Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, and metastasis is the major cause of cancer morbidity and mortality. Therefore, it is urgent to discover novel therapeutic targets and develop effective treatments for this lethal disease. Circulating tumor cells (CTCs) are considered “seeds of metastasis”. Compared to single CTCs, our previous studies have demonstrated that CD44 homophilic interaction mediates CTC aggregation to enhance the stemness, survival and metastatic ability of aggregated cells. Importantly, the presence of CD44+ CTC clusters correlates with a poor prognosis in breast cancer patients. Here, we further investigated the underlying mechanism of how CD44-mediated cell aggregation promotes TNBC metastasis. We found that cell detachment, which mimics the condition when tumor cells detach from the extracellular matrix (ECM) to metastasize, induces lipid raft disruption in single cells, but lipid rafts integrity is maintained in aggregated cells. We further found that lipid rafts integrity in aggregated cells is required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promotes CD44 cleavage and generates CD44 intracellular domain (CD44 ICD) to enhance stemness. Consequently, lipid rafts disruption inhibited Rac1 activation, CD44 ICD generation and metastasis. These data reveal a new mechanism of cell aggregation-mediated TNBC metastasis via maintaining lipid raft integrity after cell detachment. The finding provides a potential therapeutic strategy to prevent CTC cluster-initiated metastasis by disrupting lipid raft integrity and its-mediated downstream pathways.

Accumulating evidence have demonstrated that circulating tumor cell (CTC) clusters have higher metastatic ability than single CTCs, and correlates with worse cancer patient’s outcomes. The heterotypic CTC clusters such as neutrophil-CTC clusters recently were identified in both cancer mouse models and cancer patients, leading to more efficient metastasis formation compared with homotypic CTC clusters. However, the mechanism by which neutrophils are associated with CTCs remains elusive. In this study, we found that the intercellular adhesion molecule (ICAM-1) on triple-negative breast cancer (TNBC) cells mediates their binding with CD11b+ neutrophils, and CD11b deficiency inhibited TNBC metastasis In vivo. Additionally, CD11b mediated H2O2 production of neutrophils. Further studies indicated that ICAM-1 promotes uPAR secretion, which functions as a chemoattractant for neutrophils. Knockdown of uPAR in ICAM-1+ TNBC cells reduced lung-infiltrating neutrophils, and lung metastasis. The bioinformatics analysis showed uPAR is highly expressed in TNBCs, which positively correlates with higher neutrophil infiltration and negatively with breast cancer patient’s survival. Together, our findings discover a novel chemoattractant role of suPAR in TNBC metastasis, and provides a rationale for targeting ICAM-1-uPAR-CD11b axis to block CTC-neutrophil cluster formation, and its-mediated metastasis.