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

• Main Author: Dovmark, Tobias Højgaard
• Other Authors: Vaughan-Jones, Richard D. ; Swietach, Pawel
• Published: University of Oxford 2017
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.757706

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.