Investigation and therapeutic targeting of the metabolic relationship between pancreatic stellate cells and pancreatic ductal adenocarcinoma

Abstract

Ph. D. ThesisIntroduction Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive solid organ malignancies with persistently poor survival despite advancements in chemotherapy and surgical techniques. Pancreatic stellate cells (PSCs) are key pro-­‐tumourigenic players within the inflammatory microenvironment/stroma of PDAC, and thus represent an attractive therapeutic target. This project aimed to examine the under-­‐investigated relationship of PSCs to cancer metabolism, and identify novel translational treatment approaches. Methodology Human PSC and PDAC (Panc1, Miapaca2 and Bxpc3) cell lines were utilised in numerous in vitro transwell co-­‐culture experiments to investigate the effect on activity and more specifically metabolic phenotype, including the use of qPCR, western blot, metabolic assays, and seahorse technology. The impact of drugs targeting enzymes and transporters specific to lactate metabolism were then examined. In addition immunohistochemistry (IHC) was performed on resected tumours to examine the expression patterns and prognostic relevance of certain metabolic markers within the tumour microenvironment. Results Initial co-­‐culture experimentation revealed PSCs and PDAC impact on lactate metabolism, with a significant pro-­‐glycolytic effect of PSCs on the PDAC cell lines. In keeping with this, a notable upregulation in the monocarboxylate transporters 6 MCT1 and MCT4 was also observed, indicating an impact on lactate flux. PSC interactions with the PDAC lines were influenced by the underlying genetic/metabolic phenotype of each cell line. IHC staining revealed distinct MCT expression patterns, with a positive correlation between the tumoural and stromal compartments, whilst expression of MCT1 and MCT4 within the stroma and tumour respectively correlating with reduced overall survival. The MCT1 inhibitor AZD3965 exerted a KRAS-­‐dependent de-­‐activating effect on PSCs, with a reduction in αSMA and IL6 expression. AZD3965 also exerted a KRAS-­‐ dependent anti-­‐proliferative effect on the PDAC lines, a cell line-­‐specific PSC-­‐ dependent reduction in PDAC metabolic processes, as well as a reduction in PSC lactate transporter expression. Inhibition of all LDH isoforms with Galloflavin revealed a mixed effect on both PDAC proliferation and apoptosis, although an over-­‐riding reduction in lactate acidification. Conclusion This project has proven a metabolic symbiosis exists between PDAC and PSCs, which appears to be inextricably related to lactate metabolism. Inhibition of lactate transporters or enzymes has demonstrated effects on both cancer cells and PSCs which, in combination with the correlation to poor prognosis in resected specimens, holds great promise in a potentially beneficial translational impact for patients. The heterogeneity of results observed between cell lines, postulated to be due to either KRAS status or underlying metabolic phenotype, suggests a patient/tumour-­‐specific personalized approach to MCT or LDH inhibition should be adopted in any future clinical trials