Fig 2: Cells cultured in Plasmax approximate the in vivo tumor metabolome better.(A) Schematic representation of the experimental setup applied to compare the untargeted metabolic profiling of CAL-120 cells cultured in vitro (as 2D monolayers or 3D spheroids) in Plasmax and DMEM-F12, with CAL-120–derived orthotopic tumors. (B) PCA of three independent experiments or n = 3 mice (nine tumor fragments) performed as detailed in (A). (C) Weighted distance between each culture condition and the mean values of tumor samples, as calculated from the PCA reported in (B). P values refer to a two-tailed t test for unpaired homoscedastic samples. a.u., arbitrary units. (D to F) Relative abundance and ratio of urea cycle intermediates in CAL-120 cells grown in Plasmax and DMEM-F12 as 2D and 3D cultures and orthotopic mammary tumors. Means ± SEM; n = 3 independent experiments or n = 3 mice. P values refer to a two-tailed t test for unpaired homoscedastic samples comparing the in vitro conditions to the tumor samples. (G to I) Micrographs of CAL-120–derived mammary tumor tissue showing (G) hematoxylin and eosin (H&E) staining and IHC for (H) Ki67 and (I) ASS1. Arrows indicate stromal cells, whereas the arrowhead denotes vessel endothelium. T, tumor; FP, fat pad.

Fig 3: ASS1 is indeed the target of SPA and LM-2I.a Crystal violet assay for the viability of MDA-MB-231 cells treated with LM-2I or LM-2I pre-incubated with different concentrations of ASS1 recombinant protein (mean ± s.d., n = 6 biologically independent experiments). ***P < 0.001, NS: not significant (P > 0.05) from two-tailed unpaired Student’s t-tests. b Western blot for ASS1 expression in control or stable ASS1 knockout (ASS1-KO) MDA-MB-231 cells (top). Crystal violet assay for the viability of control and ASS1-KO MDA-MB-231 cells treated with LM-2I (bottom) (mean ± s.d., n = 3 biologically independent experiments). c Western blot for ASS1 expression in control or stable ASS1 knockdown (ASS1-KD) MCF-7 cells (top). Crystal violet assay for the viability of control and ASS1-KD MCF-7 cells treated with LM-2I (bottom) (mean ± s.d., n = 3 biologically independent experiments). d Western blot for ASS1 expression in control or stable ASS1 overexpression (ASS1-OE) MDA-MB-231 cells (top). Crystal violet assay for the viability of control and ASS1-OE MDA-MB-231 cells treated with LM-2I (bottom) (mean ± s.d., n = 3 biologically independent experiments). e The correlation between the relative ASS1 expression in cells and the IC50 of these cells treated with LM-2I (n = 3 independent experiments from n = 14 cell lines). P values of mean ± s.d. were determined by Pearson Coefficient. f LC-MS analysis of ASP, UMP, CMP, and TMP in MDA-MB-231 cells treated with SPA, LM-2I or vehicles for 24 h (mean ± s.d., n = 6 biologically independent experiments). **P < 0.01, ***P < 0.001 from two-tailed unpaired Student’s t-tests. g Crystal violet assay for the viability of MDA-MB-231 cells preincubated with pyrimidines or purines, followed by treatment with SPA or LM-2I (mean ± s.d., n = 6 biologically independent experiments). ** P < 0.01, ***P < 0.001, NS: not significant (P > 0.05) from two-tailed unpaired Student’s t-tests.Source data are provided as a Source Data file.

Fig 4: ASS1 functions as a breast tumor suppressor.a Western blot for ASS1 expression in breast cancer cell lines (top). The relative quantification of ASS1 expression in breast cancer cell lines (ASS1 expression in MDA-MB-231 cells was defined as 1) (bottom) (mean ± s.d., n = 3 biologically independent experiments). Western blot for the expression of indicated proteins in MDA-MB-231 cells transfected with ASS1 expression plasmid (ASS1-OE) or empty vector (b, top), and in MCF7 cells infected with control or ASS1 shRNA lentivirus (ASS1-KD) (c, top). Crystal violet assay for the proliferation rate of MDA-MB-231 cells transfected with ASS1 expression plasmid (ASS1-OE) or empty vector (b, bottom), and for MCF7 cells infected with control or ASS1 shRNA lentivirus (ASS1-KD) (c, bottom) (mean ± s.d., n = 3 biologically independent experiments). **P < 0.01, ***P < 0.001 from two-tailed unpaired Student’s t-tests.d Colony formation of MDA-MB-231 cells transfected with ASS1 expression plasmid (ASS1-OE) or empty vector, and of MCF7 cells infected with control or ASS1 shRNA lentivirus (ASS1-KD). 1 × 106 MDA-MB-231 cells stably expressing ASS1 (ASS1-OE) or control vector (NC) were injected subcutaneously into the right flank of BALB/c nu/nu female mice. Tumor sizes were measured every 2 or 3 days (e). 36 days later mice were sacrificed, tumors were dissected and weighted (f). Data represented as mean ± s.d. (n = 7 mice per group). ***P < 0.001 from two-tailed unpaired Student’s t-tests. g Representative imagines of ASS1 immunohistochemical staining in paraffin-embedded human breast cancer tissue sections. Scale bars are 50 μm. h Comparing the expression levels of ASS1 in recurrent (n = 60) and no recurrent (n = 83) human breast cancers. **P < 0.01 from two-tailed unpaired Student’s t-tests. Source data are provided as a Source Data file.

Fig 5: SPA and LM-2I target ASS1 at C97 site.a The recombinant ASS1 protein was incubated with Biotin-SPA in the absence or presence of SPA, SPAH and LM-2I for 1 h at room temperature, followed by western blot using streptavidin-HRP or ASS1 antibody (n = 3 biologically independent experiments). b Recombinant WT and mutant ASS1 proteins were incubated with Biotin-SPA or Biotin-SPAH for 30 min, followed by western blot using streptavidin-HRP or ASS1 antibody (n = 3 biologically independent experiments). MS/MS analysis for the C97-containing peptide of recombinant ASS1 pre-incubated with vehicle (c), SPA (d), or LM-2I (e). (SPA), combined with SPA; (LM-2I), combined with LM-2I; (c), carbamidomethylation; (d), deamidation. f Binding model of LM-2I-ASS1. The left panel is the global view of the entire ASS1 structure, the right panel is the focused view of LM-2I in the binding site (ASS1 in gray while LM-2I in green). Source data are provided as a Source Data file.