Tumor pH and its measurement

Cause and consequences of tumor acidity

The physiological microenvironment of solid tumors is normally characterized by poor perfusion and high metabolic rates. As a consequence, many regions within tumors are transiently or chronically hypoxic and acidic. Acidity is likely related to glucose consumption rates. High fluorodeoxyglucose (FDG) consumption has been consistently shown to be a poor prognostic indicator in human cancers. The pH of cells and tissues is the result of the balance between the metabolic processes, proton transport and buffering. Catabolism of sugars or fats results in a net production of acid, whether through fermentation or respiration (1) (FIGURE 1). The pyruvate made by glycolysis or amino acids can either enter the tricarboxylic acid (TCA) cycle through pyruvate dehydrogenase or be reduced to lactate. The carbonic acid arises by hydration of CO₂ formed by oxidation in the TCA cycle, whereas lactic acid is the product of the glycolytic pathway in the absence of oxygen. Excess lactate is exported via monocarboxylate transporters, MCT-1, -2, and sometimes -4, which can also carry H⁺. Protons themselves are exported via a number of transport systems such as Na⁺/H⁺ exchange (NHE), vacuolar H⁺ ATPases and Na⁺/HCO₃^- exchanges. All three systems perform redundant functions and can substitute for one another in the presence of inhibitors. Oxidatively produced CO₂ can exit the cell through aquaporins, where it is hydrated by membrane-bound carbonic anhydrases, to yield bicarbonate and a hydrogen ion.

Maintenance of acid-base homeostasis is critical. However this balance in solid tumors is vulnerable. In tumor cells, the increased glucose catabolism results in significant production of lactate and H⁺. Although tumor cells have increased acid production, they maintain a normal or alkaline pHi relative to normal cells. The major acid load is transported outside the cells, which cannot be removed by the vasculature. The capacity of primary extracellular buffer is limited; as a result extracellular space becomes acidic. Tumor cells seem well adapted to acidic microenvironments. In fact, in vitro studies have shown that maximum tumor cell proliferation occurs at a pHe of 6.8 instead of 7.3 in normal cells. The excess H⁺ ions diffuse along concentration gradients from the tumor into adjacent normal tissue resulting in a chronically acidic microenvironment for neighboring normal cells. Recent therapeutic approaches have been designed to target the tumor pH either through low pH activation of pro-drugs, low pH activated release of drug from micelles and nanoparticles, or by drugs that raise pH of acidic tumors. Thus, it will become increasingly important to be able to measure pH with accuracy, precision and high spatio-temporal resolution in experimental preclinical systems as well as in human beings.

PET

PET has been used for measuring tissue pH since 1970s. Techniques used with moderate success employ the distribution of radio-labeled DMO, which distribute according to the pH gradient across semi-permeable membranes via ‘ion trapping’. Although this represents the first noninvasive in vivo pH measurement, it is inaccurate and imprecise, since DMO distribution depends on the plasmalemmal pH gradient and the fractional volumes of intra- and extracellular space, both of which are unknown. Recently, an innovative technique was discovered to selectively target acidic tissues in vivo using pH low insertion peptide (pHLIP), a peptide that predominantly inserts across a lipid bilayer as a monomeric alpha-helix at acidic extracellular environment but not at normal physiological pH. Vāvere, et al., extended this method with PET imaging to investigate the acidic environment in prostate tumors using ⁶⁴Cu conjugated to the pHLIP (⁶⁴Cu-DOTA-pHLIP) (2). This is the first time a pHe-sensitive peptide based PET agent has been reported for the delineation of the pHe of tumors. Although the affinity of pHLIP to cell membrane is low at normal physiologic pH, it may result in an inaccurate pHe measurement.

MRS and MRI

In vivo MRS and MRI have been used for more than three decades to monitor metabolic and physiologic processes. Both endogenous and exogenous NMR-active compounds have been used to measure pH in vivo (3). MRS methods are generally based on a difference in chemical shifts between pH-dependent and independent resonances. A number of isotopes have been evaluated to determine tissue pH with MRS. ³¹P-MRS provides a robust technique for simultaneously measuring pHi from the chemical shift of endogenous inorganic phosphate (P_i) and pHe from the chemical shift of exogenous indicators, such as 3-aminopropyl phosphonate (3-APP). An improvement measurement was achieved using ¹H-MRS with pH-sensitive H2 resonance of 2-imidazole-1-yl-3-ethoxycarbonyl propionic acid (IEPA). Although these studies showed that the tumors’ pHe was heterogeneous, they are still limited in spatial and temporal resolution.

Optics

Optics is another powerful tool of noninvasive pH measurement in tumor and surrounding tissue. The varying fluorescent properties of the probes, which are associated with local pH, can be measured optically and be converted to pH distribution accordingly. Currently, there are two main ways for measuring tumor pH: fluorescence ratio imaging microscopy (FRIM), and fluorescence lifetime imaging microscopy (FLIM), which are accomplished by the measurements of emission spectra and lifetimes of fluorophores, respectively. In fluorescence ratiometric imaging, the emission spectra of some pH probes undergo a pH-dependent wavelength shift, thus allowing the ratio of the fluorescence intensities from the dyes at two emission wavelengths to be used for accurate determinations of pH. However, the association of pH indicators to intracellular proteins and cytosolic constituents was found to modify the ratio of intensities in two emission bands. Moreover, FRIM is based on fluorescence amplitude methods and is thus susceptible to photobleaching and to variation in light scattering and absorption of the sample, which may also introduce the bias in the results of pH measurement. FLIM is an experimental technique in which the fluorescence decay is measured at each spatially resolvable location within a fluorescence image. The pH probes used in FLIM display a pH-dependent shift in fluorescence lifetimes, which can be converted to pH map of tumor accordingly with construction of proper calibration curves. Fluorescence lifetime techniques are generally classified into two types: time-domain and frequency-domain. Compared to frequency-domain methods, time-domain measurements have higher signal-to-noise ratio (SNR) and are widely used for pH imaging (16). Although lifetime-based pH imaging has often been found to be more convenient than conventional ratiometric methods due to a simpler pH calibration, it was pointed out that this method is not necessarily a straightforward approach to measure pH in the absence of correction for the effect of probe binding.

Summary

Techniques for measuring the pH of organelles, cytosol and extracellular fluid in vivo have been consistently improving and converging. Optical techniques based on expression of pH-sensitive fluorescent dye are revolutionizing pH measurements in vitro. With improved detection platforms, optical methods hold promise to be applied in vivo. MR techniques have been developed to measure pHi with endogenous indicators, albeit with low sensitivity. Methods based on exogenous compounds have clearly shown that high resolution and highly sensitivity measurements of pHe (and possibly pHi) are tractable in vivo. Hyperpolarized ¹³C-labelled bicarbonate technique may potentially be used in the clinic and new generations of pH-sensitive PET tracers are under improvement. All of these developments are a testament to the emerging view that tissue pH is a biomedically important parameter of tumor metabolism.