A modular and flexible ESC-based mouse model of pancreatic cancer

Abstract

Genetically engineered mouse models (GEMMs) have greatly expanded our knowledge of pancreatic ductal adenocarcinoma (PDAC) and serve as a critical tool to identify and evaluate new treatment strategies. However, the cost and time required to generate conventional pancreatic cancer GEMMs limits their use for investigating novel genetic interactions in tumor development and maintenance. To address this problem, we developed flexible embryonic stem cell (ESC)-based GEMMs that facilitate the rapid generation of genetically defined multiallelic chimeric mice without further strain intercrossing. The ESCs harbor a latent Kras mutant (a nearly ubiquitous feature of pancreatic cancer), a homing cassette, and other genetic elements needed for rapid insertion and conditional expression of tetracycline-controlled transgenes, including fluorescence-coupled shRNAs capable of efficiently silencing gene function by RNAi. This system produces a disease that recapitulates the progression of pancreatic cancer in human patients and enables the study and visualization of the impact of gene perturbation at any stage of pancreas cancer progression. We describe the use of this approach to dissect temporal roles for the tumor suppressor Pten and the oncogene c-Myc in pancreatic cancer development and maintenance.

c-Myc downregulation is required for preacinar to acinar maturation and pancreatic homeostasis

“…In addition, incomplete acinar differentiation resulting from genetic inactivation of Gata6 and Ptf1a in the pancreas cooperates with mutant KRas in PDAC development 45 46 . KRas- activating mutations lead to EGFR–c-Myc cross-regulation and increased c-Myc expression47–49 to levels similar to those of Ela1-Myc mice, and c-Myc is required for mutant KRas -driven tumours in the pancreas 50. c-Myc can also participate at later stages of PDAC progression, mainly through gene amplification leading to its overexpression 51 52…”

Stromal biology and therapy in pancreatic cancer: a changing paradigm

“…Using this next-generation dual recombinase system, Schonhuber et al provided first evidence that mast cells that had traditionally been regarded as oncogenic players are surprisingly dispensable for tumour formation. Besides the Cre-loxP , Flp-FRT DRS, tetracycline-inducible expression systems,43 44 novel embryonic stem cell-based PDA models45 and recombination systems such as Dre-Rox system are valuable additions to the genomic toolbox that will spur the field. Notably, Kras alone is insufficient to induce PDA, and several transposon-based insertional mutagenesis screens in mice have led to the discovery of genetic alterations that cooperate with oncogenic K-ras to promote tumorigenesis and have not previously been implicated in pancreatic carcinogenesis 46–48…”

Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

“…Existing mouse models typically fail to model the adult onset of pancreatic cancer and induce genomic alteration in nearly every cell in the pancreas. In the decade since the first genetically engineered PDAC models were developed, few technical advances have been made, and generating the mice required to investigate a gene of interest in the established PDAC models remains a time-consuming and costly endeavor (Aguirre et al 2003;Hingorani et al 2003Hingorani et al , 2005Saborowski et al 2014).…”