Development of a cell-based fluorescent assay to measure LAT1 uptake

The Large Neutral Amino Acid Transporter (LAT1) facilitates the transport of large branched and aromatic amino acids and nutrients into cells. LAT1 is ubiquitously expressed throughout the body with abundant expression in immune cells and at the blood-brain barrier. Due to the high metabolic needs of cancer cells for nutrients as they rapidly proliferate, LAT1 is upregulated in several cancer cells. LAT1 shows promise as a candidate for the carrier-mediated transport of therapeutic cancer drugs conjugated to LAT1 substrates. To develop LAT1-based chemotherapeutic strategies, a simplified high-throughput method to evaluate LAT1 substrate transport is needed. We report a cell-based fluorescence assay to measure LAT1 substrate uptake using a tryptophan analog, 6-azatryptophan. This assay measured the transport of 6-azatryptophan in several cancer cell lines, which was dependent on the levels of LAT1 expression. We showed that 6-azatryptophan transport was mediated through LAT1 by using LAT1-specific chemical and genetic inhibitors. The results show that 6-azatryptophan is a detectable LAT1 substrate and can be used for LAT1-mediated drug delivery.