Preclinical and clinical studies indicate that circulating tumor cells (CTCs) are crucial in the metastatic cascade. CTC clusters, aggregates of tumor cells that detach from primary tumors, are often associated with poor clinical outcomes. While it is believed that cells within these clusters cooperate during dispersal, dissemination, and colonization, the extent of genomic heterogeneity within CTC clusters and their relationship to primary tumor and metastatic clones remains largely unexplored.

To investigate these questions, we established mouse xenografts using the triple-negative breast cancer cell line MDA-MB-231. We collected tissue samples from primary tumors and metastases and blood samples to isolate both single CTCs and CTC clusters. Whole-exome sequencing was performed on these samples, enabling the identification of single-nucleotide and copy-number variants through a custom bioinformatic pipeline.

Our analysis revealed ample genomic heterogeneity among CTCs and CTC clusters at the single nucleotide variant level. We identified a set of genes more frequently mutated in CTC and CTC clusters than in primary tumors and metastases, suggesting that they might play a role in the formation and survival of CTCs and CTC clusters.

This study offers new insights into the genomic landscape of CTCs and CTC clusters, identifying novel genes that might play a significant role in CTC biology.