Solid cancer cells commonly enter the blood and disseminate systemically but are highly inefficient at forming distant metastases for poorly understood reasons.,We studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NSG mice.,All 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 inefficient metastasizers.,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 upon NADPH-generating enzymes in the folate pathway.,Anti-oxidants 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 tumors in the same mice.,Oxidative stress thus limits distant metastasis by melanoma cells in vivo.
Oncogenic BRAF mutation had been considered to be a founder event in the formation of melanocytic tumours; however, we recently argued against this notion by showing marked polyclonality of BRAF mutations in acquired melanocytic nevi (Lin et al, J Natl Cancer Inst., 2009; 101:1423-7).,Here, we tested whether similar heterogeneity of BRAF mutations exists in primary melanomas.,We isolated and sequenced single melanoma cells from five primary melanoma tissues using antibodies against human high-molecular-weight melanoma-associated antigen.,We also examined 10 primary melanomas by the sensitive Mutector assay detecting the BRAFV600E mutation, as well as by cloning and sequencing of separated alleles.,Furthermore, we estimated the frequency of BRAF mutant alleles in paired samples of primary tumour and recurrence or metastasis in three patients.,Single-cell mutation analyses revealed that four of five primary melanomas contained both BRAF-wild-type and BRAF-mutant tumour cells.,Tumour heterogeneity in terms of BRAF mutations was also shown in 8 of 10 primary melanomas.,Selection of BRAF mutant alleles during progression was demonstrated in all the three patients.,Acquisition of a BRAF mutation is not a founder event, but may be one of the multiple clonal events in melanoma development, which is selected for during the progression.