Extended Data Figure 5. Secondary validation that extracellular asparagine availability affects invasion and metastasis.

a, Tumour volumes resulting from the orthotopic injection of parental 4T1 cells. Half of the mice received l-asparaginase and the other half received an equivalent volume of PBS at the same injection rate (n = 10 mice per condition, edges of the box are the 25th and 75th percentiles and error bars extend to the values q3 + w(q3 − q1) and q1 − w(q3 − q1), in which w is 1.5 and q1 and q3 are the 25th and 75th percentiles, which is also the case for b–g). b, Quantification of lung metastases detected in the animals described in a (rank-sum P < 0.001). c, Representative H&E-stained lung sections as described in b. d, Quantification of the lung metastases described in Fig. 3b, in which Asns-silenced and -expressing 4T1-T cells were injected into mice. Half of the mice received l-asparaginase and the other half received an equivalent volume of PBS at the same injection rate (n = 10 mice per condition, rank-sum P < 0.0005 for l-asparaginase versus control for each line and for Asns-silenced versus -unsilenced cells in each drug condition). e, Tumour volumes corresponding to the lung metastases described in d (rank-sum P < 0.005 for Asns-silenced versus -expressing cells in l-asparaginase-treated mice). f, Lung metastases resulting from the orthotopic injection of ASNS-silenced and -expressing MDA-MB-231 cells and subsequent treatment of the injected animals with l-asparaginase or PBS (n = 10 mice per cell line, rank-sum P < 0.05 for ASNS-silenced versus -expressing cells in both conditions and for silenced cells in treated versus untreated mice). g, Tumour volumes corresponding to the mice described in f (rank-sum P < 0.05 for Asns-silenced versus -expressing cells under both treatments and for PBS versus l-asparaginase-treated animals for each cell line). See Source Data.