cell NEC cell lines.

<p>DNA was isolated from the cell lines, primary tumors (PT) and hepatic or lymphatic metastases (M) and genetic aberrations were analyzed by aCGH analysis. Amplitudes over the midline reflect chromosomal gains, amplitudes under the midline losses. M I and M II represent the atypically resected liver metastases of the gastroesophageal NEC.</p

Immunohistochemical expression analyses of the cell lines NEC-DUE1 and NEC-DUE2.

<p>NSE neuron-specific enolase, VMAT vesicular monoamine transporter, CD56 cluster of differentiation 56, NCAM Neural Cell Adhesion Molecule, SSTR somatostatin receptor, CK cytokeratin, CEA carcinoembryonic antigen, Ca 19.9 carbohydrate antigen 19-9, TTF1 thyroid transcription factor 1, CDX2 caudal type homeobox 2.</p

NEC cell lines express general and specific neuroendocrine markers, somatostatin receptors and transcription factors.

<p>RNA from cultured NEC cell lines was isolated and RT-PCR analyses performed for (<b>A</b>) general neuroendocrine markers, (<b>B</b>) somatostatin receptors, (<b>C</b>) specific neuroendocrine markers and (<b>D</b>) transcription factors as indicated. The colon cancer cell line HCT116 served as control cell line.</p