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. Author manuscript; available in PMC: 2013 Aug 12.
Published in final edited form as: NMR Biomed. 2011 Mar 8;24(6):582–591. doi: 10.1002/nbm.1644

Imaging pH and Metastasis

Arig Ibrahim Hashim 1,*, Xiaomeng Zhang 1,*, Jonathan W Wojtkowiakk 1, Robert J Gillies 1
PMCID: PMC3740268  NIHMSID: NIHMS385819  PMID: 21387439

Abstract

Metastasis is a multistep process that culminates in the spread of cells from a primary tumor to a distant site or organs. For tumor cells to be able to metastasize, they have to locally invade through basement membrane into the lymphatic and the blood vasculatures. Eventually they extravasate from the blood and colonize in the secondary organ. This process involves multiple interactions between the tumor cells and their microenvironments. The microenvironment surrounding tumors has a significant impact on tumor development and progression. A key factor in the microenvironment is an acidic pH. The extracellular pH of solid tumors is more acidic in comparison to normal tissue as a consequence of high glycolysis and poor perfusion. It plays an important role in almost all steps of metastasis. The past decades have seen development of technologies to non-invasively measure intra- and/or extracellular pH. Most successful measurements are MR-based, and sensitivity and accuracy have dramatically improved. Quantitatively imaging the distribution of acidity helps us understand the role of the tumor microenvironment in cancer progression. This review discusses different MR methods in measuring tumor pH along with emphasizing the importance of extracelluar tumor low pH on different steps of metastasis; more specifically focusing on Epithelial to Mesenchymal transition (EMT), and anti cancer immunity.

Keywords: Tumor pH, Metastasis, Tumor microenvironment

Metastasis and its sequelae are the leading causes of cancer morbidity and mortality. As shown in FIGURE 1, metastasis involves a series of discrete steps from local invasion and intravasation through to extravasation and colonization (1). Despite intense research over the past few decades, defining the exact molecular mechanisms involved in these steps has remained elusive. Defining metastasis is made more difficult by the large molecular heterogeneity and redundancy in these pathways, combined with the low probability of forming successful metastatic lesions. In typical experimental settings, up to 500,000 cancer cells are injected intraveneously, which leads to the formation of only 50 or 100 metastatic lesions. In aggressive metastatic human cancers, there can be up to 23 million circulating tumor cells at any one time (2).

Figure 1. Stages of Metastasis.

Figure 1

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Primary carcinomas arise from epithelia which face ductal lumens and are separated from stroma by a basement membrane. After disruption of basement membrane integrity the locally invasive cells gain access to lymphatic- and heme-vasculature wherein they form circulating tumor cells, CTCs. At secondary sites (right) the tumor cells extravasate into parenchyma, where they colonize and become vascularized either through co-option or neo-angiogenesis (reprinted by permission. Scientific American.)

The physical microenvironment of tumors (pH, pO2) can have a great impact on the successful formation of metastasis. Solid tumors are believed to contain volumes that are highly acidic (3). This low extracellular pH (pHe) has been associated with tumorigenic transformation, chromosomal rearrangements, extracellular matrix breakdown, increased migration and invasion, and induction of growth factors and proteases (4,5).

Tumor acidity is a consequence of high rates of glucose metabolism, combined with poor perfusion. Experimentally induced hyperglycemia acidifies the tumor microenvironment (6-8) coincident with an increase in the glycolytic rate (9) which can lead to a reduction in extracellular pH, pHe, from 7.4 to 6.7 (10). The end product of fermentative glucose metabolism is lactate, and tumor lactate levels are independently correlated with metastasis, low pH and reduced survival (11,12). However, low pH is not equivalent to lactate, as the hydrogen ion in glycolysis is released at an earlier enzymatic step, namely glyceraldehyde phosphate dehydrogenase, GAPdH. Additionally, movement of carbons through the pentose phosphate pathway can also produce acids. Studies with glycolysis-deficient cells indicate that Embden-Meyerhoff glycolysis is not the only source of extracellular acid in solid tumors. CO2 may be a significant source of acid, as oxidative energy metabolism results in the production of the volatile acid H2CO3 from CO2 might lead to a net H+ production within a solid tumors (13). Furthermore CO2 , may be derived from elevated glutaminolysis (14). A wealth of research evidence showed that acidosis is caused by reactions other than lactate production. In muscles the increased lactate production is coincides with cellular acidosis and remain a good indirect marker for cell metabolic conditions that include metabolic acidosis (15).

Poor perfusion in tumors no only reduces the ability to remove tumor-derived acid; it also leads to regional hypoxia, which can exacerbate fermentative metabolism. It has been proposed that this leads to selection for cells that are resistant to acid and hypoxia-mediated toxicities (16). Notably, hypoxia leads to expression of carbonic anhydrase IX and the sodium-hydrogen exchanger, which can promote the movement of intracellularly produced H+ to the extracellular space (17).

Finally, it must be appreciated that lactate and acidity may be uncoupled also in their effects on cancer cells. As described above, acidosis can occur with or without high lactic acid. Lactate dehydrogenase, LDH-A, and the glucose transporter, GLUT-1, are both regulated by hypoxia via the transcription factor, HIF-1α. As a consequence, tumor cells can produce lactate and co-transport it along with H+, leading to maintenance of pHi and a lowering of the tumor pHe, yet these products can also be transported independently. Gene expression studies using primary human mammary epithelial cells suggest that lactic acidosis and general (HCl) acidosis trigger similar genetic responses, which are distinct from genetic responses to lactate and hypoxia (18). Furthermore, lactate can be a potent transcriptional regulator, even in the absence of acidosis. For example, it has been shown to stabilize and lead to elevated HIF-1α levels (19).

In this review, we will briefly discuss MR-based methods for measuring tumor pH, which was recently reviewed (20,21), and then discuss evidence relating tumor acidity to promotion of metastasis.

Imaging pH with MR

Magnetic resonance spectroscopy (MRS)

MRS and magnetic resonance spectroscopy imaging (MRSI) can provide accurate and precise noninvasive measures of pH in vivo with reasonable spatial resolution. An ideal MR pH indicator should have the following criteria: large pH sensitivity, a pKa in the physiological range, non-toxic at useful concentrations, metabolically stable and provide a high SNR within a short time period. Both endogenous and exogenous NMR-active compounds have been used to measure pH in vivo (TABLE 1). Measuring pH with MRS/MRSI is generally based on a difference in chemical shifts between pH-dependent and independent resonances. If they are in the same compartment, protonation reactions are in fast exchange, and thus the chemical shift can be use to predict the pH. If there are intervening membranes, exchange is slow and compounds at different pHs will resonate at distinct frequencies, giving rise to multiple peaks.

TABLE 1.

Indicators for measurement of pH in vivo with MRS(I) .

Isotope Frequency
(rel 1H)		 % Natural
abundance		 Sensitivity
(rel 1H)		 Compounds Application
1H 1.00 99.98 100 2-imidazole-1-yl-3-
ethoxycarbonylpropionic acid (IEPA)		 pHe
19F 0.94 100 83 3-[N-(4-fluor-2-trifluoromethylphenyl)-
sulphamoyl]-propionic acid
(ZK-150471)		 pHe
Vitamin B6 derivatives pHi pHe
31P 0.41 100 6.6 Inorganic phosphate (Pi) pHi
3-aminopropylphosphonate (3-APP) pHe
13C 0.25 1.1 0.016 Hyperpolarized 13C-labelled
bicarbonate		 pHe
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31P

Phosphorous is an abundant and sensitive MR active nucleus. The 31P isotope is 100% natural abundance, and 31P spectra exhibit a wide variety of compounds. Many small phosphorylated metabolites exist within cells have concentrations in the range of 0.1-10 mM. The most significant NMR-visible endogenous phosphorous signals from mammalian tissues come from NTPs, inorganic phosphate (Pi), and phosphocreatine (PCr). 31P MRS provides a noninvasive technique for simultaneously measuring intracellular pH, pHi, from the chemical shift of endogenous Pi and pHe from the chemical shift of exogenous indicators, such as 3-aminopropyl phosphonate (3-APP) (22). In tumors, the chemical shift of Pi, with a pKa ≈ 6.8, is usually referenced to that of glycerophosphoryl choline (at 0.49 ppm); or α peak from NTP (at −10.05 ppm), and the resulting frequency can be used to estimate pH within 0.05 pH unit after correction. Numerous studies have shown that the Pi measured by in vivo MRS is primarily intracellular and thus, the resonance frequency of this nucleus provides a measure of the intracellular pH (pHi). This has been confirmed by comparing the pH measured by Pi to that measured by intracellulary trapped 2-deoxy glucose 6-phosphate (23,24). These and subsequent studies have shown that pHi of tumors remains neutral to alkaline, even when the extracellular tumor pH is acidic (20). In order to measure pHe by MRS, it is necessary to introduce an exogenous reagent. Although a number of phosphonate-based probes have been developed for measurement of pH, only a few have demonstrated their qualities. Phenylposphonic acid has been used as a 31P-MRS indicator of extracellular pH in perfused tissues (25). Szwergold has also developed a number of phosphonate-based probes, some of which were reported to be extracellular (26). 3-APP is an exogenous indicator widely used to measure the pHe of tumors in animal models. It was developed in 1994 and shown to be nontoxic and membrane impermeant. These studies and others simultaneously measure the pHe and pHi in bioreactors and in vivo using the frequencies of 3-APP and Pi, respectively (27) (FIGURE 2). This approach has been used by numerous groups to measure the pHi and pHe in a variety of tumor xenograft models (28). These studies have demonstrated unequivocally that the pHe of tumors is significantly more acidic than that of normal tissues.

Figure 2. pHe and pHi of tumors and normal tissues by 31P MRS of 3-APP and Pi respectively.

Figure 2

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Subsequent measurements have shown unequivocally that the pHe of tumor xenografts is acidic, whereas the pHi is neutral-to-alkaline.

19F MRS

Similar to 31P, 19F can also measure a pH-induced chemical shift, but with higher sensitivity and less compartment artifacts. Hunjan and Mason developed fluorinated vitamin B6 derivatives as 19F NMR pH indicators that readily across cell membrane providing the measurement of pHi and pHe gradient in vivo (29). Griffiths et al compared 19F probe, 3-[N-(4-fluor-2-trifluoromethylphenyl)-sulphamoyl]-propionic acid (ZK-150471) with 3-APP in three solid tumor type and yields consistent pHe values (30). A drawback common to both 19F and 31P, however, is that MRS is limited in spatial resolution, even at high field.

1H MRSI

Protons are the most abundant and sensitive NMR-active isotope in biological systems. Their sensitivity derives from their high gyromagnetic ratio and isotopic abundance. The most predominant peak in the NMR spectrum comes from the 110 molar protons found in water. However, with solvent suppression techniques, the water resonance can be reduced and 1H resonances from metabolites and indicators can be measured. Thus, compared to 31P, 1H MRS allows for data to be collected in smaller voxels in less time. In general, 1H MRS approaches to measure tumor pHe have relied on compounds, such as imidazoles and aromatics, which resonate at higher frequencies (downfield) compared to endogenous metabolites. Rabenstein and Isab first proposed using imidazoles as extrinsic pHe indicators in 1982 (31). Ballesteros and colleagues have suggested several modifications of the basic structure of the imidazole molecule to improve its performance as an extrinsic pH probe (32). The most popular exogenous imidazole for 1H NMR-sensitive pHe indicator is the pH-sensitive H2 resonance of 2-imidazole-1-yl-3-ethoxycarbonyl propionic acid (IEPA), which has been shown to be non-toxic and to remain extracellular (33). Using magnetic resonance spectroscopic imaging, IEPA has been used to measure pHe in breast cancer tumors and gliomas with spatial resolution up to 1 × 1 × 1 mm3 (FIGURE 3). García-Martín et al. compared the pHe map generated from IEPA MRSI and distribution of metabolites in C6 gliomas to investigate the correlations of pHe and lactate (34). This strategy has been improved by introducing new imidazol probe ISUCA [(±)2-(imidazol-1-yl)succinic acid] which had less effects on tumor metabolism (35). These studies showed that the tumor pHe was heterogeneous, with differences of as much as 0.5 pH units across 8 mm in distance. The relationship between the low pHe and perfusion has been inferred through co-registration with contrast-enhanced images, yet cannot be directly tested by this method.

Figure 3. pHe map of breast cancer xenograft.

Figure 3

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MDA-mb-435 flank tumors were infused with IEPA and pH was measured by 1H MRS.

Hyperpolarized 13C bicarbonate

Although previous isotopes have good pH-sensitive chemical shift dispersion, many others are limited by low sensitivities due to low magnetic energy of nuclear spins at room temperature, even at the high magnetic field. For example, carbon-13 is intrinsically low sensitivity as it has only 1.1% naturally abundant. Hoffman and Henkens have shown that 13C had potential to estimate intracellular CO2 hydration and HCO3− dehydration (36). Arus et al. have investigated pH of superfused frog muscle with natural abundance 13C MRS with a high concentrations of labeled bicarbonate (37). The sensitivity of these applications can be dramatically improved by dynamic nuclear hyperpolarization (DNP), which transfers the high spin polarization of unpaired electrons to neighboring nuclei by microwave irradiation. This has been shown to be capable of enhancing the MR signal of 13C NMR by more than a factor 40,000. Brindle’s group has recently measured tumor pH with hyperpolarized 13C bicarbonate (38). A Henderson-Hasselbach equation was used to calculate tissue pH from the voxel-wise ratio of H13CO3− to 13CO2 following injection of hyperpolarized H13CO3−. The measurement showed the pH (pHi and pHe) of a lymphoma xenograft was significantly lower (6.7± 0.1) than the surrounding normal tissue (7.1 ± 0.1). Although the dynamic pharmacokinetic distribution of bicarbonate results in an unclear average of pHi and pHe (39), the data were compared to that of 3-APP, suggesting that the majority of the 13C signal was extracellular. A limitation of this approach is the rapid signal loss due to decay of the polarization. The T1 relaxation severely restricts the number of phase encoding samples during short acquisition times, as result in limited spatial resolution. However, a fast pulse sequence combined with compressed sensing reconstruction can partially compensate for this limitation.

Magnetic resonance imaging (MRI)

MR relaxometry

Gadolinium (III) complexes are typically used as MRI contrast agents because they are the most efficient of all lanthanide ions at relaxing bulk water protons. Gadolinium-based agents primarily increase relaxivity via T1 mechanisms (1/R1) and hence result in increased signal intensity. It is possible to measure the tissue pH based on perturbing the relaxivity of water via pH-sensitive relaxation agents using MRI. One clever approach to measure pH used a polyion complex (PIC) consisting 30 Gd (III) chelates bound to a poly-ornithine chain (40). The packing conformation of this complex was pH-dependent leading to changes in water accessibility and correlation times and hence changes in R1 relaxivity. Independently, Sherry and Aime groups have synthesized gadolinium-based pH-sensitive agents whose relaxivities are pH-dependent (41,42). For one of these, GdDOTA-4AmP5−, it has been shown that the H-bonding network created by phosphonate side arm protonation provides a catalytic pathway for hydrogen exchange, making the longitudinal water proton relaxivity of this molecule pH-sensitive. Quantification of this measurement is highly dependent on accurate measurement of agent concentration in each imaging voxel (43). Raghunand et al. solved this using a two-phase injection method, involving two different gadolinium agents whose relaxivities are pH-insenstive and –sensitive, respectively. This approach was used to imaging pH in kidneys (44) and later in rat brain glioma (45). An assumption was made that the two agents have identical pharmacokinetics and tissue biodistributions, the distribution of the pH-insensitive agent can be used to predict the concentration of the pH-sensitive agent. Although this approach was useful, it is time consuming and thus limited in its potential for clinical utility. A single injection method has been developed to overcome these drawbacks (46). The pH-sensitive T1 agent GdDOTA-4AmP5− was mixed in a cocktail with the pH-insensitive T2* agent, DyDOTP5 and injected into rats bearing glioma tumors. The ΔR2* effects were used to determine [DyDOTP5−] which could be used to predict [Gd] according to their mix ratio. The concentration thus determined was used to convert ΔR1 of the pH-sensitive CA to pHe maps (FIGURE 4). The primary advantage of this protocol over previous studies is the rapidity of the pHe measurement. A drawback is the need to perform the calibrations in vivo, as the ΔR2* effects are caused by susceptibility changes, and these are tumor-dependent. Nonetheless, a high-resolution pHe map can be generated with acceptable propagated errors at relatively modest concentrations of contrast agents within 16 minutes after start of infusion. In principle, this method is capable of yielding pHe maps following a single bolus injection, which would be appropriate in a clinical setting.

Figure 4.

Figure 4

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In vivo tumor pHe maps were generated using relaxivity-based single injection method, a combination of T1-weighted imaging and spectroscopic imaging in a C6 rat glioma model. A1 and B1 shows T1-W contrast enhancement after CA infusion in two different animals. A2 and B2 are the corresponding tumor pHe maps.

Chemical Exchange Saturation Transfer (CEST)

A novel approach to measure tissue pH is based on pH-sensitive chemical exchange-dependent magnetization transfer. This effect can be described by a simple two pool chemical exchange model, wherein the magnetizations for a labile proton and bulk water are derived by two groups of Bloch equations coupled by chemical exchange (47). The process involves the effect of pre-saturation that is undergoing chemical exchange of a nucleus from one site to a chemically different site. When the rate of exchange is less than the difference in frequency between the exchanging proton and bulk water, a selective pre-saturation pulse saturates the exchangeable hydrogen on exogenous or endogenous compounds, result in the transfer of hydrogen saturation state into bulk water via chemical exchange, thereby reducing the available water signal detected in the imaging experiment. Besides several MRI parameters such as bulk water T1 and RF irradiation power, CEST contrast highly depends on pH, exchangeable site concentration, temperature and other factors influencing the chemical environment of the exchangeable protons. This provides an opportunity to investigate the in vivo pH using CEST effect. pH imaging has become a important direction for CEST and is being evaluated by many groups. For a pH measurement, the hydrogens exchange more slowly at low pH due to base catalysis of proton exchange. Hence, the intensity of the water resonance can be perturbed via pH-sensitive exchange between water protons and those of slowly exchanging ionizable groups. There are three main categories of CEST imaging: diamagnetic CEST (DIACEST), paramagnetic CEST (PARACEST) and amide proton transfer (APT). Wolff and Balaban [46] first demonstrated the possibility of DIACEST imaging in which RF saturation is transferred from exchangeable solute protons to water. Hwang et al introduced a method to obtain accurate chemical exchange rates between water and -NH protons in vivo by avoiding magnetization transfer signal losses for solvent suppression (48). Van Zijl and colleagues improved the signal sensitivity caused by pH changes between 5-10 ppm (49). At same time, Zhang et al and Aime et al reported several PARACEST agents containing both highly shifted pH-insensitive and pH-sensitive exchangeable protons by enlarging the frequency range of exchanging sites (50,51). Most recently Zhou et al. demonstrated an amide proton transfer (APT) effect that is based on the magnetization exchange between bulk water and labile endogenous amide protons. Given this hydrogen exchange is exponentially proportional to physiological pH, the pH contrast can be generated by measuring the exchange rates. pH-sensitive APT imaging has served as a new metabolic biomarker to clinical imaging for brain tissue (52). A drawback to CEST imaging remains the high concentrations required (>10 mM) and the need for strong MR irradiation pulses for pre-saturation, which are limited by power deposition limitations, or SAR. Pagel’s group has recently developed a new PARACEST agent, Yb-DO3A-oAA, with two pH-responsive CEST centers that have different MR frequencies and different dependencies on pH (53). This allows quantification without saturating irradiation and mitigates the SAR problem The ratio of the two PARACEST effects can be used measure the entire physiological range of pHe from 6.1-8.0 with acceptable RF powers.

pH and Metastasis

Low pH effect on different Stages of metastasis

Metastasis involves the dissemination of cancer cells from the primary tumor to distant organs. Although genetically heterogeneous, the stages of metastasis are common in all cancers. It involves detachment of cells from the primary tumor, local invasion following proteolysis of extracellular matrix, disruption of basement membrane, intravasation in to the blood stream to yield circulating tumor cells. Circulating tumor cells, CTCs, are commonly observed in metastatic carcinomas, with a mean value of 60 and a maximum of 23,000 CTCs per 7.5 mL (2). The absolute numbers and molecular phenotypes of CTCs have prognostic value (54). To form metastases, CTCs must extravasate from the blood vessels at a secondary site. Following extravasation, the metastatic cells either co-opt existing vasculature, or induce neoangiogenesis, which coincides with conversion from a micro- to a macrometastasis. Several methods are currently available to follow metastasis in vivo. A valuable technique is optical biolumenesce imaging, which is non-invasive and semi-quantitative. Tumor cells expressing luciferase can be monitored longitudinally, beginning at the time of injection. Thus, these images can monitor the incidence and progression of metastatic lesion, Ex vivo images can also be obtained at the end of experiment to confirm and quantify results (Figure 5)

Figure 5. Measurement of metastasis in vivo and in vitro.

Figure 5

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Experimental Metastases in Intravenous Model - SCID-beige injected with5×10 6 of PC-3M-luc-C6 cells. A. Lung signals are visible after cell injection on day 0. Signals drop as lungs clear between day 1 to week 2. Metastatic signals reappear in week 3-4 and all mice develop multiple signals by week 5. B. Ex vivo imaging confirms metastases in bone and lungs. C. tumor bioluminescence expressed as photons/sec.

pH has shown to affect most of the steps in the metastatic cascade, either directly or indirectly. One of the essential steps in the growth of primary tumors is generation of new blood vessels (angiogenesis). In some systems, low pH can promote angiogenesis through induction of angiogenic growth factors, such as IL-8 and VEGF. Although the data support a generalized acid stimulation of IL-8 production (55), the data for VEGF are more equivocal, with some systems showing acid stimulation of VEGF (56) and others showing acid inhibition (57). Acid pH inversely correlates with VEGF promoter activity in human glioblastoma cells in vitro and xenografts in vivo, the induction of VEGF in Glioblastoma cells is mediated by Ap-1 and apparently ERK1/2 MAPK is required (56,58). Matrix metaloproteases, MMPs, are a family of zinc –binding endopeptidases that play a critical role in tissue remodeling during wound healing and metastasis. Although they have pH optima near pH 7.0, they are secreted as pro-enzymes whose activity is regulated by their conversion to active proteolytic forms. Acid pH can promote the conversion of MMPs. Low pH pre-treatment of human melanoma cell lines (A-07, D12 and T22) promotes experimental pulmonary metastasis in athymic nude mice by upregulating the expression of the proteolytic enzymes MMP-2, MMP-9, cathepsin B and cathepsin L (59). We have observed that low pH induces the release of active cathepsin B from MDA-mb-231 breast cancer cells (unpublished). In vitro, melanoma cells maintained under acidic conditions showed a greater motility and increased invasion when re-acclimated to normal pH (60). Imaging Proteases in cancer using optical (both fluorescent and bioluminescence), Positron emission tomography (PET), magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) was reviewed. Optical imaging and specifically fluorescent is by far the most widely used technique for imaging protease activity in vivo including cathepsin family, and the matrix metalloprotease (MMP) family. Most of the fluorescent probes used are called “activatable probes”, which can change their optical properties after protease cleavage. They are stable in vivo and show high specific labeling (61,62). The effect of acidity on tumor cells intravasation and extravasation has been investigated. The mechanism(s) of extravasation are somewhat controversial, i.e. whether it occurs through lodging of circulating tumor cells in small vessels or whether there is an interaction with post-capillary endothelia followed by active extravasation (63). More recent data support the latter hypothesis. Real-time intravital microscopy of an intracranial window chamber has shown that metastases grew only after a successful active extravasation of single cancer cells from the blood stream into the brain parenchyma (64). Cells were observed in the process of extravasation with intra- and extravascular parts and a narrowing at the vascular wall, suggesting that the cells extravasated through fenestrae in the vascular wall. Acidity may play role in this process of extravasation. In orthotopic breast cancer xenografts, inhibition of tumor acidity by chronic sodium bicarbonate appeared to have a greater effect on extravasation and colonization compared with intravasation (65). The number of circulating tumor cells did not appear to be altered by the bicarbonate treatment, although this conclusion was tempered due to low numbers of circulating tumor cells in this system. In further studies, also it has also been observed that chronic alkalinization with bicarbonate or other buffers will inhibit the formation of experimental lung metastases following tail vein injection in some, but not all tumor models (unpublished).

Epithelial to mesenchymal transition (EMT)

EMT is defined as the reversible change from non-motile epithelial cells to motile mesenchymal cells. This increases invasiveness via the disassembly of cell-to-cell contact, loss of cell polarity, and significant cytoskeletal reorganization. EMT plays a key role in tumor progression and metastasis, and is capable of inducing invasiveness in even normal mammary epithelia (66). Most studies of EMT have been performed in vitro, but a direct role of EMT in vivo has been demonstrated using a transgenic mouse model of (MYC- driven) mammary carcinogenesis (67). The hallmark of EMT is the inactivation of E-cadherin via transcriptional repression of the E-cadherin promoter, chromosomal deletion of the E-cadherin gene, or proteolytic cleavage of the E-cadherin protein (68). The binding of E-cadherin with catenins and actin cytoskeleton is essential for the formation of strong cell-cell adhesion and any event that perturbs this relationship leads to destabilization of cell-cell adhesion and reorganization of the actin cytoskeleton (69). Acid pHe induces c-Src dependent tyrosine phosphorylation of β Catenin, leading to decrease the binding of E-cadherin to β Catenin, and consequently can lead to an increase cell migration and invasion (70).

Does acidosis or lactic acidosis impair anticancer immunity?

An additional component of the metastatic cascade is innate immunity. Cancer cells are synthesizing aberrant proteins which are presented via the major histocompatibility complex (MHC-I/II) and can be potent inducers of immune surveillance. However, by various mechanisms, cancers are known to inhibit immune function. Lactic and general acidosis can affect immune function. Lactic acid impairs immune function including inhibition of polymorphonuclear leukocytes, chemotaxis, respiratory activity, suppression of proliferation and cytokine production of human cytotoxic T lymphocytes, as well as the T- Cell response to tumor -associated antigen (71). On the other hand, acidic extracellular pH can suppress human a subset of cytolytic T cells acting like NK cells in a non–MHC-restricted manner (72). There is also evidence that low pHe impairs the T cell response to antigen in vivo (73). Tumor derived lactate and acidification can inhibit monocyte cytokine (e.g. tumor necrosis factor) production by blocking lactate export and glycolysis efflux (74) . These observations suggest that inhibiting acidosis in tumors could restore immune cell function and anti-tumor response.

Can inhibition of tumor acidity inhibit metastasis?

Acidosis is a commonly observed phenotype, a “hallmark”, of cancer and is thus an attractive therapeutic target. Targeted treatment with proton pump inhibitors disrupt transmembrane pH gradients and can inhibit growth in human B cell leukemia (75). Alternatively, acidosis can be inhibited directly with the ingestion of alkaline buffers, such as bicarbonate (76). Chronic tumor alkalization by bicarbonate reduced spontaneous and experimental tumor metastases in human breast and prostate cancer mouse models (65). These effects were not generalized, however, as fast-growing tumor cell lines showed resistance. Reaction diffusion models predict that other buffers with higher pKa should be more efficacious (77) and preliminary data indicate that alternative buffers can reduce experimental metastasis in a prostate cancer model.

Conclusion

Solid tumors invariably contain volumes that are highly acidic. While a number of techniques have been developed to image pH in vivo, improvements are needed with higher spatial and temporal resolution and that can be applied in human patients. In animal models the more acidic tumors are more aggressive and there has long been thought that the low pH of tumors stimulated tumor progression and metastases. Recent data would appear to support this, as ingested buffers can neutralize tumor acidity and lead to a reduction in metastases. However, the mechanisms by which acidity inhibits metastasis are elusive. There are multiple steps of metastasis that could each exhibit pH-sensitivity. Newer tools of molecular and functional imaging will play an important role in identifying and characterizing these mechanisms.

Abbreviations

EMT

Epithelial to Mesenchymal transition

pHe

extracellular pH

pHi

intracellular pH

MRS

Magnetic resonance spectroscopy

MRSI

magnetic resonance spectroscopy imaging

Pi

inorganic phosphate

3-APP

3-aminopropyl phosphonate

IEPA

2-imidazole-1-yl-3-ethoxycarbonyl propionic acid

DNP

Dynamic Nuclear Hyperpolarization

CEST

Chemical Exchange Saturation Transfer

APT

amide proton transfer

PARACEST

paramagnetic CEST

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