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Many studies have shown that the acidity of solid tumors contributes to local invasion and metastasis. Oral pH buffers can specifically neutralize the acidic pH of tumors and reduce the incidence of local invasion and metastatic formation in multiple murine models. However, this effect is not universal as we have previously observed that metastasis is not inhibited by buffers in some tumor models, regardless of buffer used. B16-F10 (murine melanoma), LL/2 (murine lung) and HCT116 (human colon) tumors are resistant to treatment with lysine buffer therapy, whereas metastasis is potently inhibited by lysine buffers in MDA-MB-231 (human breast) and PC3M (human prostate) tumors. In the current work, we confirmed that sensitive cells utilized a pH-dependent mechanism for successful metastasis supported by a highly glycolytic phenotype that acidifies the local tumor microenvironment resulting in morphological changes. In contrast, buffer-resistant cell lines exhibited a pH-independent metastatic mechanism involving constitutive secretion of matrix degrading proteases without elevated glycolysis. These results have identified two distinct mechanisms of experimental metastasis, one of which is pH-dependent (buffer therapy sensitive cells) and one which is pH-independent (buffer therapy resistant cells). Further characterization of these models has potential for therapeutic benefit., (Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.)

The pH of solid tumors is acidic due to increased fermentative metabolism and poor perfusion. It has been hypothesized that acid pH promotes local invasive growth and metastasis. The hypothesis that acid mediates invasion proposes that H(+) diffuses from the proximal tumor microenvironment into adjacent normal tissues where it causes tissue remodeling that permits local invasion. In the current work, tumor invasion and peritumoral pH were monitored over time using intravital microscopy. In every case, the peritumoral pH was acidic and heterogeneous and the regions of highest tumor invasion corresponded to areas of lowest pH. Tumor invasion did not occur into regions with normal or near-normal extracellular pH. Immunohistochemical analyses revealed that cells in the invasive edges expressed the glucose transporter-1 and the sodium-hydrogen exchanger-1, both of which were associated with peritumoral acidosis. In support of the functional importance of our findings, oral administration of sodium bicarbonate was sufficient to increase peritumoral pH and inhibit tumor growth and local invasion in a preclinical model, supporting the acid-mediated invasion hypothesis. Cancer Res; 73(5); 1524-35. ©2012 AACR., (©2012 AACR.)

Acid-base balance is altered in a variety of common pathologies, including COPD, ischemia, renal failure, and cancer. Because of robust cellular pH homeostatic mechanisms, most of the pathological alterations in pH are expressed as changes in the extracellular, systemic pH. There are data to indicate that altered pH is not simply an epiphenomenon of metabolic or physiologic imbalance but that chronic pH alterations can have important sequelae. MRSI and MRI measurements indicate that pH gradients of up to 1.0 pH unit can exit within 1-cm distance. Although measurement of blood pH can indicate systemic problems, it cannot pinpoint the lesion or quantitatively assess the magnitude of excursion from normal pHe. Hence, there is a need to develop pHe measurement methods with high spatiotemporal resolution. The two major approaches being investigated include magnetization transfer methods and relaxation methods. pH-dependent MT effects can observed with endogenous signals or exogenously applied CEST agents. While endogenous signals have the advantage of being fully noninvasive and relatively straightforward to apply, they lack a full biophysical characterization and dynamic range that might be afforded by future CEST agents. pH-dependent relaxivity also requires the injection or infusion of exogenous contrast reagents. In both MT and relaxographic approaches, the magnitude of the effect, and, thus, the ability to quantify pHe, depends on a spatially and temporally varying concentration of the CR. A number of approaches have been proposed to solve this problem and, once it is solved, pH imaging methods will be applicable to human clinical pathologies.

Ion-trapping theory predicts that alkalinization of tumor extracellular pH will enhance the anti-tumor activity of weak-base chemotherapeutics. We have previously demonstrated that chronic and acute treatment of tumor-bearing mice with sodium bicarbonate results in tumor-specific alkalinization of extracellular pH. Furthermore, bicarbonate pretreatment enhances the anti-tumor activity of doxorubicin and mitoxantrone in two different mouse tumor models. Previous work has indicated subtle, yet significant differences between the pH sensitivities of the biodistribution and anti-tumor efficacies of doxorubicin and mitoxantrone in vitro. The present study demonstrates that systemic alkalinization selectively enhances tumor uptake of radiolabeled mitoxantrone, but not doxorubicin. Results using these two drugs are quantitatively and qualitatively very different, and can be explained on the basis of differences in the octanol-water partition coefficients of their charged forms. These results suggest that inducing metabolic alkalosis in patients would have a positive effect on response to mitoxantrone therapy. However, the therapeutic index would not increase if sodium bicarbonate also caused increased retention of mitoxantrone in susceptible normal tissues in the host. The major dose-limiting organ systems for mitoxantrone are heart, liver, bone marrow, spleen and blood cells. Bicarbonate was found to have no significant effect on the distribution of mitoxantrone to any of these tissues except for spleen. However, neither spleen weights nor lymphocyte counts were adversely affected by NaHCO(3) pretreatment, indicating that this co-therapy does not enhance myelosuppression due to mitoxantrone therapy. These findings suggest that metabolic alkalosis would produce a net gain in mitoxantrone therapeutic index.

The unique physiological environment of solid tumors, frequently characterized by areas of poor flow, hypoxia, high lactate and low extracellular pH (pHe), influences vascularization, invasion and metastasis. Thus, vascularization and the physiological and metabolic environment play permissive (and conversely preventive) roles in invasion and metastasis. By using a multi-parametric approach of combined vascular and spectroscopic imaging, we can begin to evaluate which combinations of vascular, metabolic and physiological regions in a solid tumor represent the highest 'metastatic threat'. Here, we present measurements of pHe, vascular volume and permeability from co-localized regions within a solid tumor. These studies were performed for a group of metastatic (MDA-MB-231) and non-metastatic (MCF-7) human breast cancer xenografts. In this study, we have demonstrated the feasibility of such an approach, and presented methods of analyses to detect differences in patterns of combined parameters obtained from spatially co-registered regions in a solid tumor., (Copyright 2002 John Wiley & Sons, Ltd.)

In vivo pH measurements by magnetic resonance spectroscopy reveal the presence of large regions of acidic extracellular pH in tumours, with the intracellular pH being maintained in the neutral-to-alkaline range. This acid-outside plasmalemmal pH gradient acts to exclude weak base drugs such as the anthracyclines and vinca alkaloids, a behaviour that is predicted by the decrease in octanol-water partition coefficients of mitoxantrone and doxorubicin with decreasing solution pH. This pH gradient can be reduced or eliminated in mouse models of breast cancer by systemic treatment with sodium bicarbonate. We have demonstrated tumour alkalinization following chronic ad libitum administration of NaHCO3 and acute intraperitoneal administration of NaHCO3 to tumour-bearing mice. Chronic treatment of tumour-bearing SCID mice with NaHCO3 results in an enhancement in MCF-7 tumour xenograft response to doxorubicin. Intraperitoneal administration of NaHCO3 to tumour-bearing C3H/Hen mice prior to treatment with mitoxantrone results in a greater-than 4.5-fold increase in cell-kill in the syngeneic C3H mammary tumour model. Most combination chemotherapy regimens include at least one weak base drug. Our results suggest that agents such as sodium bicarbonate, Carbicarb and the diuretic furosemide--which are known to induce metabolic alkalosis in humans--may be useful in enhancing the efficacy of these treatment regimens in humans.

Investigation into the causes and consequences of aberrant pH regulation is entirely dependent on the ability to measure this parameter with specificity and sensitivity. Since their beginnings in the 1950s, techniques for measuring cell and tissue pH have undergone a number of significant advances. Following each of these advances, new investigators have been brought into the field, and new discoveries have been made. From microelectrode and dye distribution studies, measurement of pH underwent a revolution with the advent of pH-sensitive dyes that could be loaded into the cytosol. A further significant advance came from the measurement of cell and tissue pH in whole organisms by magnetic resonance spectroscopy (MRS). Frontiers in pH measurement exist in the development of pH-sensitive proteins and pH-sensitive MR contrast agents. These are predicted to bring even more people into this fascinating field, and generate more important discoveries.

Quantification of metabolite or drug concentrations in living tissues requires determination of intra- and extracellular volumes. This study demonstrates how this can be achieved non-invasively by 31P magnetic resonance spectroscopy (MRS) employing dimethyl methylphosphonate (DMMP) as a marker of total water space, 3-aminopropylphosphonate (3-APP) as a marker of extracellular space and P and 3-APP as markers of intracellular pH (pH) and extracellular pH (pHe) respectively. The MRS measurements of the tumour volumes were validated by classic radiolabelling methods using 3H2O and [14C]inulin as markers of total and extracellular space respectively. The extracellular volume fraction measured by radiolabelling of RIF-1 tumours was 23 +/- 0.83% (mean +/- s.e.m. n = 9), not significantly different (P > 0.1) from that found by MRS (27 +/- 2.9%, n = 9, London, and 35 +/- 6.7, n = 14, Baltimore). In untreated RIF-1 tumours, pH was about 0.2 units higher than pHe (P < 0.01). 5-Fluorouracil (5FU) treatment (165 mg kg(-1)) caused no significant changes in either pHe or per cent extracellular volume. However significant increases in pH, 48 h after treatment (P < 0.01) correlated with decreased tumour size and improved bioenergetic status [NTP/inorganic phosphate (Pi) ratio]. This study shows the feasibility of an MR method (verified by a 'gold standard') for studying the effects of drug treatment on intra- and extracellular spaces and pH in solid tumours in vivo.

As a consequence of poor perfusion and elevated acid production, the extracellular pH (pHex) of tumors is generally acidic. Despite this, most in vitro experiments are still performed at the relatively alkaline pHex of 7.4. This is significant, because slight changes in pHex can have profound effects on cell phenotype. In this study we examined the effects of mildly acidic conditions on the in vitro invasive potential of two human melanoma cell lines; the highly invasive C8161, and poorly invasive A375P. We observed that culturing of either cell line at acidic pH (6.8) caused dramatic increases in both migration and invasion, as measured with the Membrane Invasion Culture System (MICS). This was not due to a direct effect of pH on the invasive machinery, since cells cultured at normal pH (7.4) and tested at acidic pH did not exhibit increased invasive potential. Similarly, cells cultured at acidic pH were more aggressive than control cells when tested at the same medium pH. These data indicate that culturing of cells at mildly acidic pH induces them to become more invasive. Since acid pH will affect the intracellular pH (pHin) and intracellular calcium ([Ca2+]in), we examined the effect of these parameters on invasion. While changes in [Ca2+]in were not consistent with invasive potential, the changes in pHin were. While these conditions decrease the overall amount of gelatinases A and B secreted by these cells, there is a consistent and significant increase in the proportion of the activated form of gelatinase B.

The extracellular pH (pHex) of tumors is generally acidic. However, it is only recently that noninvasive magnetic resonance spectroscopic (MRS) measurements have determined that the intracellular pH (pHin) of tumor cells in situ is neutral or slightly alkaline compared with that of normal tissues. Thus cells in tumors maintain larger pH gradients than do cells in nontumor tissues. To date, measurements of pHex in tumors have been made using microelectrodes, which preclude measurement of pHex and pHin within the same preparation. In addition, microelectrodes are invasive and have the potential to alter the measured pH values. The present communication describes simultaneous measurement of pHex and pHin in vitro in bioreactor culture and in vivo using 31P-MRS analyses of 3-aminopropylphosphonate (3-APP) and inorganic phosphate. In vitro results indicate that 3-APP is not toxic and that its resonant frequency is sensitive to pH and not significantly affected by temperature or ionic strength. Bioreactor experiments indicate that this compound is neither internalized nor metabolized by cells. Experiments in vivo indicate that 3-APP can be administered intraperitoneally and that RIF-1 tumors maintain a steady-state pHin of 7.25 and a pHex of 6.66. These data have significance to basic tumor cell physiology and to the design of approaches to cancer chemotherapy and hyperthermic therapy, because both of these modalities exhibit pH sensitivity. It is also likely that these techniques will be applicable to localized MRS of other organ systems in vivo.

There is abundant evidence implicating a role for intracellular pH (pHin) in the proliferative response of many cells to mitogenic agents. In mammalian cells, pHin is generally regulated by two systems: Na+/H+ exchange and HCO3- transport. Activation of Na+/H+ exchange is one of the earliest responses of mammalian cells to mitogens. In the absence of HCO3-, this activation raises the pHin. However, in the presence of HCO3-, the effect of mitogens on the pHin is unclear. HCO3- regulates pHin via mechanisms which can either acidify or alkalinize the cytosol, depending on the cell type and tissue of origin. BALB/c 3T3 mouse embryo cells are employed in the present study because they are used extensively in investigations of mammalian cell proliferation. Since these cells are of indefinite origin, there is no way to predict which HCO3- transporting system is operable in these cells and, hence, what effect HCO3- will have on the pHin and the response of pHin to mitogens. In the present article, we examine the mechanism and effect of HCO3(-)-based pHin regulation. Our results indicate that HCO3(-)-dependent pHin regulation in BALB/c 3T3 cells occurs via Na-HCO3/HCl exchange which raises pHin under physiological conditions. This activity can raise the pHin to above the set point of the activated Na+/H+ exchanger, consequently attenuating the mitogen-induced Na+/H+ exchange-mediated increases in pHin.

One of the earliest events in the response of mammalian cells to mitogens is activation of Na+/H+ exchange, which increases intracellular pH (pHin) in the absence of HCO3- or at external pH values below 7.2. The proliferative response can be blocked by preventing the pHin increase; yet, the proliferative response cannot be stimulated by artificially raising pHin with weak bases or high medium pH. These observations support the hypothesis that optimal pHin is a necessary, but not sufficient, component of the proliferative-response sequence. This hypothesis has recently been challenged by the observation that transfection of NIH 3T3 cells with yeast H(+)-ATPase renders them tumorigenic. Although previous measurements indicated that these transfected cells maintain a higher pHin in the absence of HCO3-, whether H(+)-ATPase transfection raised the pHin under physiologically relevant conditions was not known. The current report shows that these transfected cells do maintain a higher pHin than control cells in the presence of HCO3-, supporting the possibility that elevated pHin is a proliferative trigger in situ. We also show that these cells are serum-independent for growth and that they glycolyze much more rapidly than phenotypically normal cells.

The phenomenon of intracellular pH (pHin) regulation in cultured Ehrlich ascites cells was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. Measurements were made with a Bruker WH 360 wide bore NMR spectrometer at a 31P frequency of 145.78 MHz. Samples at a density of 10(8) cells ml-1 were suspended in a final volume of 2 ml of growth medium in 10 mm diameter NMR tubes. Intracellular pH was calculated from the chemical shifts of either intracellular inorganic phosphate (Piin) or intracellular 2-deoxyglucose-6-phosphate (2dG6Pin). The sugar phosphate was used as a pH probe to supplement the Piin measurements, which could not always be observed. When available, the pHin calculated from the Piin peak was identical within experimental error to the pHin calculated from the 2dG6Pin peak. Intracellular pH was measured to be more alkaline than the medium at an external pH (pHex) below 7.1. Typical values were pHin = 7.00 for pHex = 6.50. These measurements were constant for times up to 165 min using well-energized, respiring cells. This pH gradient was seen to collapse immediately upon onset of anaerobic shock. Above a pHex of 7.2 there was no significant difference between pHin and pHex. These results unequivocally demonstrate the steady state nature of the pH regulation and its dependence upon energization.

We have analyzed changes in intracellular pH and phosphate metabolism during the cell cycle of Saccharomyces cerevisiae (NCYC 239) by using high-resolution 31P NMR spectroscopy. High-density yeast cultures (2 x 10(8) cells per ml) were arrested prior to "start" by sequential glucose deprivation, after which they synchronously replicated DNA and divided after a final glucose feeding. Oxygenation of arrested cultures in the absence of glucose led to increased levels of sugar phosphates and ATP and an increase in intracellular pH. However, these conditions did not initiate cell cycle progression, indicating that energization is not used as an intracellular signal for initiation of the cell division cycle and that the cells need exogenous carbon sources for growth. Glucose refeeding initiated an alkaline intracellular pH transient only in the synchronous cultures, showing that increased intracellular pH accompanies the traversal of start. Changes in phosphate flow and utilization also were observed in the synchronous cultures. In particular, there was increased consumption of external phosphate during DNA synthesis. When external phosphate levels were low, the cells consumed their internal polyphosphate stores. This shows that, under these conditions, polyphosphate acts as a phosphate supply.

Techniques to determine intracellular pH generally report the average pH of population and do not indicate whether or not there is significant variance among cells within the population. Population variance is important to ascribe pH changes on a per cell basis. The magnitude of the pH change in individual cells is important to ascribe physiological function to changes in pH. To determine the variability of cell responses, we have used dual wavelength fluorescence emission spectroscopy of intracellular dicyanohydroquinone monitored with flow cytometry to determine the pH of normal and transformed 3T3 cells in response to serum or serum components. All cells were mechanically harvested from subconfluent cultures. Large differences in pH were observed between serum-deprived and serum-conditioned normal, but not transformed, cells. Addition of serum caused cytosolic alkalinization, with the serum-deprived cells responding more slowly. Titration of cells with submaximal doses of serum indicate that the response of pH is graded, that all cells respond in similar manner, and that the relative affinity of transformed cells for the serum components causing the pH effect is about twice that of normal cells.