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

Clinical and molecular characterization of the CRC cell lines.

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

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

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