Simple SummaryThe majority of cancer-related deaths are attributed to distant metastases—the spread of cancer to different parts of the body. Accurate and early prediction of metastasis and response to therapy is key for the better management of cancer. Although metastases were initiated by circulating tumor cells (CTCs) shed into the bloodstream, only a small subset of CTCs bears metastatic capacity. Since cellular morphology has been shown to reflect cell state and function, we aim to detect the metastasis-initiating subpopulation of CTCs through a simple image analysis. We performed deep morphometric analyses of CTCs and determined their metastatic potential in mice. We identified a subgroup of CTCs with small size, large mitochondria and rough membrane texture to have the highest tumorigenic potential. Our new findings provide a simple image-based identification of CTC subpopulations with elevated aggressiveness, which is expected to provide a more accurate prediction of breast cancer patient survival than total CTC numbers.AbstractUsing previously established CTC lines from breast cancer patients, we identified different morphometric subgroups of CTCs with one of them having the highest tumorigenic potential in vivo despite the slowest cell proliferation in vitro. This subgroup represents 32% of all cells and contains cells with small cell volume, large nucleus to cell, dense nuclear areas to the nucleus, mitochondria to cell volume ratios and rough texture of cell membrane and termed “Small cell, Large mitochondria, Rough membrane” (SLR). RNA-seq analyses showed that the SLR group is enriched in pathways and cellular processes related to DNA replication, DNA repair and metabolism. SLR upregulated genes are associated with poor survival in patients with ER+ breast cancer based on the KM Plotter database. The high tumorigenic potential, slow proliferation, and enriched DNA replication/repair pathways suggest that the SLR subtype is associated with stemness properties. Our new findings provide a simple image-based identification of CTC subpopulations with elevated aggressiveness, which is expected to provide a more accurate prediction of patient survival and therapy response than total CTC numbers. The detection of morphometric and transcriptomic profiles related to the SLR subgroup of CTCs also opens opportunities for potential targeted cancer treatment.