CBX3 confers ferroptosis resistance during blood-borne metastasis

The survival mechanisms of circulating tumor cells (CTCs) remain poorly understood while these rare cell populations transit through the blood stream and colonize distant organs including the brain. Using single-cell RNA-seq of microfluidically-enriched CTCs and patient-matched brain metastatic tumor cells of lung adenocarcinoma (LUAD), we revealed CTC-selective upregulation of core gene signatures associated with ferroptosis. The malignancy of CTCs was confirmed through comparative genomic instability and copy number variation (CNV) analyses of paired CTCs and metastatic tumor cells using both transcriptomic and whole-exome sequencing. Among the transcription factors elevated in CTCs, Chromobox 3 (CBX3) was the top hit, which was tightly correlated with GPX4 expression. Functionally, CBX3 co-operated with EP300 to protect CTCs from ferroptosis by upregulating GPX4 expression. Genetic depletion of CBX3 triggered ferroptosis and substantially reduced tumor cell survival and invasiveness in two independent cancer cell models. Conversely, CBX3 overexpression was sufficient to promote tumor growth, migration, and invasion in vitro, and metastatic progression in vivo. Clinically, CBX3 expression was significantly correlated with TNM stage and was predictive of both progression-free interval and overall survival in LUAD. In a prospective cohort of LUAD and melanoma patients, CTCs co-expressing CBX3 and GPX4 were selectively elevated in the blood samples of metastatic cases compared to the non-metastatic group, highlighting the clinical association between ferroptosis-resistant CTCs and metastatic progression.