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.

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 ∼300 μm) 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 (∼6.3) and reduced pH_e (∼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 = ∼6.6), whereas extracellular acidity was enhanced (pH_e = ∼6.6), 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 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 pH_e 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 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.

A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid–base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the misinterpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO₂/HCO₃⁻ and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems.

Purpose: Bevacizumab, an anti-VEGFA antibody, inhibits the developing vasculature of tumors, but resistance is common. Antiangiogenic therapy induces hypoxia and we observed increased expression of hypoxia-regulated genes, including carbonic anhydrase IX (CAIX), in response to bevacizumab treatment in xenografts. CAIX expression correlates with poor prognosis in most tumor types and with worse outcome in bevacizumab-treated patients with metastatic colorectal cancer, malignant astrocytoma, and recurrent malignant glioma. Experimental Design: We knocked down CAIX expression by short hairpin RNA in a colon cancer (HT29) and a glioblastoma (U87) cell line which have high hypoxic induction of CAIX and overexpressed CAIX in HCT116 cells which has low CAIX. We investigated the effect on growth rate in three-dimensional (3D) culture and in vivo, and examined the effect of CAIX knockdown in combination with bevacizumab. Results: CAIX expression was associated with increased growth rate in spheroids and in vivo. Surprisingly, CAIX expression was associated with increased necrosis and apoptosis in vivo and in vitro. We found that acidity inhibits CAIX activity over the pH range found in tumors (pK = 6.84), and this may be the mechanism whereby excess acid self-limits the build-up of extracellular acid. Expression of another hypoxia inducible CA isoform, CAXII, was upregulated in 3D but not two-dimensional culture in response to CAIX knockdown. CAIX knockdown enhanced the effect of bevacizumab treatment, reducing tumor growth rate in vivo. Conclusion: This work provides evidence that inhibition of the hypoxic adaptation to antiangiogenic therapy enhances bevacizumab treatment and highlights the value of developing small molecules or antibodies which inhibit CAIX for combination therapy. Clin Cancer Res; 18(11); 3100–11. ©2012 AACR.