Manipulating extracellular tumour pH: an effective target for cancer therapy

The pH in tumour cells and the tumour microenvironment has played important roles in cancer development and treatment. It was thought that both the extracellular and intracellular pH values in tumours are acidic and lower than in normal cells. However, recent progress in the measurement of pH in tumour tissue has disclosed that the intracellular pH (pHi) of cancer cells is neutral or even mildly alkaline compared to normal tissue cells. This review article has summarized the recent advancement in the measurement pHi and extracellular pH (pHe) in cancer cells, and the effect of pHi and pHe on proliferation, migration and biological functions of cancer cells. This paper has also elaborated recent treatment strategies to manipulate pHi and pHe for cancer treatment. Based on the recent progress in pHi and pHe manipulation in cancer treatment, we have proposed potential nanoparticle-based strategies to manipulate pHi and pHe to effectively treat cancer.


Introduction
Cancer is one of the most severe diseases in the world. According to statistics, in total 8.8 million people died from cancer in 2015, accounting for 17% of the total deaths. 1,2 Researchers have made great efforts to understand the pathogenesis and properties of cancer in order to develop effective treatments for clinic application. As known, extracellular and intracellular pHs in tissues affect the function of the cells and play an important role in cancer development and treatment. As reported, the extracellular pH (pH e ) affects the proliferation of human T cells and the expression of the interleukin-2 receptor. 3 It is widely accepted that the pH e of cancer cells is more acidic than normal cells. [4][5][6] Generally, pH e values of the normal tissues (brain tissues, subcutaneous tissues, etc.) are in the rage of 7.2-7.5. However, pH e of tumour cells is mildly acidic in the range of 6.4-7.0. Since Warburg et al. rst reported the abnormal anaerobic glycolysis in tumour cells, they measured the glucose and lactic acid in tumor veins and found more lactic acid and less glucose on the tumour tissue than on the normal tissue due to the fermentation process in the tumour side, which may affect pH e and pH i in tumour. 7 Consequently, it has been assumed that pH e and pH i in cancer cells should be more acidic than those in normal cells during 1930s to 1980s. 7,8 With the progress on sensing technologies, several techniques have been developed to measure pH i and pH e in cancer cells including pH-sensitive nuclear magnetic resonance spectroscopy (MRS), positron emission tomography (PET) radiotracers, magnetic resonance imaging (MRI) and optical imaging (Optics). 9 It has been found that pH i in cancer cells is actually mildly alkaline or near neutral, similar to normal cells. 10,11 These new ndings subvert the traditional assumption that the pH i in cancer cells is more acidic than normal cells. More extracellular acidity and more intracellular alkalinity means a smaller ratio of pH e /pH i .
Subsequently, researchers have investigated the mechanisms of pH controls in cancer cells and microenvironments. Numerous membrane transporters across tumour cells have been found for pH hemeostasis in cancer cells, and further been used to manipulate pH e and pH i . 2,[12][13][14][15][16][17] These novel strategies have been developed to control the pH e /pH i ratio in cancer microenvironments and cells to induce apoptosis of cancer cells, improving the treatment efficiency. 18 In this review, we have summarized the recent progress on the studies of pH e and pH i in tumour tissues and their corresponding normal tissues. Then, we have further outlined the mechanisms of pH e /pH i maintenance in cancer cells and the developed therapeutics to manipulate the pH e /pH i in cancer tissues. In the outlook, the potentials of new strategies using state-of-art nanotechnology to manipulate the pH e /pH i in cancer tissues have been proposed for cancer treatment.
2. pH e /pH i in tumour tissues versus normal tissues
Although several novel MRI and optical imaging agents (probes) have been developed and applied for in vivo pH measurement, there is not adequate data of the pH values measured by the same MRI or Optics method for comparable analysis. Thus, for the consistency of the comparison, the pH measured by POT or MRS were collected and compared in Section 2.2 and 2.3.

Extracellular pH (pH e )
According to the literature reports, pH e of eight types of tumour tissues and the corresponding normal tissues has been summarized in Fig. 1. These data were selected based on the measurements using pH-sensitive electrodes. 4,6,[34][35][36][37][38][39] As shown in Fig. 1, pH e of cancer cells is 0.3-0.7 pH unit lower than that of corresponding normal cells. For example, malignant melanoma tissues have an average pH e of 6.96 while the average pH e in normal skin cells is 7.39, 4 which is 0.43 difference. The average pH e in vulvar tumours is 7.26, 0.7 pH unit less than in normal vulvar tissues (with an average pH e of 7.96). 6 Uterine tumour tissues also have a lower average pH e (6.92) than normal uterus, whose average pH e is 7.64. 6 Although the average pH e of two kinds of brain tumours is slightly different, they are both more acidic than normal brain tissues. 38 Similar results have also been observed in other tissues, such as lung, 34 breast, 39 and skeletal muscle. [35][36][37] Thus, it is very clear that most cancer cells usually have a more acidic pH e than their corresponding normal cells, and the differences vary from 0.3-0.7.
Warburg et al. proposed that tumour cells used glycolysis rather than oxidative phosphorylation to acquire energy, even in the presence of oxygen. 7 Excess anaerobic glycolysis has been considered as the major reason for the extracellular acidity of tumour tissues. 10,40 For most animal cells, there are two different pathways for glucose metabolism, i.e. aerobic and anaerobic glycolysis. The detailed processes of glucose metabolism in the cells have been briey outlined in Fig. 2. There are two possible pathways for glucose metabolism in the cells: aerobic and anaerobic pathway. Generally, one glucose molecule is metabolized to two pyruvate molecules, producing two ATP molecules as the energy. In the aerobic pathway, two pyruvate molecules react with CoA-SH and form acetyl-CoA by releasing CO 2 . Subsequently, the produced acetyl-CoA undergoes the citric acid cycle, nally degrading into CO 2 and producing 30 ATP molecules. In the anaerobic process, two pyruvate molecules transfer into two lactate molecules with the assistance of lactate dehydrogenase, but this transfer only produce 2 ATP. The overall reactions of these two ways are briey expressed as follows: In the normal cells, most glucose is fully metabolized to produce carbon dioxide, water and the energy via the aerobic pathway. However, in the tumour cells, the glucose is mostly metabolized through the anaerobic pathway, which produces a large amount of lactate and releases limited energy due to a high level of pyruvate and hypoxia in the tumour environment. During the process, the tumour growth requires a large amount of energy compared to the normal tissue, which produces more CO 2 and lactic ions in tumour. The produced CO 2 was excreted extracellularly, resulting in the acidic condition in the tumour microenvironment, i.e. 0.3-0.7 pH units lower than the average pH e of normal tissues.

Intracellular pH i : acidic or not?
Interestingly, pH i of cancer cells is not acidic, not as postulated previously. Since the 1980's, more research outcomes have demonstrated that pH i of cancer cells is around neutral and even mildly alkaline. 10,11 Fig. 3 has displayed the pH i of six kinds of tumour tissues and their corresponding normal tissues collected from MRS method. 11,[41][42][43][44][45] Very surprisingly, the average pH i of these tumour cells is slightly higher than that in their corresponding normal cells, although the difference is less than 0.1 pH unit and not signicant. For example, the average pH i of brain tumours is 7.31, slightly higher than normal brain cells (7.24). 11,42,46 Redmond et al. reported that the intracellular environment of osteosarcoma cells is also mildly more alkaline than in normal cells. 43 Furthermore, this weak alkalinity of the intracellular environment in tumour cells has also been discovered in many other types of tumours, such as hepatoblastoma 11 and squamous cell carcinoma. 44,45 These evidences thus clearly indicate that pH i of tumour cells is near neutron or even more alkaline. Thus, the discrepancy of pH e and pH i in tumour cells is much larger than in normal tissues.

How cancer cells maintain their unbalanced pH e /pH i ratio?
For most cells, the maintenance of neutral (or mild alkaline) pH i is achieved by transporting respiratory end-products (such as CO 2 and lactate) across the cell membrane. When the extracellular concentration of acidic respiratory end-products is lower than intracellular, the excess CO 2 can passively across the cell membrane by diffusion. 47 However, in most cases, the CO 2 and lactate generated from glucose metabolise is accumulated in extracellular tumour site due to low blood ow rate, resulting in development of acidic microenvironments in tumour. 48,49 In this situation, the release of CO 2 and lactate in microenvironments mainly relies on numerous special membrane proteins, such as carbonic anhydrase enzymes (CA2, CA9 and CA12). More relevant pH regulators are listed in Table 2 and discussed in Section 3. Overall, the maintenance of pH e and pH i is based on passive diffusion and active membrane transporters. Table 2 briey summarizes some major pH regulators in tumours and their main functions, including anion exchangers (SLC4A1, SLC4A2, and SLC4A3), proton transporter vacuolar ATPase (V-ATPase), mono-carboxylate transporters (MCT1, MCT2, MCT3, and MCT4), sodium ion based chloride/bicarbonate exchanger (SLC4A8) and Na + /H + exchanger 1 (SLC9A1). [50][51][52][53][54][55][56][57][58][59] In the last two decades, several complicated mechanisms have been revealed about how cancer cells maintain the alkaline pH i and acidic pH e . 60-65 Among them, the mechanism for the import of weak bases (e.g. bicarbonate) and the extrusion of weak acids (e.g. CO 2 , H 2 CO 3 , and lactate) with the assistance of proteins in tumour cell membrane has been clearly demonstrated. 66 Apart from this, the intracellular protons have been pumped out of tumour cells in three different ways, including direct discharge from the cells, exchange with other extracellular cations (e.g. Na + ), and extrusion by the vacuolar ATPase. 56,67 3. The effect of pH e and pH i on tumour activity As discussed above, the difference of pH e and pH i in tumour cells is much larger than in normal cells. The maintenance of pH e and pH i in the tumour mainly relies on some specic proton pumps and intracellular buffer systems. 10,68,69 For instance, the balance of HCO 3 À /CO 3 2À buffer system in tumour is administrated by carbonic anhydrase enzymes CA2, CA9 and CA12. 12,70,71 Besides, the Na + /H + buffer system is manipulated by Na + /H + exchangers, such as SLC9A1. 72 The regulation of pH e and pH i depends on the synergic effect of all of these pumps and buffer systems. It is known that even the little change of pH e /pH i ratio may severely affect many biological and chemical processes in the cells, and eventually result in the proliferation and aggressiveness of cancer cells. 60 For example, the incubation of melanoma in the acidic environment can signicantly enhance its metastasis, aggressiveness and migratory activity in vitro. 73 Martinez-Zaguilan reported that C8161 and A375P cells were cultured in acidic medium (pH 6.8) for 3 weeks and then transferred to the membrane invasion culture system (MICS) chambers. 73 They found that C8161 cells and A375P cells treated in acidic  medium have signicantly enhanced migration and invasion, as shown in Fig. 4. Moellering et al. also reported that acidictreated C8161 cells cultured in normal medium (pH 7.4) showed higher aggressiveness than those cultured in acidic environment (low pH group) and control (native group), as shown in Fig. 5. 74 The C8161 cells cultured in lower pH medium (6.7) has shown the inhibition of the cell invasion, indicating less aggressiveness. These results have demonstrated that the regulation of pH e and pH i ratio in the tumour is highly important for metastasis, aggressiveness and migratory activity. Fine control of pH e and pH i in tumour may improve the cancer treatment.   Furthermore, the slight change of pH e and pH i may also disorder the function of some proteins (such as tenascin and bronectin), particularly in cancer cells. 75,76 For example, mild change of environmental pH by 0.7 pH unit dramatically affected the RNA alternative slicing. The major expression of tenascin-C (TN-C) isoforms was 8 kb TN mRNA in human skin broblasts at pH 7.4, while 6 kb TN mRNA isoform was the majority of TN-C expression at pH 6.7 (see Fig. 6).
Tumour microenvironment triggers the tumour heterogeneity during the cancer development. It is well known that acidic condition and hypoxia are important characteristics in the tumour microenvironment. The homeostasis of pH e and pH i is very important for all kinds of cells. As discussed above, compared with normal cells, cancer cells have a more acidic pH e and more alkaline pH i , suggesting that the pH homeostasis regulation of tumour tissues may be more complex and involve in more proteins and buffer systems. The pH environment may inuence the growth and function of the cells in two main ways. On the one hand, the 0.1 alteration in the ratio of pH e /pH i may affect many essential biochemical processes in the cell metabolism system, such as ATP synthesis, cell proliferation, aggressiveness, migration and diffusion, and the function of some membrane proteins. 60 On the other hand, the tiny disturbance of pH e may activate the mechanism of alternative splicing of constituents in extracellular matrix to produce isoform of tenascin and bronectin, which specically occur in cancer cells rather than in normal cells. 75,76 Although the isoforms of these alternatively spliced proteins do not involve in the manipulation of tumour's pH e /pH i ratio, they may provide binding sites for antigen-based cancer therapy. 18 4. Strategies to manipulate the pH e / pH i ratio As discussed above, the small change in pH e /pH i ratio of tumour cells may disturb many biological functions, including proliferation, aggressiveness, and migration. This relationship demonstrates that adjusting the pH e /pH i ratio in the tumour tissues may halt cancer progress or even completely inhibit cancer growth. In recent years, several approaches have been developed to manipulate pH e /pH i ratio for cancer treatment. These approaches can be classied as direct manipulation and indirect manipulation. Direct manipulation is to regulate the pH e /pH i ratio of tumour cells by using acidic/alkaline drugs and indirect manipulation is based on operating the pH regulators of tumour cells.

Direct manipulation using small molecule drugs
The drugs for direct manipulation are mainly small molecular substances (such as bicarbonates). This approach is to directly increase pH e of tumour tissues to the normal level (0.3-0.7 pH unit). It can be achieved by oral administration of alkaline agents or even by simple adjustment of diet habit.
The alkaline agents include sodium bicarbonate and trisodium citrate. 77 In practice, it seems difficult to maintain the mildly alkaline microenvironment near tumour tissues via oral administration, as a high dose and continuous intake of the alkaline substrate is required. Based on the breast cancer study, White et al. investigated the exact daily dose of sodium bicarbonate needed for breast cancer treatment. 78 The calculated daily dose for a normal human (with 70 kg weight) would be 31.75 g sodium carbonate or 32.5 g trisodium citrate. 79 Another example is the Tris-base buffer to inhibit tumour progression and metastasis. 80 The size of the pancreatic tumour in the mouse model was signicantly decreased aer 200 mM of Trisbuffer treatment. Based on their data, the daily dose for the mice can be calculated as 18.2 g of Tris-base buffer per kg, equivalents to 31 g of Tris-base intake per day for an adult (70 kg). Although it is possible for a cancer patient to intake more than 30 g alkaline agents (such as sodium carbonate or trisodium citrate) with daily drinking water, it would be more efficient to deliver alkaline agent to the tumour tissues rather than to the whole body. A recent non-randomized controlled study investigated the efficacy of local infusion of alkaline agent. 81 Researchers found that there was a 6.4-fold difference of geometric mean of viable tumour residues (VTR) when the hepatocellular carcinoma patients were treated with transarterial chemoembolization (TACE) accompanied with or without locally infusing bicarbonate (LIB) into tumour (Table  3). Such a local administration may be a better strategy for anticancer therapy.
The adjusted diet could be low in protein but high in potassium and/or magnesium. [82][83][84] It has been proved that potassium can effectively neutralize mineral acidity and even mildly alkaline pH of urine via KHCO 3 generation or glutamine sparing. 85 The pH i may be altered by a large change of the intake of potassium due to its fundamental physiologic and metabolic importance. 85 Based on another big data analysis (based on more than 300 000 cases), the risk of suffering from pancreatic cancer decreased by 18% for each 100 mg increase of magnesium intake per day by men on the continuous scale. 86 These results may provide a diet-based way to manipulate the pH environment in vivo and assist cancer treatment.

Indirect manipulation: proton pump inhibitors
The second alternative strategy to administrate the pH e /pH i ratio is to inhibit the functional proton pumps. It is well known that the maintenance of high pH e /pH i ratio in tumour tissues relies on many proton regulators (pumps) on the cell membrane. Most of these proton pumps on the tumour cell membrane have a few specic isoforms that do not exist on the normal cell surface. Thus these isoforms may provide some specic target sites for cancer therapy. Once these functional proton pumps are inhibited, the pH balancing system of tumour cells may be disordered and the pH e /pH i ratio may increase. The abnormal proton transportation and change of the pH e /pH i ratio may affect the behaviour of tumour cells. Recent research reports have demonstrated that the inhibition of proton regulators have suppressed the proliferation and promoted the programmed cell death in some tumour cell lines. [87][88][89][90][91][92] For example, treatment with proton pump inhibitors led to the induction of apoptosis in many types of gastric cancer cells, which involves in the regulation of tumour pH. 89 Besides, the inhibition of proton extrusion by Na + /H + exchanger inhibitors 72 or V-ATPase inhibitors 93 may make cancer cells susceptible or vulnerable. Now a few proton pump inhibitor drugs have been used in the clinical stage. Table 4 lists some inhibitors and their target proton pumps. 90,[94][95][96][97][98][99][100][101][102] As seen in Table 4, the current inhibitor drugs mainly focus on two major pH regulators (V-ATPase and SLC9A1) and only one of these drugs, cariporide, has been successfully developed to phase III clinical trial.
Interestingly, decreasing pH i may increase hyperthermia efficacy (over 42 C) and the programming cell death response to TNF (tumour necrosis factor) induced by apoptosis ligand, also known as TRAIL. 96,[103][104][105] For example, both balomycine A1 (an inhibitor of V-ATPase) and EIPA (an inhibitor of the Na + /H + exchanger) increased the thermo-sensitivity of the AsPC-1 tumours (grown in nude mice) by individually mildly decreasing pH i , and the thermo-sensitivity was markedly enhanced by the sharp decrease in pH i , resulting from the synergetic effect of the combination of these two therapies. 96 Fig . 7 outlines the functions of some specic pH regulators and relatively main inhibitors for tumour cells. The functions of these ion exchangers and proton pumps, and their main inhibitors have been described in Tables 3 and 4 The overall process of ion exchangers is the cellular intake of HCO 3 À and cellular exhaust of CO 2 and Cl À , which both lead to pH e decrease and pH i increase. Proton pumps (or more exactly Na + / H + exchangers) directly exchange intracellular H + with extracellular Na + . To conclude, more targeting sites and relevant inhibitors need to be explored in order to more efficiently manipulate pH e / pH i in tumour cells for potential and effective cancer therapy.

Alternative methods to manipulate the pH e /pH i ratio
Several research reports have showed that the apoptosis of tumour cells can be boosted by the adequately large decrease of their pH i . [87][88][89][90][91][92] One way to achieve the reduction of pH i in tumour cells is to promote cancer glycolysis to the utmost extent by maximizing the glucose supplement. The extremely high rate of glycolysis may break the capacity of proton pumps in tumour cells, which means that tumour cells cannot timely transport acidic metabolites (such as H + , H 2 CO 3 , lactate etc.) outside and hence decreases pH i . For example, a very high glycolysis rate was observed in human melanoma cells (cultured in the medium containing high amount of glucose) when DNP, an uncoupling agent, was added. 106 The programmed cell death can also be activated by the sharp reduction of pH i , nally leading to cell death. 102 This may result from several different mechanisms, one of which is the reduction of glycolysis metabolism. 107 For example, the enzymatic functions of hexokinase, one of the vital enzymes for the maintenance of the high level of glycolysis metabolism in tumours cells, was strongly inhibited (activity decreased from 82 AE 3.2% to 31.2 AE 5.7) with the sharp decrease (from 8 to 6 respectively) of pH i in SNB-19 glioma cells. 108 The tumour glycolysis can be promoted by inhibiting the production of mitochondrial ATP, which requires some specic inhibitors. meta-Iodobenzylguanidine is one of the inhibitors of mitochondrial complex 1, acting as proton extrusion inhibitors (or hyperglycemia) and then decreasing pH i in cancer cells. 100,[109][110][111] However, this drug is normally used as a radioiodine therapy agent, and the dose used for radioiodine therapy is not high enough to perform a strong inhibition on proton transportation. Dinitrophenol (DNP), a new type of chemotherapeutic drugs, has also shown a remarkable enhancement in glycolysis with the increase of blood pressure at a low dose. It has been reported that mM-level DNP can inhibit the proliferation of cancer cells and lead to apoptosis in the human pulmonary adenocarcinoma Calu-6 cell line. 112 Overall, even though there are some drugs (such as metaiodobenzylguanidine and DNP) that have shown their ability to decrease pH i by boosting the glycolysis rate in tumour cells, the hyperglycemia-reliable mechanism restricts the feasibility of this cancer therapy strategy.

Conclusions and future prospective
In this review, pH e and pH i in tumour cells have been summarized and the ways to manipulate cellular pH in cancer cells have been discussed. It is clear that tumour cells have a more acidic pH e (0.3-0.7 lower) than normal cells, and pH i in tumour cells is neutral or even more alkaline than that in normal cells. The abnormally high ratio of pH e /pH i in tumour cells is due to the high rate of glycolysis in tumour cells, which produces numerous acidic products (such as H 2 CO 3 and CO 2 ). The maintenance of pH e /pH i relies on several special proton pumps on tumour cell membranes, such as SLC9A1 and V-ATPase. Then the mechanisms of these proton pumps are discussed and two potential pH manipulating strategies are presented, including direct manipulation by delivering small molecule drugs and indirect manipulation sing proton pump inhibitors.
It has been demonstrated that treatment of cancers (halting its proliferation, aggressiveness and even inducing programmed cell death) is very possible by manipulating pH e /pH i ratio in tumour. A future potential method is to combine 2 or 3 inhibitors so that pH e /pH i can be well controlled, which may signicantly enhance the efficacy of the cancer treatment.
The other future approach to manipulating the pH e /pH i ratio for cancer treatment is to use functional nanoparticle delivery systems to efficiently transport the known inhibitors. Compared to small molecular inhibitors, nanoparticles could have more advantages. For example, nanoparticles can be accumulated around tumour tissues through enhanced permeability and retention effect (EPR effect). 113 Of course, inhibitor-loaded nanoparticles can be further functionalized with target ligands, which may enhance the accumulation in the tumour tissues and manipulate the pH e /pH i ratio.
Another potential way to direct pH manipulation can be achieved by target delivery of alkaline nanoparticles to the tumour tissues by virtue of EPR effect. Thus, accumulated alkaline nanoparticles neutralize the extracellular acids and efficiently increase pH e . Moreover, some alkaline nanoparticles can be modied as a carrier for delivering anticancer drugs to more efficiently treat cancers. 114,115

Conflicts of interest
There are no conicts to declare.