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. 2020 Sep;585(7823):113-118.
doi: 10.1038/s41586-020-2623-z. Epub 2020 Aug 19.

Lymph protects metastasizing melanoma cells from ferroptosis

Jessalyn M Ubellacker  1 , Alpaslan Tasdogan  1 , Vijayashree Ramesh  1 , Bo Shen  1 , Evann C Mitchell  1 , Misty S Martin-Sandoval  1 , Zhimin Gu  1 , Michael L McCormick  2 , Alison B Durham  3 , Douglas R Spitz  2 , Zhiyu Zhao  1 , Thomas P Mathews  1   4 , Sean J Morrison  5   6   7
Affiliations

Lymph protects metastasizing melanoma cells from ferroptosis

Jessalyn M Ubellacker et al. Nature. 2020 Sep.

Abstract

Cancer cells, including melanoma cells, often metastasize regionally through the lymphatic system before metastasizing systemically through the blood1-4; 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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Figures

Extended Data Figure 1.
Extended Data Figure 1.. Representative flow cytometry gates for the isolation of melanoma cells from the blood and lymph and representative bioluminescence images of visceral organs to quantitate metastatic disease burden. Related to Table 1, Figures 1, 2 and 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 hematopoietic 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 tumor-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 microliter of blood and tumor-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 in source data files.
Extended Data Figure 2.
Extended Data Figure 2.. The effect of Liproxstatin-1 on the growth of subcutaneous tumors and the effect of other inhibitors on metastasis. Related to Figures 1 and 2.
(a) Addition of 2-mercaptoethanol (2ME) to M405 melanoma cells isolated from subcutaneous tumors 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 tumors formed by human (d-f) or mouse (g, h) melanomas. (i) Pre-treatment 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 pre-treated 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 panels 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 in source data files.
Extended Data Figure 3.
Extended Data Figure 3.. The effect of Gpx4 deficiency on the survival and proliferation of mouse melanomas in culture. Related to Figure 2.
(a) Western blot analysis of GPX4 in parental or Gpx4-deficient 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 Figure 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 tumors formed by Gpx4-deficient or parental control melanomas cells (n=6 mice per melanoma in two independent experiments). f-i, Growth of primary subcutaneous tumors (f, g) and frequency of circulating melanoma cells in the blood (h, i) of NSG mice transplanted with parental or Gpx4-deficient melanomas (n=4–5 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 in the source data files.
Extended Data Figure 4.
Extended Data Figure 4.. Lipid species in plasma and lymph. Related to Figure 3.
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 (TG) 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 modeling 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 in the source data files.
Extended Data Figure 5.
Extended Data Figure 5.. Acsl3 is required for oleic acid incorporation into phospholipids and the protective effect of oleic acid against ferroptosis. Related to Figure 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 deficient mouse melanomas (representative of 2 independent experiments). Uncropped western blots are in Supplementary Figure 1. c-d, Relative levels of oleic acid in phospholipids from Acsl3-deficient and parental control melanomas. In some cases, wild-type (Acsl3OE) or catalytically dead mutant Acsl3 (mut. Acsl3OE) were over-expressed 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/C11Oxidized+C11Non-oxidized ratio) levels in mouse melanoma cells from subcutaneous tumors 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-deficient 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 deficient melanomas in culture with oleic acid and with or without Erastin. In some cases, wild-type (Acsl3OE) or catalytically dead mutant Acsl3 (mutant Acsl3OE) were over-expressed in YUMM1.7 (g) or YUMM3.3 (h) melanomas. (i) Metastatic disease burden in mice intranodally injected with Acsl3-deficient 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 in source data files.
Extended Data Figure 6.
Extended Data Figure 6.. Expression of potential ferroptosis regulators by melanoma cells. Related to Figure 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 Figure 1. (g) FSP1 transcript levels by qRT-PCR in human melanoma cells isolated from subcutaneous tumors, blood, or lymph of xenografted mice (3 replicates (each replicate was pooled from 4–5 mice) per melanoma). (h) RNAseq 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 tumors 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 panels g and 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 in source data files.
Figure 1.
Figure 1.. Melanoma cells in lymph experience less oxidative stress as compared to melanoma cells in the blood.
a-b, Efficiently metastasizing human (a) or mouse (b) melanomas were transplanted subcutaneously into NSG (a) or C57BL (b) mice. After the tumors reached ~2 cm in diameter the numbers of melanoma cells per microliter of tumor-draining blood or lymph (Drain.) or blood from cardiac puncture or contralateral lymph (Dist.) were quantified by flow cytometry. c-f, ROS levels (c, d) or GSH/GSSG ratios (e, f) in melanoma cells from subcutaneous tumors, the blood, and the lymph of NSG mice transplanted with patient-derived melanomas (c, e) or C57BL mice with mouse melanomas (d, f). g-j, GSH concentration (g), GSSG concentration (h), GSH/GSSG ratio (i), and 8-OHdG concentration (j) in plasma or lymph fluid from NSG or C57BL mice. The number of replicates (each replicate was pooled from 6–10 mice) 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 correlated-samples two-way ANOVAs, paired t-tests or Wilcoxon tests (a, b), correlated-samples two-way ANOVAs (g-j), or two-way ANOVAs (c-f). Multiple comparisons were adjusted using Holm-Sidak’s (a, b) or Tukey methods (c-f).​ For all panels, statistical tests were two-sided where applicable and *p<0.05, **p<0.01, ***p<0.001. Exact p-values are in source data files.
Figure 2.
Figure 2.. Melanoma cells undergo increased ferroptosis in blood as compared to lymph.
a-b, Lipid ROS (BODIPY-C11Oxidized/C11Oxidized+C11Non-oxidized ratio) levels in melanoma cells from subcutaneous tumors, blood, and lymph of NSG mice with patient-derived melanomas (a) or C57BL mice with mouse melanomas (b). c-d, Human or mouse melanomas were treated in culture with the ferroptosis promoter, Erastin, and/or the ferroptosis inhibitor, Liproxstatin-1 (Liprox). e, Free iron concentrations in plasma or lymph fluid from NSG or C57BL mice. f-i, Human (f, h) or mouse (g, i) melanomas were pre-treated with Liproxstatin-1 then injected intravenously (f, g) or intranodally (h, i) into NSG (f, h) or C57BL (g, i) mice and metastatic disease burden was assessed 1–3 months later by bioluminescence imaging. j-k, Mouse melanomas were pre-treated with N-acetyl-L-cysteine (NAC) or Trolox then injected intravenously (j) or intranodally (k) into C57BL mice and metastatic disease burden was assessed 1–2 months later by bioluminescence imaging. l-n, Percentage of mice transplanted with parental or Gpx4-deficient melanomas that formed metastatic tumors after subcutaneous (l), intravenous (m), or intranodal (n) injection. 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 two-way ANOVAs (a, d, f), correlated-samples two-way ANOVAs (e), Kruskal-Wallis tests (b, c, j, k), t-tests (g-i), or multiple logistic regressions (l-n). Multiple comparisons were adjusted using Tukey’s (a, d, l-n) or Dunn’s (b, c, j, k) tests. No statistically significant differences were observed in panels h, i, k, or n. For all panels, statistical tests were two-sided where applicable and *p<0.05, **p<0.01, ***p<0.001. Exact p-values are in the source data files.
Figure 3.
Figure 3.. Oleic acid levels are higher in lymph, and in melanoma cells from lymph, and oleic acid protects against ferroptosis.
(a) Principal component analysis of metabolomic profiling of melanoma cells from blood or lymph of the same mice xenografted with M481, M405, or UT10 melanomas. (b) The most enriched pathways among metabolites that significantly differed (p<0.001 and >1.5-fold change) between melanoma cells from blood and lymph. (c, d) Human (c) and mouse (d) melanomas were cultured for 12 hours with or without oleic acid or linoleic acid before adding Erastin for 24 hours and counting cells. (e, f) Human (e) and mouse (f) melanomas were pre-treated for 12 hours with vehicle (control), oleic acid, or linoleic acid then intravenously injected into NSG (e) or C57BL (f) mice and metastatic disease burden was assessed 1–3 months later by bioluminescence imaging. g-h, Relative oleic acid abundance in the plasma and lymph of NSG (g) and C57BL (h) mice (TGs, triacylglycerols, PLs, phospholipids). i-j, Acsl3-deficient mouse melanomas were cultured as in 3d (parental control cell data) for 12 hours with vehicle, oleic acid, and/or linoleic acid before adding Erastin for 24 hours and counting cells. (k) Mouse melanoma cells from primary subcutaneous tumors or lymph node metastases were injected intravenously (IV) or subcutaneously followed by intravenous retransplantation (SQ to IV) in C57BL mice and the frequency of cells that formed metastatic tumors was determined by limiting dilution analysis. Human (l) or mouse (m) melanoma cells were isolated from primary subcutaneous tumors or lymph node metastases of the same mice, then treated with Erastin for 24 hours in culture to assess their sensitivity to ferroptosis. The number of replicates in each treatment 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 MetaboAnalyst’s clustering analysis (a) and metabolite set enrichment analysis (MSEA) (b), Kruskal-Wallis tests (c, e(M405), i, j), Welch’s one-way ANOVA (d), one-way ANOVAs (e(M481, UT10), l, m), two-way ANOVAs (f, k), or correlated samples two-way ANOVAs (g, h). Multiple comparisons were adjusted using Dunn’s (c, e(M405), i, j), Dunnett’s T3 (d), Tukey’s (e(M481, UT10), f), or Sidak’s (l, m) tests. For all panels statistical tests were two-sided where applicable and *p<0.05, **p<0.01, ***p<0.001. Exact p-values are in source data files.
See this image and copyright information in PMC

Comment in

  • Cancer cells stock up in lymph vessels to survive.
    Grüner BM, Fendt SM. Grüner BM, et al. Nature. 2020 Sep;585(7823):36-37. doi: 10.1038/d41586-020-02383-5. Nature. 2020. PMID: 32814912 No abstract available.
  • Lymph: (Fe)rrying Melanoma to Safety.
    Lund AW, Soengas MS. Lund AW, et al. Cancer Cell. 2020 Oct 12;38(4):446-448. doi: 10.1016/j.ccell.2020.08.011. Epub 2020 Sep 10. Cancer Cell. 2020. PMID: 32916127
  • A cozy niche in an iron world.
    Conrad M, Novikova M. Conrad M, et al. Signal Transduct Target Ther. 2020 Nov 4;5(1):261. doi: 10.1038/s41392-020-00368-4. Signal Transduct Target Ther. 2020. PMID: 33149129 Free PMC article. No abstract available.

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