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, was employed to study progression of orthotopically transplanted syngeneic tumors, and data were compared to wild-type (WT) 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 took 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 was monitored by different approaches, and blood and tissue samples were 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 were complemented by co-culture experiments employing murine PDAC cell lines and PSC derived from WT- and UCP2-/--mice.
Specifically, we have studied 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 were 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 was selected for in-depth mechanistic studies. These targets included key regulators of senescence, also of major importance for the project as a whole, such as PAI-1, CDK5 and p16/p21.
Some key findings of the subproject have recently been published (Revskij et al. 2022) and can be summarized as follows: Measurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to UCP2-/- mice. In tumors in the knockout strain, higher levels of interferon-γ mRNA despite similar numbers of tumor-infiltrating T-cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 knockout mice than in WT animals. Accordingly, PSC from UCP2-/- mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. Taken together, our data suggests that UCP2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms. The evaluation of the various molecular findings has not yet been completed.