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

Abstract

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.

New insights into the physiological role of carbonic anhydrase IX in tumour pH regulation

“…Although addition and subsequent withdrawal of ammoniumcontaining solution alkalinises and then acidifies the intracellular compartment, only extracellular pH changes are registered by the glycocalyx-binding dye. The size of the surface pH e transient depends on the ability of extracellular CO 2 /HCO 3 À to buffer pH e changes (Swietach et al, 2009). When the experiment was repeated in the presence of the CA inhibitor acetazolamide, surface pH e transients were much larger than under control conditions.…”

“…This model predicts that respiring tissues should develop spatial gradients of pH i and pH e that are sensitive to CA activity. Moreover, these pH gradients will also depend on the site of CA activity Swietach et al, 2009). For instance, a dominance of intracellular CA activity would favour intracellular CO 2 hydration, which lowers pH i .…”

“…To test this, experiments were performed on multi-cellular spheroids that harbour a diffusionally restricted space surrounding component cells (Sutherland, 1988;Swietach et al, 2008;Swietach et al, 2009). Figure 3 illustrates an experiment performed on a cultured spheroid, comprised of CAIX over-expressing HCT116 cells, superfused with CO 2 / HCO 3 À buffer.…”

The chemistry, physiology and pathology of pH in cancer

“…Extracellular CO 2 also ionizes ( pK a ¼ 6.1) but to a much lesser degree than lactic acid. Although the spontaneous hydration reaction is very slow (time constant 5 s), it can be catalysed by exo-facial carbonic anhydrase (CA) enzymes [23][24][25], such as the tumour-associated isoforms CAIX and CAXII [26][27][28][29][30]. Catalysed conversion of CO 2 to HCO 3 À and H þ can facilitate overall CO 2 diffusion by means of a parallel flux of H þ þ HCO 3 À , a phenomenon first described in vitro by Gros & Moll [31].…”

“…This important aspect of tissue pH regulation cannot be investigated by measuring pH in suspensions or two-dimensional monolayers prepared from cultured cells. A more instructive approach to studying pH non-uniformity in tissue is to image cancer-derived multicellular three-dimensional spheroids for pH i and pH e (figure 5) [25,36,56].…”