Amino acid uptake and regulation in multicellular hepatoma spheroids

Background. Cancer cells maintained in monolayer tissue culture are frequen tly 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 environ ment, a model was developed and characterized for the growth of SK-Hep huma n hepatoma cells in suspension as multicellular tumor spheroids (MTS). The measurement of nutrient uptake in such a system has never been established. Materials and methods. SK-Hep cultures were initiated as single cell suspen sions and grown as MTS in siliconized spinner flasks. The transport of seve ral individual amino acids (arginine, glutamate, leucine, alpha-(N-methylam ino)isobutyric acid (MeAIB), and glutamine (GLN)) was measured in SR-Hep si ngle cell suspensions and MTS (0.50-0.60 mm diameter) by a radiotracer/rapi d filtration technique, as was the regulation of glutamine uptake by phorbo l esters. L-[H-3]GLN uptake was also measured in larger spheroids (0.85-1.5 mm diameter). MTS cellularity was evaluated by histological examination, a nd single cell integrity after the transport assay was confirmed by scannin g electron microscopy (SEM), Results. SB-Hep MTS displayed gradients of cellular morphology and staining , with central necrosis visible at diameters >0.8 mm. Single cell suspensio ns endured the rapid filtration technique based on functional Na(+)dependen t uptake rates and SEM analysis. Of all amino acids tested, only GLN transp ort rates were visibly affected by growth format. In small MTS, Na+dependen t GLN uptake was diminished by 40%, but was 40-53% higher in MTS >1 mm disp laying 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 tr ansport down-regulation (by 40-50%), previously established in SK-Hep monol ayers, remained operative in all cell formats tested. Conclusions. The data suggest that the tumor micro-environment differential ly impacts the uptake of specific nutrients despite the conservation of key regulatory pathways. This MTS technique may prove useful for further studi es on the role of nutrient transport in nascent tumor growth. (C) 2000 Acad emic Press.