| Abstract: | Twomitochondrion-specific fluorochromes,10-N-nonyl acridineorange (NAO) and rhodamine 123 (Rh123), were used todetermine the mechanism responsible for alterations in energymetabolism of transformed rat embryo fibroblast cells isolated fromdifferent locations within multicellular spheroids. Accumulation ofRh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thusrepresents mitochondrial distribution only. A reproducible selectivedissociation procedure was used to isolate cells from differentlocations within the spheroids. After isolation, cells weresimultaneously stained with one mitochondrial stain and the DNA dyeHoechst 33342, and several parameters, including cell volume, weremonitored via multilaser-multiparameter flow cytometry. Our dataclearly show a decrease in the uptake of Rh123 in cells from theperiphery to the inner regions of the tumor spheroids, reflecting apersistent alteration in mitochondrial function. However, NAO stainingexperiments showed no reduction in the total mitochondrial mass perunit cell volume. Because cells were exposed to stain under uniformconditions after isolation from the spheroid, these data indicate thatdownregulation of mitochondrial function is associated with cellquiescence rather than a transient effect of reduced nutrientavailability. This result, which is in accordance with data from twoother cell lines (EMT6 and 9L), might reflect a general phenomenon inmulticellular spheroids, supporting the hypothesis that quiescent cellsin the innermost viable spheroid layer stably reduce theirmitochondrial function, presumably to compensate for lower nutrientsupply and/or decreased energy demand. |
