Role of carbonic anhydrase catalysis and gap junctional coupling in regulating ph in pancreatic ductal adenocarcinoma (PDAC)

Thesis

Pancreatic ductal adenocarcinoma (PDAC) develops from the acinar cells of the exocrine pancreas. PDAC tumours are hypoxic and produce a characteristically acidic microenvironment hypothesised to favour more aggressive phenotypes. My thesis work concerned the acid-handling mechanisms in 2-D (monolayer) or 3-D (spheroid) PDAC culture, to underpin survival advantages under acid-stress. The dependence on aerobic glycolysis was assessed in PDAC cells to appreciate the metabolic lactic acid-production. Fluorescence imaging revealed variances in H⁺ buffering capacity between PDAC cell lines. Activity of intra- and extracellular carbonic anhydrase (CA) enzymes facilitating CO₂/HCO₃- buffer equilibration revealed the highest extracellular CA-activity in glycolytic Colo357 cells. Interestingly, fluorescence recovery after photobleaching (FRAP) revealed strong connexin 43 (Cx43)-mediated cell-to-cell diffusive coupling in Colo357 cells. Photolytic H+-uncaging producing local acid-disturbances at the hypoxic core of Colo357 spheroids, revealed a major Cx43-mediated acid neutralising HCO₃ flux towards the core. The secondary-active HCO3- uptake was shown to take place in the spheroid rim, rather than at the core, because NBCe1 inhibition by DIDS only affected acid-handling at the core of Cx43-coupled spheroids, but not of Cx43-uncoupled spheroids. Normoxic cells in the spheroid rim thereby demonstrated metabolic altruism by donating actively imported HCO₃- ions to hypoxic cells via gap junctions. Cx43 channels were also shown to transmit lactate, and in Colo357 spheroids, 80% of lactate at the core was vented via Cx43 channels compared to canonical MCT transport. Moreover, Cx43-coupling confers a survival advantage to cells at the proliferating spheroid rim, as demonstrated by co-culturing wild type (Cx43 positive) cells with GFP-expressing Cx43 knockdowns. Furthermore, orthotopic MiaPaCa2 xenografts models showed increased Cx43 staining at invasive sites, indicating the enrichment of a more aggressive phenotype in Cx43-positive cells. Overall, my studies have confirmed the importance of HCO₃^- as a base for neutralising cancer-derived acids and demonstrated novel pathways for venting glycolytic products.

Authors: Dovmark, T

• DOI: 10.5287/ora-gongbwa27
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford