The metastatic spread of breast cancer accelerates during sleep

The metastatic spread of cancer is achieved by the haematogenous dissemination of circulating tumour cells (CTCs). Generally, however, the temporal dynamics that dictate the generation of metastasis-competent CTCs are largely uncharacterized, and it is often assumed that CTCs are constantly shed from growing tumours or are shed as a consequence of mechanical insults1. Here we observe a striking and unexpected pattern of CTC generation dynamics in both patients with breast cancer and mouse models, highlighting that most spontaneous CTC intravasation events occur during sleep. Further, we demonstrate that rest-phase CTCs are highly prone to metastasize, whereas CTCs generated during the active phase are devoid of metastatic ability. Mechanistically, single-cell RNA sequencing analysis of CTCs reveals a marked upregulation of mitotic genes exclusively during the rest phase in both patients and mouse models, enabling metastasis proficiency. Systemically, we find that key circadian rhythm hormones such as melatonin, testosterone and glucocorticoids dictate CTC generation dynamics, and as a consequence, that insulin directly promotes tumour cell proliferation in vivo, yet in a time-dependent manner. Thus, the spontaneous generation of CTCs with a high proclivity to metastasize does not occur continuously, but it is concentrated within the rest phase of the affected individual, providing a new rationale for time-controlled interrogation and treatment of metastasis-prone cancers. Overall design: We isolated live circulating tumor cells (CTCs) from a breast cancer patient (number of CTCs = 36) and two breast cancer xenografts, NSG-CDX-BR16 (number of CTCs = 138) and NSG-LM2 (number of CTCs = 108) at different time points. Isolated CTCs were categorized into three different types : single CTC, CTC clusters and CTC-white blood cells (CTC-WBC) clusters. Amplified cDNA was prepared according to the Smart-seq2 protocol. Libraries were prepared using with Nextera XT (Illumina) and sequenced on Illumina NextSeq500 instrument in 75-bp single-read mode.