Fig. 6

Carbonic anhydrases fundamentally modify pH dynamics in breast cancer tissue, inhibiting net acid extrusion from cancer cells and lowering interstitial net H⁺ elimination from the core to the periphery. A + B Average pH traces (A) and quantified rates of acidification (B) in response to application of CO₂ to a HEPES-buffered CO₂/HCO₃^–-free solution. We show how lysates of ErbB2-induced murine breast cancer tissue and the carbonic anhydrase inhibitors acetazolamide (ATZ, 100 µM) and 4-(aminoethyl)benzenesulfonamide (AMB, 30 µM) influence the rate of CO₂ hydration (n = 4). Data were compared by one-way ANOVA followed by Šidák’s post-test. ***P < 0.001, NS: not significantly different vs. HEPES. ^###P < 0.001 vs. lysate. C Confocal image acquired at the equatorial plane of an organoid loaded with the pH-sensitive fluorophore carboxy-SNARF-1. The white delineations illustrate exemplary regions of interest used to quantify pH dynamics in the organoid core and periphery. D-H Extracellular (surface) pH dynamics in response to acute exposure of murine ErbB2-induced (D–F, n = 8–14) and human (G + H, n = 8–9) breast cancer organoids to CO₂/HCO₃^– relative to the initial mean value in absence of CO₂/HCO₃^–. Panel F shows quantified pH_o differences between the core and periphery of organoids and how they are modified by CO₂/HCO₃^– and carbonic anhydrase inhibition; panel H shows similar information regarding the pH_o differences between the organoid core and experimental bath. FC5-207A was used at a concentration of 200 µM. *P < 0.05, ***P < 0.001 vs. CO₂/HCO₃^–-free. ^#P < 0.05, ^###P < 0.001 vs. CO₂/HCO₃^–. ^##P < 0.01 vs. slope = 0 by linear trend analysis. Purple “NS” (= not significantly different) report the results of statistical tests comparing the effect of ATZ to that of AMB in the presence of CO₂/HCO₃^– as indicated by the purple arrows. I-N Intracellular pH dynamics in response to acute exposure of murine ErbB2-induced (I–K, n = 6–26) and human (L-N, n = 8) breast cancer organoids to CO₂/HCO₃^– relative to the initial mean value in absence of CO₂/HCO₃^–. Panel K and N show pH_i differences between the core and periphery of organoids and how they are modified by CO₂/HCO₃^– and carbonic anhydrase inhibition. ***P < 0.001 vs. CO₂/HCO₃^–-free. ^##P < 0.01 vs. CO₂/HCO₃^–. NS: not significantly different vs. slope = 0 by linear trend analysis (panel K) or vs. CO₂/HCO₃^– (panel N). Purple stars (*P < 0.05, **P < 0.01) report the results of statistical tests comparing the effect of ATZ to that of AMB in the presence of CO₂/HCO₃^– as indicated by the purple arrows. Data in panel F, H, K, and N were compared by one-way ANOVA followed by Šidák’s post-test or linear trend analysis. The ATZ- and AMB-induced pH-changes were compared by unpaired two-tailed Student’s t tests