脂肪肝与结直肠癌肝转移生长模式

Fig. 1 | Liver steatosis increases the frequency of replacement metastases and proline levels within these lesions. a, Fraction of patients with CRC with pure replacement and encapsulated metastases, with or without liver steatosis (Rotterdam cohort, n = 155 patients; Göttingen cohort, n = 51 patients). Chi-square test. b, Percentage of CMT93 replacement metastases in mice fed with control diet (CD; n = 3 mice) or HFD (n = 8 mice). Unpaired two-tailed t-test with Welch correction. c–f, Size (c), P5CS mRNA (d), P5CS protein (e) and intracellular proline levels (f) of PDOs treated with ethanol (EtOH) control or a combination of fatty acids (FA; 66 µM palmitate and 50 µM oleate). E1, E2 and E3 indicate three individual PDO lines established from three patients with encapsulated metastases. In c, each treatment condition in each PDO line is repeated independently 6 (E1, E3) or 7 times (E2). In d–f, each PDO line is treated as a biological replicate (n = 3 PDO lines). Two-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test (c); unpaired two-tailed t-test (d–f). g, Proline levels of CMT93 metastases in mice fed with control diet or HFD determined with matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI–MSI) (50-μm pixel size) with corresponding H&E staining. Metabolite signals are shown after root-mean-square normalization. Scale bars, 500 µm. E, encapsulated metastasis; L, liver; R, replacement metastasis; S, stromal rim. All data are shown as mean ± s.d.

Fig. 2 | Formation of replacement metastases depends on proline-fuelled collagen synthesis regulated by MYC. a,b, Percentage of CMT93 replacement metastases in control diet or HFD mice injected intrasplenically with cells expressing control shRNA (shControl) or shRNA targeting P5cs (shP5cs) (a; shControl in HFD, n = 5 mice; other conditions, n = 4 mice), or P5cs- overexpressing (OE) (n = 12 mice) or control cells (n = 11 mice) (b). Two-way ANOVA with Šídák’s multiple comparisons test (a); unpaired two-tailed t-test with Welch correction (b). Control overexpression cells are also shown as control in i. c, Relative COL1A1 mRNA expression in PDOs treated with EtOH or a combination of fatty acids (66 µM palmitate and 50 µM oleate). E1, E2 and E3 are PDO lines established from three patients with encapsulated metastases (n = 3 PDO lines). Unpaired two-tailed t-test. d, Linearized collagen in CMT93 liver metastases from control diet or HFD mice using Picrosirius red staining. Collagen I fibres appear red and collagen III fibres appear green in polarized light. Collagen fibres appear red under bright-field illumination. Scale bars, 200 µm (overview) and 50 µm (zoom). E, encapsulated metastasis; L, liver; R, replacement metastasis; S, stromal rim. e,f, Percentage of CMT93 replacement metastases in control diet or HFD mice injected intrasplenically with cells transduced with shControl or shRNA targeting Col1a1 (shCol1a1) (n = 5 mice) and control diet mice injected with P5cs-OE or control cells transduced with shCol1a1 or control shRNA (n = 8 mice). Two-way ANOVA with Šídák’s multiple comparisons test. Control overexpression cells are also shown as control in j. g, Total and acetylated MYC (K323) protein in PDOs treated with EtOH or fatty acids (n = 3). Unpaired two-tailed t-test. h, MYC staining of CMT93 liver metastases in mice on control diet or HFD. Scale bars, 100 µm. EPCAM staining is also shown in Extended Data Fig. 2d. i,j, Percentage of CMT93 replacement metastases in control diet mice injected intrasplenically with Myc-OE (n = 7 mice) or control (n = 11 mice) cells (i), or control diet mice injected with Myc-OE and/or shCol1a1 cells (j; n = 8 mice). Unpaired two-tailed t-test with Welch correction (i); two-way ANOVA with Šídák’s multiple comparisons test (j). k, Total and acetylated MYC (K323) protein from CMT93 liver metastasis of mice on control diet or HFD (n = 4 mice). l, Percentage of CMT93 replacement metastases in control diet or HFD mice injected intrasplenically with cells transduced with shControl or shRNA targeting Cpt1a (shCpt1a) (shControl plus control diet, n = 4 mice; other conditions, n = 6 mice). Two-way ANOVA with Šídák’s multiple comparisons test. All data are shown as mean ± s.d.

Fig. 3 | Targeting P5CS and COL1A1 impairs growth of replacement metastases. a, Proline levels in CMT93, MC38 and CT26 liver metastases determined using MALDI–MSI (50-μm pixels), with corresponding H&E staining. Metabolite signals are shown after root-mean-square normalization. Scale bars, 500 µm. E, encapsulated metastasis; L, liver; R, replacement metastasis; S, stromal rim. b, Linearized collagen fibres in CMT93, MC38 and CT26 liver metastases using Picrosirius red staining. Collagen I fibres appear red and collagen III fibres appear green in polarized light. Collagen fibres appear red under bright-field illumination. Scale bars, 200 µm (overview) and 50 µm (zoom). c,d, Total metastatic area in livers of mice injected intrasplenically with shControl (n = 25 mice), shP5cs (n = 12 mice) or shCol1a1 (n = 13 mice) MC38 cells (c) or with shControl (n = 5 mice), shP5cs (n = 6 mice) or shCol1a1 (n = 6 mice) CT26 cells (d). One-way ANOVA with Dunnett’s multiple comparison test. All data are shown as mean ± s.d.

Fig. 4 | Replacement metastases from patients with CRC exhibit increased proline and collagen synthesis. a,b, Proline levels in encapsulated or replacement metastases from PDX models (a; n = 4) and in encapsulated or replacement metastases from patients with CRC with steatotic or non- steatotic livers (b; n = 19) determined with MALDI–MSI (50-μm pixels) with corresponding H&E staining. Metabolite signals are shown after root-mean- square normalization. Scale bars, 200 µm (a) and 2 mm (b). E, encapsulated metastasis; L, liver; R, replacement metastasis; S, stromal rim. c, Collagen deposition in encapsulated or replacement patient CRC liver metastases (n = 51) visualized using Masson trichrome staining. Scale bars, 100 µm (overview) and 50 µm (zoom). d, Linearized collagen in frozen encapsulated or replacement patient CRC liver metastases in steatotic or non-steatotic livers using Picrosirius red staining (n = 19). Collagen I fibres appear red and collagen III fibres appear green in polarized light. Collagen fibres appear red under bright-field illumination. Scale bars, 200 µm (overview) and 50 µm (zoom). e, P5CS (red), COL1A1 (brown), MYC (yellow) and pan-cytokeratin (PanCK, white) staining using fluorescent immunohistochemistry and the corresponding H&E staining in encapsulated or replacement liver metastases from patients with CRC with steatotic or non-steatotic livers (n = 14). Scale bars, 100 µm.

Fig. 5 | Pharmacological MYC inhibition shows selective efficacy against replacement metastases. a, Total liver metastatic area in mice injected intrasplenically with MC38 or CT26 cells (n = 5 mice) and treated with MYCi975 (MYCi; 100 mg kg−1 (body weight) day−1) or vehicle. Unpaired two-sided t-test with Welch correction. b, Relative size of PDO treated with DMSO (control) or MYCi975 (2 µM for R1 (control, n = 8; MYCi, n = 6 independent replicates), E1 (control, n = 7; MYCi, n = 6 independent replicates), E2 (control, n = 5; MYCi, n = 4 independent replicates) and E3 (control, n = 5; MYCi, n = 4 independent replicates) and 2.5 µM for R2 and R3 (n = 3 independent replicates)). R1, R2 and R3 are PDO lines derived from three patients with replacement metastases; E1, E2 and E3 are PDO lines derived from three patients with encapsulated metastases. Two-way ANOVA with Šídák’s multiple comparisons test. c, Bioluminescence-based tumour growth in mice with intrahepatically implanted PDX from a patient with replacement metastasis that were treated with MYCi975 (40 mg kg−1 (body weight) day−1, n = 8 mice) or vehicle (n = 10 mice). F-test comparing tumour growth. d,e, Correlation of dose of the MYC inhibitor OMO-103 (d) and liver steatosis as indicated by liver/spleen ratio, which anti-correlates with liver fat content (values lower than 1 indicate liver steatosis) (e) with therapy response, as indicated by change in target lesion diameter (positive values indicate tumour progression and negative values indicate tumour regression) in nine patients (five with CRC, two with pancreatic ductal adenocarcinoma (PDAC), one with sarcoma and one with non-small cell lung cancer (NSCLC)). Two-tailed Pearson’s correlation analysis. f, Schematics of the identified mechanism of a fatty liver microenvironment contributing to replacement metastases via MYC-mediated proline and collagen metabolism. Data are shown as mean ± s.d. (a,b) or mean ± s.e.m. (c).