ICAM1-ICAM1 interactions initiate CTC cluster formation and trans-endothelia migration in lung metastasis of triple negative breast cancer

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency compared to single cells and are associated with lower overall survival in breast cancer. However, the cellular heterogeneity and molecular mechanisms underlying CTC cluster formation and metastasis have yet to be fully elucidated. Combining single-cell RNA sequencing and flow cytometry, we compared the profiles of sorted tumor cells from the primary breast tumor and lung metastases of triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs). The expression levels of intercellular adhesion molecule 1 (ICAM1) increased 60- to 200-fold in the lung metastases of four PDXs. ICAM1 was also specifically enriched in the clustered CTCs versus the single CTCs of patients with advanced breast cancer. Depletion of ICAM1 abrogated lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation whereas overexpression of ICAM1 accelerated spontaneous metastasis of PDXs. While the known ligands of ICAM1 were not detectable in breast cancer cells, machine learning-based algorithms further assisted identification of ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, both among neighboring tumor cells and between tumor cells and endothelial cells, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promoted metastasis by activating and maintaining cellular pathways related to the cell cycle and stemness, such as CDK6 levels. Finally, blocking ICAM1 interactions with an anti-ICAM1 neutralizing antibody significantly inhibited TNBC CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 could serve as a novel therapeutic target in the early stages of metastasis initiation as well as regenerative tumor growth of TNBC.