Oxidative stress inhibits distant metastasis by human melanoma cells

Abstract

Solid cancer cells commonly enter the blood and disseminate systemically, but are highly inefficient at forming distant metastases for poorly understood reasons. Here we studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NOD-SCID-Il2rg−/− (NSG) mice. We show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas. Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours. Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence on NADPH-generating enzymes in the folate pathway. Antioxidants promoted distant metastasis in NSG mice. Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumours in the same mice. Oxidative stress thus limits distant metastasis by melanoma cells in vivo.

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Figure 1: Metastasizing melanoma cells experience high levels of oxidative stress.
Figure 2: Melanoma cell metastasis, but not subcutaneous tumour growth, is promoted by antioxidants in vivo.
Figure 3: During metastasis, some melanoma cells reversibly increase their expression of folate pathway enzymes that generate NADPH and folate pathway inhibition selectively impairs metastasis.

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Acknowledgements

S.J.M. is a Howard Hughes Medical Institute (HHMI) Investigator, the Mary McDermott Cook Chair in Pediatric Genetics, the director of the Hamon Laboratory for Stem Cells and Cancer, and a Cancer Prevention and Research Institute of Texas Scholar. We thank K. Correll and M. Gross for mouse colony management; N. Loof and the Moody Foundation Flow Cytometry Facility. We thank N. Meireles and the University of Michigan Melanoma Biobank, for Biobank database and melanoma clinical data management.

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Authors

Contributions

E.P. and S.J.M. conceived the project, designed, and interpreted experiments. M.A. and R.J.D. participated in the design and interpretation of experiments related to metabolic mechanisms. E.P. performed all the experiments with technical assistance from S.E.H., metabolomics assistance from M.A and imaging assistance from M.M.M. Z.H. and M.A developed metabolomics methods. Z.Z. assisted with statistical analyses. A.M.L. and T.M.J. provided melanoma specimens and associated clinical data. E.P., M.A. and S.J.M wrote the manuscript.

Corresponding author

Correspondence to Sean J. Morrison.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Expression of melanoma markers by xenografted melanomas.

a, M405, M481, M514, M528, M498, M597, M610 and UT10 tumours were consistently positive for S100, a marker used clinically to diagnose melanoma. b, Flow cytometric analysis of xenografted tumour cells that were HLA-ABC+ and negative for mouse CD31/CD45/Ter119 showed that these cells were usually positive for melanoma cell adhesion molecule (MCAM) and melanoma-associated chondroitin sulphate proteoglycan (MCSP). Both of the tumours that lacked MCSP staining (M514 and M597) were heavily pigmented and expressed other melanoma markers (such as S100 and MCAM).

Extended Data Figure 2 Clinical data on the melanomas used in this study and summary of their metastatic behaviour in NSG mice.

a, Summary of the clinical characteristics of the melanomas used in this study at the time of banking, as well as patient outcome after banking, and metastasis patterns upon transplantation of banked tumours into NSG mice. Melanomas were stratified into efficiently and inefficiently metastasizing melanomas. Efficiently metastasizing melanomas formed distant metastases in patients and in NSG mice, whereas inefficiently metastasizing melanomas did not. The latter group did form micrometastases in the lung, but not outside of the lung in the period of time it took for subcutaneous tumours to grow to 2 cm in diameter (when the mice had to be euthanized in these experiments27). Nonetheless, most of the inefficiently metastasizing melanomas have the ability to form macrometastases if given enough time (data not shown). b, Growth rates of subcutaneous tumours in NSG mice after subcutaneous transplantation of 100 cells. Statistical significance was assessed using two-tailed Student’s t-test. c, Clinical characteristics of the patients from whom melanomas were obtained at the time of banking and upon subsequent clinical follow up. The tumours were confirmed to be melanomas by clinical dermatopathology. The tumours were independently confirmed to be melanomas after xenografting in mice by histological and flow cytometric analysis of melanoma markers (Extended Data Fig. 1) as well as examination by a clinical dermatopathologist.

Extended Data Figure 3 Barriers to distant metastasis in vivo.

a, Live human melanoma cells were identified by flow cytometry based on the expression of DsRed (all melanomas in this study stably expressed DsRed) and human HLA and the lack of expression of mouse CD45, CD31 and Ter119 (to exclude mouse haematopoietic and endothelial cells). Human melanoma cells were observed in the blood of NSG mice bearing efficiently metastasizing melanomas. b, Mice xenografted with efficiently metastasizing melanomas (n = 43 mice with tumours derived from four patients) had significantly higher frequencies of CMCs in their blood than mice xenografted with inefficiently metastasizing melanomas (n = 13 mice with tumours derived from four patients) or control mice that had not been xenografted (n = 18 mice). Blood was collected by cardiac puncture. Statistical significance was assessed using ANOVA followed by Tukey’s test for multiple comparisons. **P < 0.005. cf, Bioluminescence analysis of total photon flux (photons s−1) from mouse organs after intravenous injection (c, d) or intrasplenic injection (e, f) of luciferase-tagged melanoma cells derived from efficiently metastasizing (c, e) or inefficiently metastasizing (d, f) melanomas. Each melanoma was derived from a different patient and was studied in an independent experiment. g, Schematic of the experiment shown in Table 2a. h, Schematic of the experiment shown in Table 2b. i, Summary of mean limiting dilution frequencies of tumour-forming cells after subcutaneous, intravenous, or intrasplenic transplantation into NSG mice.

Extended Data Figure 4 Unsupervised clustering suggests that melanoma cells undergo reversible metabolic changes during metastasis.

a, b, Hierarchical clustering from two independent experiments reflecting subcutaneous tumours and metastatic nodules from the liver, pancreas, lung and kidneys of mice transplanted with melanomas M405, M481 and M514 (a) (see Extended Data Table 1 for data on individual metabolites) and subcutaneous tumours and metastatic nodules from the liver, pancreas and kidneys of mice transplanted with melanomas M405, M481 and UT10 (n = 2–3 mice per melanoma in each experiment; see Extended Data Table 2 for data on individual metabolites) (b). c, Hierarchical clustering of metabolites extracted from flow cytometrically sorted human melanoma cells isolated from subcutaneous tumours or metastatic nodules (UT10, M481, n = 3 mice per melanoma in two independent experiments). d, e, Hierarchical clustering of metabolites extracted from subcutaneous tumours and metastatic nodules from mice transplanted subcutaneously with either subcutaneous, circulating or metastatic melanoma cells (n = 4 mice for each melanoma in two independent experiments). f, GSH/GSSG ratios from each of the experiments that compared subcutaneous tumours and metastasizing cells. (i) Metabolites were extracted in the presence of 0.1% formic acid to inhibit spontaneous oxidation42 in two independent experiments comparing subcutaneous and metastatic tumours from mice with three different melanomas in each experiment (M405, M481 and UT10). (ii) and (iii) GSH/GSSG ratios from the experiments shown in a and b, respectively. (iv) GSH/GSSG ratios in melanoma cells isolated by flow cytometry from subcutaneous tumours and the blood (circulating melanoma cells) of mice bearing M405 and M481. (v) Metabolites were extracted in the presence of 0.1% formic acid in two independent experiments in which melanoma cells were isolated by flow cytometry from subcutaneous and metastatic tumours (M405 and M481). While the GSH/GSSG ratio was always significantly higher in melanoma cells from subcutaneous tumours as compared to circulating cells or metastatic nodules the ratio varied among experiments as a result of technical differences in cell isolation and metabolite extraction as well as differences in mass spectrometry sensitivity to GSH and GSSG. g, GSH/GSSG ratios in subcutaneous tumours that arose from the transplantation of melanoma cells obtained from subcutaneous tumours or metastatic nodules, as well as the metastatic nodules from the same mice (M405; n = 2 to 3 mice per treatment in one experiment). These data suggest that the decline in GSH/GSSG ratio in metastasizing melanoma cells is reversible upon subcutaneous transplantation. h, Histogram showing mitochondrial mass in subcutaneous tumour cells that arose from the transplantation of subcutaneous cells (SQ from SQ), subcutaneous tumour cells that arose from the transplantation of metastatic cells (SQ from Mets), and metastatic cells (metastases). These histograms reflect the data shown in Fig. 1g. All error bars represent s.d. Statistical significance was assessed using, two-tailed Student’s t-tests (f and g). *P < 0.05.

Extended Data Figure 5 Metastatic nodules exhibited increased enrichment of labelled serine and glycine as compared to subcutaneous tumours.

In vivo isotope tracing of uniformly 13C-labelled lactate (M + 3) (a), 3-phosphoglycerate (M + 3) (b), serine (M + 3) (c), and glycine (M + 2) (d) in subcutaneous tumours versus metastatic nodules from the same mice (UT10, n = 3–4 mice per time point in two independent experiments). The fractional enrichment of labelled lactate, and 3-PG did not significantly differ among plasma, subcutaneous tumours or metastatic tumours at any time point. By contrast, the fractional enrichment of labelled serine and glycine were significantly higher in metastatic as compared to subcutaneous tumours. This is consistent with increased de novo serine synthesis in metastatic tumours but could also reflect altered serine/glycine exchange with circulating serine/glycine pools in metastatic as compared to subcutaneous tumours. e, NADPH/NADP ratios in subcutaneous tumours and metastatic nodules from the same mice shown in Fig. 2d, e. f, g, Western blot of ALDH1L2 (f) and MTHFD1 (g) protein after shRNA knockdown in melanoma cells. Uncropped western blots are shown in Supplementary Fig. 1. h, i, Amount of GSH (h) and GSSG (i) per mg of subcutaneous or metastatic tumour as measured by LC–MS (M405, M481 and UT10, n = 2–3 mice per melanoma in two independent experiments). All data represent mean ± s.d. Statistical significance was assessed using two-tailed Student’s t-tests (e, h and i) and one-way ANOVAs followed by Dunnett’s tests for multiple comparisons (ad). *P < 0.05; ***P < 0.0005. j, Schematic of the folate pathway including NADPH generating (green box) and NADPH consuming (red box) reactions.

Extended Data Figure 6 Immunofluorescence analysis of ALDH1L2 in subcutaneous melanoma as well as metastatic nodules in the liver, pancreas and lung.

ad, Melanoma cells can be distinguished from host stromal cells based on staining for the melanoma marker, S100.

Extended Data Table 1 Fold change and statistical significance corrected for multiple comparisons of metabolites detected by LC–MS/MS in the experiment in Extended Data Fig. 2a.
Extended Data Table 2 Fold change and statistical significance corrected for multiple comparisons of metabolites detected by LC–MS/MS in the experiment shown in Extended Data Fig. 2b.
Extended Data Table 3 Treatment with the antioxidant NAC increased tumour formation by intravenously transplanted melanoma cells.
Extended Data Table 4 Fold increase in metastatic nodules as compared to subcutaneous tumours of transcripts involved in antioxidant responses and NADPH generation.

Supplementary information

Supplementary Information

Supplementary Figure 1 shows the western blots for Figures 3a, 3h and Extended Data Figures 6f, 6g. Tumour measurements for Figures 2a, 3b, 3e and 3i are also shown. (PDF 1751 kb)

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Piskounova, E., Agathocleous, M., Murphy, M. et al. Oxidative stress inhibits distant metastasis by human melanoma cells. Nature 527, 186–191 (2015). https://doi.org/10.1038/nature15726

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