Spatiotemporal pH Heterogeneity as a Promoter of Cancer Progression and Therapeutic Resistance

Abstract

Dysregulation of pH in solid tumors is a hallmark of cancer. In recent years, the role of altered pH heterogeneity in space, between benign and aggressive tissues, between individual cancer cells, and between subcellular compartments, has been steadily elucidated. Changes in temporal pH-related processes on both fast and slow time scales, including altered kinetics of bicarbonate-CO₂ exchange and its effects on pH buffering and gradual, progressive changes driven by changes in metabolism, are further implicated in phenotypic changes observed in cancers. These discoveries have been driven by advances in imaging technologies. This review provides an overview of intra- and extracellular pH alterations in time and space reflected in cancer cells, as well as the available technology to study pH spatiotemporal heterogeneity.

Keywords: acidosis; carbonic anhydrase; hyperpolarized 13C MRI; interstitial pH; lactic acid; magnetic resonance imaging; positron emission tomography; tumor heterogeneity; tumor microenvironment.

Figure 1

Spatiotemporal pH heterogeneity in cancer. Upper right inset: Proton extrusion mechanisms (red arrows and labels) employed by tumor cells include transport proteins such as monocarboxylate transporter 4 (MCT4), sodium-proton exchanger 1 (NHE1), or vacuolar-type ATPase. Alternatively, protons can be titrated with imported bicarbonate (HCO₃-), which then diffuses out of the cell as CO₂. Carbonic anhydrase 9 (CAIX) and potentially other extracellular isoforms catalyze bicarbonate-CO₂ exchange in order to reduce CO₂ back-diffusion into cells and induce interstitial proton release. High proton extrusion flux leads to an acidic pH_e. Cells may also experience systemic fluctuations in CO₂ (green arrows and labels), which induces pH_i fluctuations in cells expressing intracellular carbonic anhydrase isoforms. Upper left inset: Variations in pH lead to alterations in protonation states of proteins with pH-sensitive amino acid residues, thereby causing structural changes that affect protein function. Middle inset: Cancer cells may alter pH on a subcellular basis. Intracellular and extracellular pH spatial heterogeneity can promote focal adhesion formation and/or degradation for cellular migration. Additionally, altered lysosomal pH can facilitate drug resistance. An acidic pH_e is associated with immune cell anergy, drug localization to the extracellular space, and extracellular matrix remodeling. Lower section: pH heterogeneity may also exist on the level of tissues. Certain tumor cells may lower their pH_i in order to reduce proliferation and maintain capacity for differentiation. pH_e gradients can be sculpted in a tumor depending on metabolic differences between cells (e.g., glycolytic vs. oxidative metabolism) in combination with the proton extrusion mechanisms employed. Finally, systemic CO₂ fluctuations can alter pH depending on CA expression and localization.

Figure 2

Mechanisms of pH measurement in cells and tissues. As a general rule, a pH-sensing agent must contain at least one functional group with a pK_a within the physiological range of detection to generate image contrast. (a) Agents may demonstrate pH-dependent cell binding or uptake. In this case, a pH decrease can trigger a change in cell permeability, membrane binding, or release of a prodrug agent which can bind to cells. Importantly, absolute pH quantification is not possible. This approach is used primarily for pH imaging with positron emission tomography (PET) or single-photon emission computerized tomography (SPECT). (b) MR-based agents may interact with water protons in a pH-dependent manner, in which pH induces changes in relaxivity or in exchange (as in acido-chemical exchange saturation transfer, acidoCEST). (c) The protonated and deprotonated states of an agent may emit different electromagnetic frequency waves. In this case, the ratio of emission between the two wavelengths can be used to determine the pH. This approach is relevant to fluorescent-based pH probes as well as hyperpolarized (HP) [¹³C]bicarbonate. (d) If the kinetic rate of protonation–deprotonation is much faster than the absolute frequency difference between emission wavelengths, the agent will exhibit a frequency shift rather than two distinct emission wavelengths. The observed frequency depends on the relative populations (ρ) of protonated and deprotonated states, thereby giving the pH. This approach describes pH imaging with MR spectroscopic techniques (¹H, ³¹P, HP ¹³C).

Figure 3

Hyperpolarized imaging of pH_e, lactate conversion, and perfusion can be performed in a single imaging study. Data are shown for a transgenic adenocarcinoma of the mouse prostate (TRAMP) animal model displaying a consolidated, high-grade tumor confirmed with histology. HP images of (a) extracellular pH, (b) lactate-to-pyruvate (Lac/Pyr) ratio, and (c) urea signal intensity are shown overlaid on ¹H anatomical images, enabling voxel-to-voxel correlations in order to study the interplay between metabolism and acidification.