The Role of Carbonic Anhydrase 9 in Regulating Extracellular and Intracellular pH in Three-dimensional Tumor Cell Growths

We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of similar to 300 mu m) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pH(i)) and pH in the restricted extracellular spaces (pH(e)). With no CA9 expression, spheroids developed very low pHi (similar to 6.3) and reduced pH(e) (similar to 6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pH(i) = similar to 6.6), whereas extracellular acidity was enhanced (pH(e) = similar to 6.6), so that radial pHi gradients were smaller and radial pHe gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pHe indicates the enzyme is facilitating CO2 excretion from cells (by converting vented CO2 to extracellular H+), rather than facilitating membrane H+ transport (such as H+ associated with metabolically generated lactic acid). This latter process requires titration of exported H+ ions with extracellular HCO3-, which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pHe will depend on the cellular CO2/lactic acid emission ratio (set by local oxygenation and membrane HCO3- uptake). Our results suggest that CO2-producing tumors may express CA9 to facilitate CO2 excretion, thus raising pHi and reducing pHe, which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.