GoKawiila. Representative images and size of CMT93 spheroids with P5cs or/and Col1a1 overexpression (OE) or OE of an empty vector as the control (n = 4 biological replicates). Scale bar 200 µm. Data are normalized to the control without any OE. Two-way ANOVA with Šídák’s multiple comparisons test. Only relevant comparisons are shown. b. Quantification of linearized collagen in CMT93-derived liver metastases from mice on CD or HFD (n = 3 mice) using Picrosirius red staining. The birefringence signal of linearized collagen of the conditions is first normalized to the area covered by tumor and then normalized to the CD-encapsulated metastasis, which is set to 1. Two-way ANOVA with Dunnett multiple comparison test. The corresponding representative images are shown in Fig. 2d. c. Percentage of CMT93 replacement metastases in mice intrasplenically injected with P5cs overexpression (OE) or/and Col1a1 OE CMT93 cells in control diet mice (n = 7 mice for conditions without P5cs OE, 8 mice for conditions with P5cs OE) based on the length of the liver-metastasis interfaces of all metastases showing either a replacement growth or a encapsulated growth. Two-way ANOVA with Šídák’s multiple comparisons test. Only relevant comparisons are shown. d. Venn diagram overlapping the shared transcription factors for COL1A1 (yellow circle), P5CS (blue), PYCR1 (red), PYCR2 (purple) from the public CHIP-seq database ChEA. Numbers in the circles indicate the total number of transcription factors for each gene. MYC is the only transcription factor shared by all four genes. e. Gene set enrichment analysis enrichment plot for the ‘Myc-targets’ gene set based on differential expression analysis in CMT93 spheroids treated with ethanol (EtOH, control) compared to 200 µM palmitate (PA) treatment (n = 3 biological replicates). P - value indicates the significance of the enrichment score (unpaired one-tailed t-test). f. Protein levels of total and acetylated-c-MYC at K323 in three patient-derived organoid (PDO) lines treated with ethanol (EtOH) as the control or a combination of the fatty acids (FA, palmitate (66 µM) and oleate (50 µM)). E1, E2, E3 indicate three individual PDO lines established from three patients with encapsulated metastases (n = 3 PDO lines). The corresponding quantification is shown in Fig. 2g. g. Protein levels of c-MYC in CMT93 spheroids treated with ethanol (EtOH) as the control, 200 µM palmitate (PA, n = 3 biological replicates), 130 µM octanoate (OcA, n = 3 biological replicates), 114 µM stearate (SA, n = 4 biological replicates) or 150 µM oleate (OA, n = 3 biological replicates). Protein expression quantifications are based on the relative band intensity after normalization to β-actin. Unpaired two-sided t-test with Welch correction. h. Quantification of c-MYC staining intensity in liver tissues with CMT93-derived liver metastases in mice on CD (n = 5 mice) or HFD (n = 4 mice). Only cancer cells are considered for the quantification. c-MYC staining intensity inside cancer cell nuclei in each condition is normalized to the signal of the encapsulated metastases in CD, which is set to 1. Brown-Forsythe Welch ANOVA with Dunnett multiple comparison test. i. Relative mRNA levels of P5cs, Pycr1, Pycr2 and Col1a1 in CMT93 spheroids with shControl or shMyc treated with ethanol (EtOH) as the control or 200 µM palmitate (PA) (n = 3 biological replicates). Data are normalized to the shControl in EtOH. Two-way ANOVA with Šídák’s multiple comparisons test. Only relevant comparisons are shown. j. Intracellular proline abundance in CMT93 spheroids with shControl or shMyc in ethanol (EtOH) as the control or 200 µM palmitate (PA) (n = 6 biological replicates for shMyc in EtOH, n = 7 biological replicates for the other conditions). Data are normalized to shControl in EtOH. Two-way ANOVA with Šídák’s multiple comparisons test. Only relevant comparisons are shown. k. Relative collagen synthesis in CMT93 spheroids with shControl or shMyc in ethanol (EtOH) as the control or 200 µM palmitate (PA) (n = 6 biological replicates) measured by 13C 5 -glutamine-based tracing into collagen-derived proline. Data are normalized to shControl in EtOH. Two-way ANOVA with Šídák’s multiple comparisons test. Only relevant comparisons are shown. l. Representative images and size of CMT93 tumor spheroids with shControl or shMyc in ethanol (EtOH) as the control, EtOH + 1.5% collagen, 200 µM palmitate (PA) or 200 µM PA + 1.5% collagen (n = 4 biological replicates). Matrigel is added to all conditions. Data is normalized to shControl in EtOH. Scale bar 200 µm. Two-way ANOVA with Šídák’s multiple comparisons test. Only relevant comparisons are shown. m. Representative images and size of PDOs treated with ethanol (EtOH) as the control, palmitate (PA, 200 µM), EtOH with the MYC inhibitor MYCi975 (2.5 µM), or PA with MYCi975 (n = 3 independent replicates). E4 and E5 indicates two PDO lines established from two patients with encapsulated metastases. Data is normalized to the control. Scale bar 200 µm. One-way ANOVA with Šídák’s multiple comparisons test. Only relevant comparisons are shown. All data are shown as mean ± SD.
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