Project Summary/Abstract Breast cancer is the most prevalent cancer among women and leads to about 40,000 metastatic related deaths annually in the United States. Circulating tumor cells (CTCs) shed from the primary tumor into the circulatory system. These CTCs are then displaced in distant organs where metastatic tumors eventually arise. Distinctive breast cancer CTCs are assumed to result from discrete patterns of mutated cancer genes. Nonetheless, the contribution of epigenetic changes to the development of individual CTC characteristics is unknown. DNA methylation at cytosines in CpG dinucleotides is critical in programming gene expression and its disruption is a typical hallmark of cancer. Vertebrate CpG islands are short spreads of DNA sequences that differ from the typical genomic pattern by being GC-rich and predominantly unmethylated. In cancer cells, some CpG islands become strongly methylated, which results in repression of gene transcription. Additionally, partially methylated domains (PMDs) are found in gene poor regions which correspond to lamina-attachment domains. PMDs are thought to be linked to gene expression, but not enough is known about why their corresponding domains exist or about their single cell composition. Methylomes have also been linked to enhancers that control a cohort of gene expression. Our lab has established several patient-derived CTC lines, allowing me to analyze the methylomes in CTCs for the first time. By studying the methylomes of CTCs using whole genome bisulfite sequencing (WGBS), I hope to understand if methylation patterns in CTCs are heterogeneous and how this variation contributes to metastasis. Findings from WGBS analysis have demonstrated that the CTC lines Brx50 and Brx61 appear to be less methylated when compared to the CTC lines Brx07 and Brx68. Intriguingly, Brx07 and Brx68 are more metastatic when compared to Brx50 and Brx61. These specific areas that vary between the CTCs’ methylomes could be potential areas of interest for metastasis.