The high metabolic rate required for tumor growth often leads to hypoxia in poorly-perfused regions. Hypoxia activates a complex gene expression program, mediated by hypoxia inducible factor 1 (HIF1alpha). One of the consequences of HIF1alpha activation is up-regulation of glycolysis and hence the production of lactic acid. In addition to the lactic acid-output, intracellular titration of acid with bicarbonate and the engagement of the pentose phosphate shunt release CO(2) from cells. Expression of the enzyme carbonic anhydrase 9 on the tumor cell surface catalyses the extracellular trapping of acid by hydrating cell-generated CO(2) into [see text] and H(+). These mechanisms contribute towards an acidic extracellular milieu favoring tumor growth, invasion and development. The lactic acid released by tumor cells is further metabolized by the tumor stroma. Low extracellular pH may adversely affect the intracellular milieu, possibly triggering apoptosis. Therefore, primary and secondary active transporters operate in the tumor cell membrane to protect the cytosol from acidosis. We review mechanisms regulating tumor intracellular and extracellular pH, with a focus on carbonic anhydrase 9. We also review recent evidence that may suggest a role for CA9 in coordinating pH(i) among cells of large, unvascularized cell-clusters.

Intracellular pH was recorded fluorimetrically by using carboxy‐SNARF‐1, AM‐loaded into superfused ventricular myocytes isolated from guinea‐pig heart. Intracellular acid and base loads were induced experimentally and the changes of pHi used to estimate intracellular buffering power (β). The rate of pHi recovery from acid or base loads was used, in conjunction with the measurements of β, to estimate sarcolemmal transporter fluxes of acid equivalents. A combination of ion substitution and pharmacological inhibitors was used to dissect acid effluxes carried on Na+‐H+ exchange (NHE) and Na+‐HCO3− cotransport (NBC), and acid influxes carried on Cl−‐HCO3− exchange (AE) and Cl−‐OH− exchange (CHE). The intracellular intrinsic buffering power (βi), estimated under CO2/HCO3−‐free conditions, varied inversely with pHi in a manner consistent with two principal intracellular buffers of differing concentration and pK. In CO2/HCO3−‐buffered conditions, intracellular buffering was roughly doubled. The size of the CO2‐dependent component (βCO2) was consistent with buffering in a cell fully open to CO2. Because the full value of βCO2 develops slowly (2·5 min), it had to be measured under equilibrium conditions. The value of βCO2 increased monotonically with pHi. In 5 % CO2/HCO3−‐buffered conditions (pHo 7·40), acid extrusion on NHE and NBC increased as pHi was reduced, with the greater increase occurring through NHE at pHi < 6·90. Acid influx on AE and CHE increased as pHi was raised, with the greater increase occurring through AE at pHi > 7·15. At resting pHi (7·04‐7·07), all four carriers were activated equally, albeit at a low rate (about 0·15 mM min−1). The pHi dependence of flux through the transporters, in combination with the pHi and time dependence of intracellular buffering (βi+βCO2), was used to predict mathematically the recovery of pHi following an intracellular acid or base load. Under several conditions the mathematical predictions compared well with experimental recordings, suggesting that the model of dual acid influx and acid efflux transporters is sufficient to account for pHi regulation in the cardiac cell. Key properties of the pHi control system are discussed.

Cell survival is conditional on the maintenance of a favourable acid–base balance (pH). Owing to intensive respiratory CO 2 and lactic acid production, cancer cells are exposed continuously to large acid–base fluxes, which would disturb pH if uncorrected. The large cellular reservoir of H + -binding sites can buffer pH changes but, on its own, is inadequate to regulate intracellular pH. To stabilize intracellular pH at a favourable level, cells control trans-membrane traffic of H + -ions (or their chemical equivalents, e.g. ) using specialized transporter proteins sensitive to pH. In poorly perfused tumours, additional diffusion-reaction mechanisms, involving carbonic anhydrase (CA) enzymes, fine-tune control extracellular pH. The ability of H + -ions to change the ionization state of proteins underlies the exquisite pH sensitivity of cellular behaviour, including key processes in cancer formation and metastasis (proliferation, cell cycle, transformation, migration). Elevated metabolism, weakened cell-to-capillary diffusive coupling, and adaptations involving H + /H + -equivalent transporters and extracellular-facing CAs give cancer cells the means to manipulate micro-environmental acidity, a cancer hallmark. Through genetic instability, the cellular apparatus for regulating and sensing pH is able to adapt to extracellular acidity, driving disease progression. The therapeutic potential of disturbing this sequence by targeting H + /H + -equivalent transporters, buffering or CAs is being investigated, using monoclonal antibodies and small-molecule inhibitors.

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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 ϳ300 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 pH i (ϳ6.3) and reduced pH e (ϳ6.9) 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 ‫؍‬ ϳ6.6), whereas extracellular acidity was enhanced (pH e ‫؍‬ ϳ6.6), so that radial pH i gradients were smaller and radial pH e gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pH e indicates the enzyme is facilitating CO 2 excretion from cells (by converting vented CO 2 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 HCO 3 ؊ , which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pH e will depend on the cellular CO 2 /lactic acid emission ratio (set by local oxygenation and membrane HCO 3 ؊ uptake). Our results suggest that CO 2 -producing tumors may express CA9 to facilitate CO 2 excretion, thus raising pH i and reducing pH e , which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.