Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. We further demonstrated that CD44 directly mediated cell aggregation through CD44 homophilic interactions. Machine learning-based computational modeling combined with experimental mutagenesis tests revealed that the extracellular domains I and II of CD44 are essential for its trans-dimerization and predicted high-score residues to be required for dimerization. Substitutions of 10 these residues in domain I (Ser-45, Glu-48, Phe-74, Cys-77, Arg-78, Tyr-79, Ile-88, Arg-90, Asn-94, and Cys-97) or 5 residues in domain II (Ile-106, Tyr-155, Val-156, Gln-157, and Lys-158) abolished CD44 dimerization and reduced tumor cell aggregation in vitro. Importantly, the substitutions in domain II dramatically inhibited lung colonization in mice. The CD44 dimer-disrupting substitutions decreased downstream PAK2 activation without affecting the interaction between CD44 and PAK2, suggesting that PAK2 activation in tumor cell clusters is CD44 trans-dimer-dependent. These results shed critical light on the biochemical mechanisms of CD44-mediated tumor cell cluster formation and may help inform the development of therapeutic strategies to prevent tumor cluster formation and block cluster-mediated metastases.


Dr. Xia liu received her PhD degree from Peking Union Medical College, China. Then I pursued her postdoctoral training at Case Western Reserve University and NOrthwestern University. In 2020, she became an Assistant Professor at the Department of Toxicology and Cancer Biology, University of Kentucky, USA. Currently, the research in her laboratory is focused on the mechanisms of breast cancer metastasis, cancer liquid biomarkers discovery, the role of innate immunity in cancer, and development of novel therapeutic strategies to treat metastatic cancers, .