Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC.

Circulating tumor cells (CTCs) are vital components of liquid biopsies for diagnosis of residual cancer, monitoring of therapy response, and prognosis of recurrence. Scientific dogma focuses on metastasis mediated by single CTCs, but advancement of CTC detection technologies has elucidated multicellular CTC clusters, which are associated with unfavorable clinical outcomes and a 20-to 100-fold greater metastatic potential than single CTCs. While the mechanistic understanding of CTC cluster formation is still in its infancy, multiple cell adhesion molecules and tight junction proteins have been identified that underlie the outperforming attributes of homotypic and heterotypic CTC clusters, such as cell survival, cancer stemness, and immune evasion. Future directions include high-resolution characterization of CTCs at multiomic levels for diagnostic/prognostic evaluations and targeted therapies. Circulating tumor cells form clusters that enhance breast cancer metastasisAlthough metastasis accounts for 90% of solid tumor-related mortality, it currently evades targeted treatments and demands a better understanding. Metastasis is a multistep process during which cancer cells spread from the primary tumor site to distant organs, starting with the primary tumor formation, where tumor cells gradually expand and locally invade the surrounding stroma and tissues, including the blood and lymphatic vessels. At this point, the intravasated tumor cells, now called 'circulating tumor cells' (CTCs) and a vital component of liquid biopsy [1,2], develop adaptive mechanisms that favor their survival in the harsh microenvironment of the circulatory system. CTCs may disseminate to distant parts of the body before they finally extravasate or get trapped within the capillaries in certain organs, form metastatic niches, and regenerate secondary tumors [3][4][5].The presence of CTCs in the blood of patients with cancer was first detected in 1869 by Thomas Ashworth [6], but, because of advances in technology, CTCs have only recently attracted great attention for their key role in tumor metastasis [6,7]. Many studies have shown that CTCs may be used to predict disease progression and prognosis in patients with metastatic cancer [4]. In metastatic cancers such as breast cancer, the currently accepted level of CTCs that correlates with worse overall survival and progression-free survival is five or more CTCs in 7.5 mL of blood [8]. CTC enumeration can be used to better stratify patients with stage IV breast cancer as stage IV aggressive, with more than five CTCs, and stage IV indolent, with fewer than five CTCs [8]. Stage IV indolent disease is associated with significantly longer overall survival, regardless of disease subtype and prior treatment [8]. The presence of CTCs in early breast cancer was also demonstrated to have prognostic impact [9,10]. Thus, CTC analysis in patients with cancer is a minimally invasive, clinically informative method of predicting disease stage and survival that is not dependent on cancer subtype or previous trea...

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Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is co-expressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.