Quantitative in-vivo characterization of intracellular and extracellular pH profiles in heterogeneous tumors

Cancer Res 2013 Jun 10 [Epub ahead of print]

4 June 2013

 

Acid production and transport are currently being studied to identify new targets for efficient cancer treatment, as subpopulations of tumor cells frequently escape conventional therapy owing to their particularly acidic tumor microenvironment. Heterogeneity in intracellular and extracellular tumor pH (pHi, pHe) has been reported, but none of the methods currently available for measuring tissue pH provides quantitative parameters characterizing pH distribution profiles in tissues. To this intent, we present here a multiparametric, noninvasive approach based on in vivo 31P NMR spectroscopy, and its application to mouse tumor xenografts. First, localized 31P NMR spectrum signals of pHi and pHe reporter molecules (inorganic phosphate, Pi, and 3-aminopropylphosphonate, 3-APP, respectively) were transformed into pH curves using established algorithms. While Pi, is an endogenous compound, 3-APP had to be injected intraperitoneally. Then, we developed algorithms for the calculation of six to eight quantitative pH parameters from the digital points of each pH curve obtained. For this purpose, each pH distribution profile was approximated as a histogram, and intensities were corrected for the nonlinearity between chemical-shift and pH. For each histogram derived from a Pi or 3-APP resonance, we obtained the following tumor pH profile parameters: weighted mean, weighted median, mode(s), skewness (asymmetry), kurtosis (peakedness), and entropy (smoothness). In addition, relative sizes of tissue volumes defined by characteristic pH ranges were estimated by integration and/or by fitting the curve to multiple modes. Our algorithms and the results obtained for animal models were validated (i) by computer simulations of 31P NMR resonances and pH profiles; and (ii) by comparison with combinations of ≤ 3 test solutions at well-defined pH values, containing the pH reporter molecule 3-APP. All calculations were performed with an EXCEL spreadsheet, thus avoiding any specialized software or hardware. Consequently, heterogeneous pHi and pHe distribution profiles in tumors can be characterized by multiple quantitative parameters derived from classical statistics, through pH distribution profiles obtained from in vivo 31P NMR spectra. This original technique is helpful in analyzing tumor tissue features with increased detail, by a single experiment also yielding information on underlying energy and phospholipid metabolism.