Dual recombinase mouse model to dissect cell type‐specific role of microRNA‐21 in pancreatic cancer

Abstract

Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by its highly aggressive cancer cells and extensive stroma reaction. This reactive stroma contains distinct tumor-restraining and tumor-promoting fibroblast subpopulations. We previously studied the effects of global loss of pro-fibrotic non-coding regulatory microRNA-21 (miR-21) in KRas-driven p53-deleted genetically engineered mouse models (GEMMs) of PDAC. The absence of tumor-restraining smooth muscle actin (SMA)–expressing myofibroblasts (myCAFs) and a massive infiltrate of immune cells were the most salient phenotypic features of global miR-21 loss. Stromal miR-21 activity was required for induction of myCAFs in in-vivo isograft transplantation experiments. Objective We hypothesize that cell-intrinsic activity of miR-21 is required for induction and maintenance of myCAFs. To test this hypothesis, we have generated a new dual recombinase mouse model to temporarily and spatially control the dose of miR-21 activity using the tamoxifen-inducible Flp/FRT system in combination with the Cre/LoxP system that activates the KRas-driven GEMMs. Methods We describe the generation of a “flirted” Mir-21 allele flanked by FRT sites using easi-CRISPR technology and the characterization of mouse strains carrying one or two flirted alleles using molecular biology and histological analyses. Results We demonstrate the efficient recombination of flirted Mir-21 allele(s) in compounded strain with constitutive flippase (Rosa26-Flpe) or with tamoxifen-inducible flippase (Rosa26-FlpoER) after a single intraperitoneal injection of 75 mg/kg tamoxifen. We are crossing individual strains to produce a compounded strain with all the genetic elements required for this model. Histological and immunohistochemical characterization of tumors arising from these animals with full or partial loss of miR-21 activity will be presented at the meeting. Conclusions This dual recombinase system will be the first to conditionally delete the Mir-21 gene in myCAFs in well-established PDAC GEMMs. The genetic components of this dual recombinase system could be of general interest to the scientific community to mechanistically dissect the role of miR-21 in other cell types in PDAC by swapping the FlpoER driver strain or in myCAFs in other KRas-driven cancer types by swapping the Cre driver. Gained knowledge of the cell-intrinsic role of miR-21 in myCAFs and other cell types will be crucial to inform best strategies for pharmacological modulation of miR-21 activity in patients for cancer interception and/or treatment.