Microscopic images of intraspheroidal pH by 1H magnetic resonance chemical shift imaging of pH sensitive indicators

Objective: We investigate microscopic pH heterogeneity within tumor spheroids using a novel H-1 NMR methodology that provides high resolution measurements of intraspheroidal pH. Material and Methods: High resolution microscopic images of intraspheroidal pH were obtained by H-1 NMR using chemical shift selective excitation of the H2 resonance of imidazole added to the incubation medium. Imidazole accumulated in the intraspheroidal space in a pH dependent manner. Maps of intraspheroidal pH could be obtained by transforming pixel by pixel (32 x 32 mu) the regional variation of imidazole H2 intensity into a relative pH scale. Results: Our analysis revealed drastic intraspheroidal pH alterations depending on the size of the spheroid, ca. 0.6 pH units more acidic in the necrotic core than in the periphery, for spheroids of 600 mu m diameter. The presence of concentric regions having similar intraspheroidal pH was consistently observed. The thickness of these regions decreased from pH 7.2 to pH 6.8 and increased below the latter pH value. Conclusion: Our observations are compatible with the general model of spheroid growth where the more external layers of cells are in active growth and depict more alkaline pH values while the inner layers remain quiescent or evolve to a necrotic core, depicting more acidic pH values.