Fig 2. SLC38A2 is the major concentrative alanine transporter in PDAC.

(A) Volcano plot of differentially expressed transporters (‘SLC’ proteins) between hPSC#1 and PANC1 cell lines. Transporters were considered differentially expressed if log₂ fold change (FC) ≥ 2 and p-value ≤ 0.05. Transporters involved in amino acid transport are highlighted with colored squares and labeled with protein name. (B) Relative protein expression across panel of non-malignant pancreatic and PDAC cell lines quantified by summing reporter ion counts of peptide-spectral matches for SLC1A4 and SLC38A2. Two tandem mass tag-labeled biological replicates were collected for each cell line. (C & D) Alanine secretion (+) and uptake (−) flux in HY19636, MiaPaCa2, and PANC1 cells cultured in either basal DMEM (C) or DMEM supplemented with 1mM L-alanine (D) for 24 hours. SLC38A2 was suppressed by CRISPR/Cas9 using two sgRNAs targeting SLC38A2 (sgSLC38A2 #1, #3) or a control sgRNA targeting Tomato (sgTom). All experiments were conducted using pools of cells within 1-2 passages after selection. Error bars depict s.d. of ≥3 technical replicate wells from one independent experiment normalized to growth of surrogate wells. (E) Diagram depicting role of SLC38A2 in mediating sodium-dependent, concentrative uptake of alanine in PDAC (top panel). Cells lacking SLC38A2 rely on passive diffusion through other transporter(s) that cannot sustain alanine influx or maintain intracellular concentrations (bottom panel). (F) Intracellular alanine levels normalized by cell number in SLC38A2-deficient (sgSLC38A2 #1) or control (sgTom) HY19636, MiaPaCa2, and PANC1 cells. Data are represented as percentage relative to control (sgTom). Error bars depict s.d. of ≥3 technical replicate wells from one independent experiment. (G) SLC38A2-deficient and control HY19636 cells were supplemented with 1mM L-alanine tert-butyl ester for 24 hours. Esterified alanine internalization, de-esterification, and secretion were measured by quantifying alanine release into conditioned media and normalizing to viable cell density over 24 hours. Error bars depict s.d. of ≥3 technical replicate wells from one independent experiment normalized to growth of surrogate wells. (H) SLC38A2 expression allows PDAC cells to actively re-uptake alanine that is passively exchanged with the environment to allow for intracellular (cytosolic) concentration and enhanced utilization (left panel). SLC38A2-deficient cells do not have another concentrative alanine transport mechanism, and intracellular alanine is passively lost to the environment causing a decrease in intracellular levels and utilization (right panel). Significance determined with two-way ANOVA using Tukey’s multiple comparisons test in C and D; two-way ANOVA using Sidak’s multiple comparisons test in F; t-test in G. * p<0.05, ** p<0.01, *** p<0.001.