Tumor-associated carbonic anhydrase 9 spatially coordinates intracellular pH in three-dimensional multicellular growths

Abstract

CA9 is a membrane-tethered, carbonic anhydrase (CA) enzyme, expressed mainly at the external surface of cells, that catalyzes reversible CO 2 hydration. Expression is greatly enhanced in many tumors, particularly in aggressive carcinomas. The functional role of CA9 in tumors is not well established. Here we show that CA9, when expressed heterologously in cultured spheroids (0.5-mm diameter, ∼25,000 cells) of RT112 cells (derived from bladder carcinoma), induces a near-uniform intracellular pH (pH i) throughout the structure. Dynamic pHi changes during displacements of superfusate CO2 concentration are also spatially coincident (within 2 s). In contrast, spheroids of wild-type RT112 cells lacking CA9 exhibit an acidic core (∼0.25 pHi reduction) and significant time delays (∼9 s) for pHi changes in core versus peripheral regions. pHi non-uniformity also occurs in CA9-expressing spheroids after selective pharmacological inhibition of the enzyme. In isolated RT112 cells, pHi regulation is unaffected by CA9 expression. The influence of CA9 on pHi is thus only evident in multicellular tissue. Diffusion-reaction modeling indicates that CA9 coordinates pHi spatially by facilitating CO2 diffusion in the unstirred extracellular space of the spheroid. We suggest that pHi coordination may favor survival and growth of a tumor. By disrupting spatial pHi control, inhibition of CA9 activity may offer a novel strategy for the clinical treatment of CA9-associated tumors. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.