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.
The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene.,With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality.,BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18).,Nineteen patients had tissues available from multiple metastatic sites.,Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR.,The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes.,To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases.,Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors.,Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation.,In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients.,Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful.