Establishment and Characterization of a Highly Tumourigenic and Cancer Stem Cell Enriched Pancreatic Cancer Cell Line as a Well Defined Model System

Abstract

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer.

Depletion of RAD17 sensitizes pancreatic cancer cells to gemcitabine

“…An individual validation confirmed strong synthetic lethality of gemcitabine and knockdown of RAD17 and CHK1. In particular, the successful validation of RAD17 in the primary cell line JoPaca-1 underlines the relevance of this finding, as the cells show a strong resemblance to their primary tumour origin (Fredebohm et al, 2012). Validation of RAD17 knockdown using three independent shRNA expression constructs minimizes the chance of any off-target effects being responsible for the observed phenotypes (Echeverri et al, 2006).…”

“…RAD17 was most significant with an SL ratio of 20.704 (log 2 ). (Fredebohm et al, 2012) using increasing concentrations of gemcitabine and CHK1 knockdown as a positive control. In all three cell lines, knockdown of RAD17 enhanced gemcitabine cytotoxicity significantly for the three independent shRNA expression constructs.…”

“…BxPC-3 and MiaPaca-2 cell lines both carry deleterious mutations in the DNAbinding domain of p53 (Petitjean et al, 2007) necessary for transcriptional regulation. JoPaca-1 cells, however, have a mutation solely at codon 72 of the proline-rich domain that is important for induction of apoptosis but not cell-cycle arrest (Baptiste et al, 2002;Fredebohm et al, 2012;Walker and Levine, 1996;Zhu et al, 1999). However, the functional DNA-binding domain of p53 in JoPaca-1 cells does not prevent sensitivity to the synthetic lethality of CHK1 and RAD17 inhibition and gemcitabine.…”

“…The primary cell line JoPaca-1 was isolated as described previously (Fredebohm et al, 2012) and used at low passage (,P10) to retain primary characteristics. HPDE cells were kindly provided by Professor Asymmetric mitosis of MiaPaca-2 cells treated with shRAD17 and shCTRL with or without 50 nM gemcitabine was quantified microscopically.…”

Stem Cells in Pancreatic Cancer

“…Increased activity of ALDH1 was associated with CSCs and has been correlated with invasion, migration and poor overall survival in patients with pancreatic cancer [16]. Therefore, ALDH (+) cells have stem and mesenchymal cell features and are more tumorigenic than CD44 + /CD24 + cells [17].…”