TP3 - SASKit
Senescence-related biomarkers and signaling pathways in blood, cells and tissues of PDAC models
This subproject is dedicated to the analysis of mechanisms and consequences of senescence in the context of PDAC and fibrosis. Therefore, an animal model of accelerated ageing, the UCP2-/- mouse, will be employed to study progression of orthotopically transplanted syngeneic tumors, and data will be compared to wild-type mice with the same genetic background, and sham-transplanted mice. These experiments are based on the hypothesis that cellular senescence plays a double-edged and context-dependent role in PDAC progression. While induction of cancer cell senescence may limit tumor progression, aging of the immune system and the host tissue is likely to display the opposite effect. Furthermore, senescence of pancreatic stellate cells (PSC), the main source of extracellular matrix proteins in the pancreas, may modulate the PDAC-typical desmoplastic reaction.
UCP2 encodes for a mitochondrial anion carrier protein and has been implicated in the separation of oxidative phosphorylation from ATP synthesis. UCP2 deletion results in increased levels of reactive oxygen species (ROS). By using UCP2-/- mice, we therefore take advantage of an established model of increased ROS and accelerated aging to selectively study the effects of cellular senescence in non-cancer cells; e.g., immune cells and PSC (the tumor cells themselves are UCP2+/+). Tumor progression will be monitored by magnetic resonance imaging (MRI), and blood and tissue samples will be subjected to histological and molecular endpoint studies to evaluate and quantify markers of senescence in the various cell types, provide protein expression data, and enable gene expression measurements. These in vivo studies will be complemented by co-culture experiments employing murine PDAC cell lines and PSC derived from WT- and UCP2-/--mice.
Specifically, we will study the senescence associated secretory phenotype (SASP) on both the PDAC cell and PSC side, depending on the origin and combination of the cells. Therefore, cell culture supernatants as well as proteins/RNA from PDAC cells and PSC will be subjected to follow-up studies, supplemented by ELISA tests to measure secreted proteins, quantification of ROS levels and biological assays to monitor cellular functions. Based on the intermediate results of this subproject and the entire consortium, a small number of molecular targets will be selected for in-depth mechanistic studies. These targets should be key regulators of senescence, but also be of major importance for the project as a whole. Default targets are PAI-1, CDK5 and p16/p21.