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.
Mammalian cells generally regulate their intracellular pH (pHi) via collaboration between Na(+)-H+ exchanger and HCO3- transport. In addition, a number of normal mammalian cells have been identified that express H(+)-adenosinetriphosphatases (ATPases) in their plasma membranes. Because tumor cells often maintain a high pHi, we hypothesized that they might functionally express H(+)-ATPases in their plasma membranes. In the first phase of the present study, we screened 19 normal and tumorigenic human cell lines for the presence of plasmalemmal H(+)-ATPase activity using bafilomycin A1 to inhibit V-type H(+)-ATPase and Sch-28080 to inhibit P-type H(+)-K(+)-ATPase. Bafilomycin A1 decreased pHi in the six tumor cell lines with the highest resting pHi in the absence of HCO3-. Sch-28080 did not affect pHi in any of the human cells. Simultaneous measurement of pH in the cytoplasm and in the endosomes/lysosomes localized the activity of bafilomycin to the plasma membrane in three cell lines. In the second phase of this study, these three cell lines were shown to recover from NH4(+)-induced acid loads in the absence of Na+. This recovery was inhibited by N-ethylmaleimide, bafilomycin A1, and ATP depletion and was not significantly affected by vanadate, Sch-28080, or hexamethyl amiloride. These results indicate that a vacuolar type H(+)-ATPase is expressed in the plasma membrane of some tumor cells.
Although considerable progress has been made toward understanding glioblastoma biology through large-scale genetic and protein expression analyses, little is known about the underlying metabolic alterations promoting their aggressive phenotype. We conducted global metabolomic profiling on patient-derived glioma specimens and identified specific metabolic programs differentiating low- and high-grade tumors, with the metabolic signature of glioblastoma reflecting accelerated anabolic metabolism. When coupled with transcriptional profiles, we identified the metabolic phenotype of the mesenchymal subtype to consist of accumulation of the glycolytic intermediate phosphoenolpyruvate and decreased pyruvate kinase activity. Unbiased hierarchical clustering of metabolomic profiles identified three subclasses, which we term energetic, anabolic, and phospholipid catabolism with prognostic relevance. These studies represent the first global metabolomic profiling of glioma, offering a previously undescribed window into their metabolic heterogeneity, and provide the requisite framework for strategies designed to target metabolism in this rapidly fatal malignancy.
Tumor pH is physiologically important since it influences a number of processes relevant to tumorigenesis and therapy. Hence, knowledge of localized pH within tumors would contribute to understanding these processes. The destructiveness, poor spatial resolution, and poor signal-to-noise ratio (SNR) of current technologies (e.g., microelectrodes, 31 P magnetic resonance spectroscopy) have limited such studies. An extrinsic chemical extracellular pH (pH e ) probe is described that is used in combination with 1 H magnetic resonance spectroscopic imaging to yield pH e maps with a spatial resolution of 1 ؋ 1 ؋ 4 mm 3 . Since the discovery of lactic acid production in tumors more than 50 years ago (1), it has generally been assumed that the pH of tumors is acidic. Indeed, numerous microelectrode measurements have shown that extracellular tumor pH (pH e ) is acidic (2). This acidic pH e of tumors has been confirmed with less invasive 31 P magnetic resonance spectroscopy (MRS) measurements (3). Although the intracellular pH (pH i ) of tumors remains neutral to alkaline (4,5), it is somewhat influenced by the pH e (6).An acidic pH e of tumors is physiologically important since it influences a number of processes relevant to carcinogenesis and therapy. Knowledge of localized pH within tumors, both intra-and extracellular, would allow more detailed study of these processes and relate them to intratumoral pH heterogeneity. For example, it has been found that low pH e in vitro causes tumorigenic transformation of primary Syrian hamster embryo cells (7) and can lead to chromosomal rearrangements in Chinese hamster embryo cells (8,9). Furthermore, culturing cells at low pH causes them to be more invasive in vitro (10) and metastatic in vivo (11). Finally, the orientation of the pH gradient across the cell membrane may influence cell drug resistance (6,12) Previously reported measurements of extracellular pH using either microelectrodes or 31 P MRS of 3-aminopropylphosphonate (3-APP) (3) have drawbacks. Microelectrodes are invasive and can destroy the membrane integrity, thereby disrupting the mechanism for maintaining the pH e . 31 P MRS does not suffer this drawback and has the additional advantage of permitting simultaneous measurements of intracellular pH. However, the limited sensitivity of 31 P MRS allows measurements of pH e only from relatively large tissue volumes. Hence, 31 P MRS provides measurements of pH ranges rather than different pH values for discrete spatial locations (13).The use of 1 H MRS, inherently more sensitive than 31 P MRS, would allow measurements of pH over smaller tissue volumes. For example, the imidazole protons of histidine have long been useful as intracellular pH indicators in NMR (14,15).Rabenstein and Isab (16) first proposed using imidazoles as extrinsic pH e indicators. Gil et al (17) suggested several modifications of the basic structure of the imidazole molecule to improve its performance as an extrinsic pH probe. To date, the most promising candidate for a 1 H nuclear magnetic resonance ...
The original version of this Article contained a typographical error in the spelling of the author Sara Carvalho, which was incorrectly given as Sara Cavalho. This has now been corrected in both the PDF and HTML versions of the Article.
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