Cancer cells, including melanoma cells, often metastasize regionally through the lymphatic system before metastasizing systemically through the blood1,2,3,4; however, the reason for this is unclear. Here we show that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood. Immunocompromised mice with melanomas derived from patients, and immunocompetent mice with mouse melanomas, had more melanoma cells per microlitre in tumour-draining lymph than in tumour-draining blood. Cells that metastasized through blood, but not those that metastasized through lymph, became dependent on the ferroptosis inhibitor GPX4. Cells that were pretreated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection. We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid and less free iron in lymph. Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumours. Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than did melanoma cells from subcutaneous tumours. Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood.
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S.J.M. is a Howard Hughes Medical Institute Investigator, the Mary McDermott Cook Chair in Pediatric Genetics, the Kathryn and Gene Bishop Distinguished Chair in Pediatric Research, the director of the Hamon Laboratory for Stem Cells and Cancer, and a Cancer Prevention and Research Institute of Texas Scholar. The research was supported by the Cancer Prevention and Research Institute of Texas (RP170114 and RP180778) and by the National Institutes of Health (NIH; U01 CA228608). A.T. was supported by the Leopoldina Fellowship (LPDS 2016-16) from the German National Academy of Sciences and the Fritz Thyssen Foundation. B.S. was supported by a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship from the National Heart, Lung, and Blood Institute (F32 HL139016). M.L.M. and D.R.S. were supported by NIH grants P01 (CA217797) and P30 (CA086862). We thank M. Dellinger for comments on the manuscript; N. Meireles for collecting human melanomas; M. Nitcher for mouse colony management; the BioHPC (High Performance Computing) core for data storage; and N. Loof and the Moody Foundation Flow Cytometry Facility.
S.J.M. is an advisor for Frequency Therapeutics and Protein Fluidics as well as a stockholder in G1 Therapeutics and Mereo Biopharma.
Peer review information Nature thanks Martin Bergo, Marcus Conrad, Sarah-Maria Fendt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Representative flow cytometry gates for the isolation of melanoma cells from blood and lymph and representative bioluminescence images of visceral organs to quantify the metastatic disease burden.
Related to Table 1, Figs. 1, 2, 3. a–d, Flow cytometry plots showing the gating strategies used to identify human melanoma cells in the blood (a) or lymph (b) of NSG mice or mouse melanoma cells in the blood (c) or lymph (d) of C57BL mice. In all cases, cells were gated on forward scatter area versus side height (FSC-H vs. FSC-A) to exclude red blood cells and cell clumps. Mouse haematopoietic and endothelial cells were excluded by gating out cells that stained positively for anti-mouse CD45, CD31, or Ter119. Human melanoma cells were selected by including cells that stained positively for HLA-ABC and mouse melanoma cells were selected by including cells that stained positively for CD146. Melanoma cells were also identified in these studies based on DsRed, which was stably expressed in all melanomas along with luciferase. e–g, Representative bioluminescence imaging of visceral organs dissected from a negative control mouse (e), and mice transplanted with luciferase-expressing human (f) or mouse (g) melanomas. h, Evan’s blue dye was injected into a subcutaneous melanoma to expose the tumour-draining blood (white arrow) and lymphatic (black arrow) vessels. i, Inefficiently metastasizing human melanomas were transplanted subcutaneously into NSG mice and the number of melanoma cells per microlitre of blood and tumour-draining lymph were determined (n = 4 or 5 mice per melanoma from two independent experiments). Statistical significance was assessed using a Kruskal–Wallis test (*P < 0.05, ***P < 0.001). Exact P values are provided in the source data files.
Extended Data Fig. 2 The effect of liproxstatin-1 on the growth of subcutaneous tumours and the effect of other inhibitors on metastasis.
Related to Figs. 1, 2. a, Addition of 2-mercaptoethanol to M405 melanoma cells isolated from subcutaneous tumours or the blood blunted the increase in ROS levels in melanoma cells from the blood. b, c, Human (b) or mouse (c) melanomas were treated in culture with erastin and/or deferoxamine. d–h, Treatment of mice with liproxstatin-1 had little or no effect on the growth of subcutaneous tumours formed by human (d–f) or mouse (g, h) melanomas. i, Pretreatment of inefficiently metastasizing human melanoma cells with Liproxstatin-1 did not significantly affect metastatic disease burden after intravenous injection into NSG mice. j, Mouse melanomas were pretreated with autophagy (3-MA), apoptosis (ZVAD), or necroptosis (GSK′872) inhibitors then injected intravenously into C57BL mice and metastatic disease was assessed 1 month later by bioluminescence imaging. The number of replicates is indicated in each panel and the number of independent experiments is shown in Supplementary Data, ‘Statistics and reproducibility’. All data represent mean ± s.d. Statistical significance was assessed using a correlated-samples two-way ANOVA followed by Sidak’s multiple comparisons adjustment (a), two-way ANOVA (i) followed by Tukey’s multiple comparison adjustment (b), Kruskal–Wallis tests followed by Dunn’s multiple comparisons adjustment (c (Y1.7), j), one-way ANOVA followed by Tukey’s multiple comparisons adjustment (c (Y3.3)), or nparLD tests (d–h). No statistically significant differences were observed in d–f, i or j. For all panels, statistical tests were two-sided where applicable and *P < 0.05, **P < 0.01, ***P < 0.001. Exact P values are provided in the source data files.
Extended Data Fig. 3 The effect of Gpx4 deletion on the survival and proliferation of mouse melanomas in culture.
Related to Fig. 2. a, Western blot analysis of GPX4 in parental or Gpx4-deleted mouse melanomas (representative of 2 independent experiments). b, Western blot analysis of GPX4 in efficiently and inefficiently metastasizing melanomas from patients as well as normal mouse brain and liver tissue. Actin was used as a loading control (representative of 2 independent experiments). Uncropped western blots are in Supplementary Fig. 1. c, d, Gpx4-deficiency did not significantly affect the growth of melanoma cells cultured in low oxygen (c) but did significantly reduce the growth of some melanomas cultured at atmospheric oxygen levels (d) (n = 6 replicate cultures per melanoma; data reflect one representative experiment of two conducted). e, Lipid ROS levels in melanoma cells from subcutaneous tumours formed by Gpx4-deleted or parental control melanomas cells (n = 6 mice per melanoma in two independent experiments). f–i, Growth of primary subcutaneous tumours (f, g) and frequency of circulating melanoma cells in the blood (h, i) of NSG mice transplanted with parental or Gpx4-deleted melanomas (n = 4 or 5 mice per melanoma per experiment from two independent experiments). All data represent mean ± s.d. Statistical significance was assessed using repeated measures two-way ANOVAs (c (Y1.7), d) or mixed-effects analysis (c (Y3.3)) followed by Dunnett’s multiple comparisons adjustment (c, d), correlated-samples two-way ANOVA (e), nparLD tests (f, g), Fisher’s LSD test (e), Welch’s one-way ANOVA followed by Dunett’s T3 multiple comparisons adjustment (h), or Kruskal–Wallis tests followed by Dunn’s multiple comparisons adjustment (i). For all panels, statistical tests were two-sided where applicable and **P < 0.01, ***P < 0.001. Exact P values are provided in the source data files.
Related to Fig. 3. a, b, Lipid species that significantly differed in abundance between the plasma and lymph of NSG (a) or C57BL (b) mice (P < 0.01). c, Relative triacylglycerol content in the ApoB+ and ApoB− fractions of blood plasma or lymph fluid (after cells were removed) from C57BL mice (two independent samples per treatment). d, Relative oleic acid abundance in the ApoB+ and ApoB− fractions of blood plasma or lymph fluid from C57BL mice (two independent samples per treatment). Statistical significance was assessed using generalized linear modelling with log-transformed, half-min imputed data replacing zeros followed by the Benjamini-Hochberg multiple comparisons adjustment using two-sided t-statistics (a and b). The number of replicates is indicated in each panel. Each panel reflects two independent experiments. All data represent means and, when present, error bars reflect s.d. Exact P values are provided in the source data files.
Extended Data Fig. 5 ACSL3 is required for oleic acid incorporation into phospholipids and the protective effect of oleic acid against ferroptosis.
Related to Fig. 3. a, Western blot analysis of ACSL3 in efficiently and inefficiently metastasizing melanomas from patients as well as normal mouse brain and liver tissue. Actin was used as a loading control (representative of 2 independent experiments). b, Western blot analysis of ACSL3 in parental control and Acsl3-deleted mouse melanomas (representative of 2 independent experiments). Uncropped western blots are in Supplementary Fig. 1. c–d, Relative levels of oleic acid in phospholipids from Acsl3-deleted and parental control melanomas. In some cases, wild-type (Acsl3OE) or catalytically dead mutant Acsl3 (mut. Acsl3OE) were overexpressed in YUMM1.7 (c) or YUMM3.3 (d) mouse melanomas. The number of replicates per treatment is indicated in each panel (two independent experiments). e, Lipid ROS (BODIPY-C11Oxidized/BODIPY-C11Oxidized + BODIPY-C11Non-oxidized ratio) levels in mouse melanoma cells from subcutaneous tumours in C57BL mice. The number of replicates per melanoma is indicated in each panel (two independent experiments). The data from parental controls cells in this experiment are also shown in Extended Data Fig. 3e. f, Growth of Acsl3-deleted and parental control melanomas in culture (4 replicate cultures per melanoma per experiment, representative of two independent experiments; no differences were statistically significant). g, h, Growth of Acsl3-deleted melanomas in culture with oleic acid and with or without erastin. In some cases, wild-type (Acsl3OE) or catalytically dead mutant Acsl3 (mut. Acsl3OE) were overexpressed in YUMM1.7 (g) or YUMM3.3 (h) melanomas. i, Metastatic disease burden in mice intranodally injected with Acsl3-deleted or control melanomas. All data represent mean ± s.d. Statistical significance was assessed using one-sided ANOVAs followed by Sidak’s and Dunnett’s (c, d) multiple comparisons adjustments, paired t-tests (e), repeated measures two-sided ANOVAs followed by Dunnett’s multiple comparisons adjustment (f), Kruskal–Wallis tests followed by Dunn’s multiple comparisons adjustment (g, h, i (Y3.3)), or Welch’s one-way ANOVA followed by Dunnett’s T3 multiple comparisons adjustment (i (Y1.7)). For all panels, statistical tests were two-sided where applicable and *P < 0.05, **P < 0.01, ***P < 0.001. Exact P values are provided in the source data files.
Related to Fig. 3. a–f, Western blot analysis of SLC7A11 (a, b), ACSL4 (c, d) and FSP1 (e, f) in efficiently and inefficiently metastasizing human melanomas (a, c, e) as well as mouse melanomas (b, d, f) (representative of two experiments). Normal mouse liver and brain, human lung, and mouse fibroblasts were sometimes included as positive or negative controls. Uncropped western blots can be found in Supplementary Fig. 1. g, FSP1 transcript levels by qRT–PCR in human melanoma cells isolated from subcutaneous tumours, blood, or lymph of xenografted mice (3 replicates (each replicate was pooled from 4 or 5 mice) per melanoma). h, RNA-sequencing analysis of the fatty acid transporters FATP1, FATP3, FATP4, and FATP5 in efficiently and inefficiently metastasizing human melanomas (2 to 3 replicates per melanoma). i, Staining with anti-CD36 or isotype control antibody in human and mouse melanomas. Data are representative of 2 tumours per melanoma. j, Elevated levels of oleic acid in lymph suppress lipid ROS accumulation and ferroptosis in metastasizing melanoma cells, increasing their ability to survive during subsequent dissemination through the blood. Image created with BioRender. No statistically significant differences were observed in g or h. All data represent mean ± s.d. Statistics were determined repeated measures two-sided ANOVA followed by Tukey’s multiple comparisons adjustment (g). Exact P values are provided in the source data files.
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Ubellacker, J.M., Tasdogan, A., Ramesh, V. et al. Lymph protects metastasizing melanoma cells from ferroptosis. Nature 585, 113–118 (2020). https://doi.org/10.1038/s41586-020-2623-z
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