Amino acid uptake and regulation in multicellular hepatoma spheroids

Cancer cells maintained in monolayer tissue culture are frequently used to study tumor biology and nutrient uptake, but there is a concern that this system may not fully reflect clinical tumor physiology. Because cells grown in a 3-D configuration more closely resemble an in vivo environment, a model was developed and characterized for the growth of SK-Hep human hepatoma cells in suspension as multicellular tumor spheroids (MTS). The measurement of nutrient uptake in such a system has never been established.

SK-Hep cultures were initiated as single cell suspensions and grown as MTS in siliconized spinner flasks. The transport of several individual amino acids (arginine, glutamate, leucine, alpha-(N-methylamino)isobutyric acid (MeAIB), and glutamine (GLN)) was measured in SK-Hep single cell suspensions and MTS (0. 50-0.60 mm diameter) by a radiotracer/rapid filtration technique, as was the regulation of glutamine uptake by phorbol esters. l-[(3)H]GLN uptake was also measured in larger spheroids (0.85-1.5 mm diameter). MTS cellularity was evaluated by histological examination, and single cell integrity after the transport assay was confirmed by scanning electron microscopy (SEM).

SK-Hep MTS displayed gradients of cellular morphology and staining, with central necrosis visible at diameters >0.8 mm. Single cell suspensions endured the rapid filtration technique based on functional Na(+)-dependent uptake rates and SEM analysis. Of all amino acids tested, only GLN transport rates were visibly affected by growth format. In small MTS, Na(+)-dependent GLN uptake was diminished by 40%, but was 40-53% higher in MTS >1 mm displaying central necrosis, when compared to single cell suspensions. Likewise, slight parallel changes in glutamine transporter ATB(0) mRNA levels were observed in Northern blot analysis. Finally, phorbol ester-dependent GLN transport down-regulation (by 40-50%), previously established in SK-Hep monolayers, remained operative in all cell formats tested.

The data suggest that the tumor microenvironment differentially impacts the uptake of specific nutrients despite the conservation of key regulatory pathways. This MTS technique may prove useful for further studies on the role of nutrient transport in nascent tumor growth.