Figure 2. ASNS-high cells grow faster and form tumors more rapidly in vivo.

(A, B) RNAseq heatmap shows clustering of cells based on ASNS expression levels, with ASNS hi, mid, and lo. (C) in vitro growth curves for 827 cells with asparagine depletion via ASNase as well as L-Asn supplementation rescue. (D) in vitro growth assays showing glutamine (Q) and glucose withdrawal. ASNS high cells were able to retain robust growth rates in the absence of L-asparagine while this depletion reduced ASNS low growth and induced significant cell death. Regression comparison of matched constructs shows significance (p<0.0001). Glutamine withdrawal had a more significant effect on ASNS high cells (GSC827, 604 ASNS-hi) compared to ASNS low cells (604, 827 shASNS). ASNS low cells were significantly more affected by loss of nutrients within 24 hrs. Rescue of survival could be seen with exogenous supplementation of 0.1 M L-Asn. Regression comparison indicates significant difference in slopes (p<0.0001). Asparagine withdrawal had a more significant effect on ASNS high cells (GSC827, 604 ASNS-hi) compared to ASNS low cells (604, 827 shASNS). Regression comparison indicates significant difference in slopes (p<0.0001). Intracranial tumor implantation model in B6/Foxn1−/− mice shows significant survival difference based on ASNS status. (E) GSC604–48 average survival of 26.5-days (HR=22.92). (F) GSC827 N.S. average survival of 46.5-days (HR=6.44). Cells injected (1×10⁶ cells/mouse) into left flank on B6.Cg-Foxn1 nu/J mice (n=2 mice/group). (G) GSC604–48 tumors were larger and grew faster than 827 control mice, although both are ASNS-high.