Metastasis is the primary cause of breast cancer-related morbidity and mortality. During metastasis, cells from primary tumors shed into the bloodstream as circulating tumor cells (CTCs). CTCs travel to distant organs to establish secondary tumors. CTCs that form clusters exhibit a drastic increase in metastatic potential compared to single CTCs. While previous studies have described E-cadherin as a key mediator of adhesion in CTC clusters and metastasis, many breast tumors, including tumors of the highly metastatic triple negative breast cancer (TNBC), express little to no E-cadherin. Thus, an alternative, E-cadherin-independent mechanism must exist to mediate clustering between CTCs, thereby promoting metastasis and disease progression. To investigate CTC clustering mechanisms, we developed an in vitro tumor cell clustering assay that incorporates physiological shear force and mimics in vivo conditions. Using this assay, we found that E-cadherin-negative metastatic breast cancer cells can form cellular interactions with characteristics similar to cell adherens junctions. Speculating that extracellular matrix (ECM) components from tumor cells may contribute to CTC clustering, we analyzed ECM components by breast cancer subtypes. We found that hyaluronic acid synthase 2 (HAS2), which is the primary enzyme responsible for hyaluronic acid (HA) production in breast cancer cells, is significantly upregulated in TNBC. We further observed that HA mediates clustering between TNBC tumor cells and confers them with the ability to overcome insults present in the bloodstream, including shear forces. Importantly, we detected HA enrichment at the cell-cell junction of interacting CTCs in TNBC patient blood specimens. Mechanistically, our preliminary results suggest that metastatic TNBC cells upregulate HAS2 expression in a chromatin looping mechanism mediated by PRDM6, a transcriptional regulator and putative histone lysine methyltransferase. Collectively, these preliminary findings lead us to hypothesize that in aggressive TNBC, high levels of HA augment CTC clustering via HA-dependent adhesive interactions between neighboring cells. We further hypothesize that the PRDM6 upregulates HA levels through epigenetic modulation of HAS2 expression, including chromatin looping interactions. We propose to study our hypotheses through two specific aims: 1) determine the role of HA in TNBC tumor cell clustering and metastasis and 2) elucidate the epigenetic regulation of HAS2 that impacts breast tumor cell clustering. To investigate our hypotheses, we will utilize novel in vitro clustering assays and in vivo mouse models along with electron microscopy to reveal the structural characteristics of CTC clusters. In addition, we will utilize integrative multi-omics data analysis to elucidate the co-regulatory network governing HAS2 expression during CTC clustering. Importantly, our results will be extensively validated in blood specimens from metastatic TNBC patients. Conse...