Extended Data Fig. 4: eIF5A hypusination results in TGFβ-mediated collagen synthesis.

a. GSEA normalized enrichment scores (NES) for the top 15 gene sets commonly upregulated in translation based on changes in the ratio of Polysomal to Subpolysomal RNA levels, between 4T1 spheroids silenced for scramble shRNA grown in lung-like medium (LLM) supplemented with aspartate and 4T1 spheroids silenced for (i) Dhps (orange symbols) or (ii) Grin2d (purple symbols) grown in LLM supplemented with aspartate, or (iii) 4T1 spheroids silenced for scramble shRNA grown in LLM without aspartate (green symbols). White dots represent the average NES over all three comparisons. Gene sets related to TGFβ signaling are highlighted in bold. b. Word cloud highlighting the top 100 most frequently found terms among enriched gene sets, based on GSEA results analogous to those used to generate Extended Data Fig. 4a, but considering all three comparisons simultaneously (see Methods). c. Phosphorylated SMAD3 levels in 4T1 spheroids silenced for Grin2d or scramble shRNA (left), HUH7 (middle) and B16F10 (right) spheroids, grown in LLM supplemented with or without aspartate. A representative image of n = 3 experiments is shown. Quantification of phosphorylated SMAD3 signal normalized over total SMAD3 signal is indicated on top of each lane. d. Phosphorylated SMAD3 levels in 4T1 (left) and MCF10A HRAS^V12 (right) spheroids grown in LLM supplemented with aspartate and treated with or without a TGFβ inhibitor. A representative image of n = 3 experiments is shown. Quantification of phosphorylated SMAD3 signal normalized over total SMAD3 signal is indicated on top of each lane. e. Total spheroid areas for 4T1 (left) and MCF10A HRAS^V12 (right) cells grown in LLM supplemented with or without aspartate and treated with or without the TGFβ inhibitor for 5 (4T1) or 3 days (MCF10A HRAS^V12). N = 6 4T1 no aspartate, n = 11 4T1 no aspartate with TGFβ inhibitor, n = 11 4T1 aspartate, n = 9 aspartate with TGFβ inhibitor; n = 7 MCF10A HRAS^V12 no aspartate, n = 6 MCF10A HRAS^V12 aspartate, n = 9 MCF10A HRAS^V12 no aspartate with TGFβ inhibitor, n = 9 MCF10A HRAS^V12 aspartate with TGFβ inhibitor. Data are presented as mean ± s.d. (n indicates independent samples). Two-way ANOVA with Tukey’s multiple-comparison tests. f. GSEA normalized enrichment scores (NES) for gene sets containing either of the terms COLLAGEN, MATRISOME, ECM, and EXTRACELLULAR_MATRIX found on cancer cells based on scRNA-seq comparing 4T1 lung metastases from mice pre-treated with control medium or TSFs. Dot colors and areas indicate FDR-adjusted P-values and gene-set sized, respectively. P-values based on fgsea’s adaptive multilevel splitting Monte Carlo approach, subject to FDR adjustment using the Benjamini-Hochberg (BH) approach. g. Relative mRNA expression of Col1a1 in 4T1 (top) and MCF10A HRAS^V12 (bottom) spheroids grown in LLM supplemented with or without aspartate. Data for each gene and cell line are normalized relative to the average of the respective control (no aspartate) condition. Bars represent averages, and single dots individual replicates. Error bars represent ± s.d. (n = 3 independent replicates). Unpaired two-tailed t-test with Welch correction. h. Box and whisker plots of relative abundance of collagen I in 4T1 (left) and MCF10A HRAS^V12 (right) spheroids grown in LLM supplemented with or without aspartate, measured by immunofluorescence. N = 10 4T1 no aspartate, n = 6 4T1 aspartate, and n = 5 MCF10A HRAS^V12 no aspartate and n = 9 MCF10A HRAS^V12 aspartate. The total fluorescence intensity was normalized over the number of DAPI-stained nuclei. Relative fluorescence intensities per cell are depicted, normalized to the mean intensity for the control condition. Box hinges indicate the 1^st/3^rd quartiles of the corresponding data, while box mid-lines represent medians, and whiskers span the range of the data. Individual data points are indicated by the white dots. Unpaired two-tailed t-test with Welch correction. Representative three-dimensional representation are depicted on the right. Blue, DAPI-stained nuclei; red, collagen I. i. Box and whisker plots of relative abundance of collagen I in 4T1 spheroids silenced for Dhps (top) or Grin2d (bottom) or scramble shRNA, grown in LLM supplemented with or without aspartate, measured by immunofluorescence. Top: n = 15 no aspartate scramble, n = 11 aspartate scramble, n = 5 no aspartate shDhps, n = 6 aspartate shDhps. Bottom: n = 6 no aspartate scramble, n = 6 aspartate scramble, n = 7 no aspartate shGrin2d, n = 6 aspartate shGrin2d. The total fluorescence intensity was normalized over the number of DAPI-stained nuclei. Relative fluorescence intensities per cell are depicted, normalized to the mean intensity for the control condition. Box hinges indicate the 1^st/3^rd quartiles of the corresponding data, while box mid-lines represent medians, and whiskers span the range of the data. Individual data points are indicated by the white dots. Two-way ANOVA with Tukey’s multiple-comparison tests. Representative three-dimensional representation are depicted in Extended Data Fig. 4j. j. Box and whisker plots of relative abundance of collagen I in MCF10A HRAS^V12 spheroids silenced for DHPS, GRIN2D or scramble shRNA, grown in LLM supplemented with or without aspartate, measured by immunofluorescence. N = 6 no aspartate scramble, n = 7 aspartate scramble, n = 8 no aspartate shDHPS, n = 6 aspartate shDHPS, n = 8 no aspartate shGRIN2D, n = 6 aspartate shGRIN2D. The total fluorescence intensity was normalized over the number of DAPI-stained nuclei. Relative fluorescence intensities per cell are depicted, normalized to the mean intensity for the control condition. Box hinges indicate the 1^st/3^rd quartiles of the corresponding data, while box mid-lines represent medians, and whiskers span the range of the data. Individual data points are indicated by the white dots. Two-way ANOVA with Tukey’s multiple-comparison tests. Representative three-dimensional representation are depicted on the right and include samples shown in Extended Data Fig. 4i. Blue, DAPI-stained nuclei; red, collagen I. k. Box and whisker plots of relative abundance of collagen I in 4T1 (left) and MCF10A HRAS^V12 (right) spheroids grown in LLM supplemented with or without aspartate and treated with or without the TGFβ inhibitor, measured by immunofluorescence. N = 5 4T1 no aspartate, n = 4 4T1 aspartate, n = 6 4T1 no aspartate with TGFβ inhibitor, n = 5 4T1 no aspartate with TGFβ inhibitor, and n = 5 MCF10A HRAS^V12 no aspartate, n = 4 MCF10A HRAS^V12 aspartate, n = 5 MCF10A HRAS^V12 no aspartate with TGFβ inhibitor, n = 6 MCF10A HRAS^V12 aspartate with TGFβ inhibitor. The total fluorescence intensity was normalized over the number of DAPI-stained nuclei. Relative fluorescence intensities per cell are depicted, normalized to the mean intensity for the control condition. Box hinges indicate the 1^st/3^rd quartiles of the corresponding data, while box mid-lines represent medians, and whiskers span the range of the data. Individual data points are indicated by the white dots. Two-way ANOVA with Šídák’s multiple-comparison tests. Representative three-dimensional representation are depicted on the right. Blue, DAPI-stained nuclei; red, collagen I. l. Relative spheroid areas for 4T1 cells silenced for Grin2d, Dhps or scramble shRNA grown in LLM with 1.5% Matrigel, supplemented with or without aspartate and with or without 1.5% Collagen-I. N = 3 no aspartate scramble, n = 3 aspartate scramble, n = 4 no aspartate scramble with Collagen-I, n = 5 aspartate scramble with Collagen-I; n = 3 no aspartate shDhps, n = 3 aspartate shDhps, n = 5 no aspartate shDhps with Collagen-I, n = 4 aspartate shDhps with Collagen-I; n = 3 no aspartate shGrin2d, n = 4 aspartate shGrin2d, n = 7 no aspartate shGrin2d with Collagen-I, n = 7 aspartate shGrin2d with Collagen-I. Data are presented as mean ± s.d, normalized to the mean spheroid areas for the respective no aspartate conditions (n indicates independent samples). Two-way ANOVA with Tukey’s multiple-comparison tests. m. Relative spheroid areas for 4T1 cells grown in LLM with 1.5% Matrigel, supplemented with or without aspartate and with or without 1.5% Collagen-I and treated with or without the TGFβ inhibitor. N = 4 no aspartate, n = 4 aspartate, n = 4 no aspartate with Collagen-I, n = 4 aspartate with Collagen-I; n = 4 no aspartate with TGFβ inhibitor, n = 4 aspartate with TGFβ inhibitor, n = 5 no aspartate with TGFβ inhibitor and Collagen-I, n = 4 aspartate with TGFβ inhibitor and Collagen-I. Data are presented as mean ± s.d, normalized to the mean spheroid area for the respective no aspartate conditions (n indicates independent samples). Two-way ANOVA with Tukey’s multiple-comparison tests. n. Top: Quantification of linearized collagen based on Picrosirius Red staining and polarized light microscopy detected in EMT6.5 lung metastases silenced for Dhps or scramble shRNA in mice pre-treated with daily injections of aspartate (20 mM, i.p., 10 days) or PBS. Significant collagen red/yellow/green increase (*) 0.0023/0.1758/0.0042 EMT6.5 aspartate scramble, n = 5 PBS and n = 5 aspartate. Significant collagen red/yellow/green decrease (**) < 0.0001/ < 0.0001/ < 0.0001 (EMT6.5 shDhps), n = 5 PBS scramble, n = 5 aspartate scramble and n = 5 aspartate shDhps. Data are normalized by metastasis area (megapixel) and expressed as mean ± SEM. Two-way ANOVA with Šídák’s multiple-comparison tests. Bottom: representative images of linearized collagen based on Picrosirius Red staining and polarized light microscopy detected in EMT6.5 lung metastases. Red color mostly indicates thick collagen I fibers and green color mostly indicates thin collagen III fibers. A representative image of n = 5 EMT6.5 PBS scramble, n = 5 EMT6.5 aspartate scramble, n = 5 EMT6.5 aspartate shDhps lung metastases is shown. Scale bars = 20 μm.

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