For cancer to metastasize to other organs, cancer cells must first break away from the primary tumor and enter the bloodstream. A study published yesterday (June 22) in Nature suggests that this dissemination mostly occurs while its host sleeps. The authors observed that circulating tumor cells (CTCs)—those that have detached from the tumor to migrate to distant organs—were found in significantly higher numbers in blood samples collected while breast cancer patients and mice were in their resting period compared to their active one.

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Catherine Alix-Panabières, a cancer researcher at University Hospital of Montpellier in France who did not participate in this study but is currently collaborating with one of the authors on a different project, says she is happy to see these new “very strong data” supporting the role of circadian rhythms in tumor dissemination. She and her colleagues had previously speculated that circulating tumor cells could be influenced by biological cycles in a 2020 Genome Biology review article. But at the time, there were no rigorous studies comparing CTCs produced during the day versus the night, she says.

Nicola Aceto, a Swiss Federal Institute of Technology in Zurich molecular oncologist and lead author of the new study, tells The Scientist over email that, prior to the new study, he and his colleagues had unexpectedly “observed high variation in CTC numbers depending on the time at which blood samples [of cancer patients] were taken.” To examine this variation more closely, they collected blood samples from 30 hospitalized breast cancer patients at 4:00 AM, when they were resting, and again the same day at 10:00 AM, comparing the samples’ CTC abundance. They found a remarkable difference between day and night collections: 78 percent of the total CTCs detected across all patients were found in the 4:00 AM blood samples. 

They repeated the experiment in four different mouse models of breast cancer, sampling the animals’ blood while they were resting (during the day) and when they were active (at night). The outcome matched the observations in humans: 87 to 99 percent of the CTCs were detected while the mice were in their resting phase. Moreover, the team compared the metastatic potential of cells gathered at each time point by injecting them into tumor-free mice and found that cancer cells released during the resting phase were significantly better at colonizing lung tissues. 

In humans, CTCs have a very short half-life and only persist for a few hours or less, explains Alix-Panabières, who holds a patent for CTC detection and consults for a pharmaceutical company called Menarini regarding similar work. So any CTCs found in the blood must have recently been released, she notes. It is important to understand when these circulating cells are generated and when they are most aggressive, she adds, as that information will help with developing therapeutics to target and kill this specific subset of cells. 

78 percent of the total CTCs detected across all patients were found in the 4:00 AM blood samples.

Why CTCs shed at night are more adept at forming metastasis is not yet entirely clear, but Aceto and colleagues conducted various analyses to get at this question. They analyzed the gene expression profiles of CTCs in both patients and mice, and discovered that those released during rest had higher expression levels of genes associated with cell division and mitosis compared to CTCs from the active phase, which instead show higher expression levels of genes involved in translation. These results hint at an increased ability to proliferate in the sleep-shed cells. 

The team also found that glucocorticoid, androgen, and insulin receptors are highly expressed in CTCs. As the ligands for these receptors are regulated by circadian rhythms, they suggest that CTCs may respond to the daily oscillations of hormones. When the researchers treated mice with dexamethasone during sleep or with a continuous release of testosterone—a glucocorticoid and an androgen ligand, respectively—the number of CTCs decreased in their blood during the rest phase to a significant extent. Meanwhile, insulin treatment during sleep inverted the proliferation cycle, reducing CTCs detected while the mice were at rest and increasing them when the mice were active. 

NYU Langone Medical Center cancer researcher Thales Papagiannakopoulos, who did not participate in this study, says that he suspects there may be “an immune component” in addition to the role of hormones. But it is hard to disentangle the role of different factors, he adds, and teasing out the whole mechanistic basis of these observations will not be easy. 

Qing-Jun Meng, a University of Manchester chronobiologist who was not involved in this study, says that the phenotype of the circulating tumor cells described in the study is “striking,” and agrees with Papagiannakopoulos that more needs to be done to understand the mechanisms behind it. For example, while Aceto and colleagues did not identify oscillatory expression of circadian clock genes in the CTCs, Meng points out that certain breast cancer subtypes do show rhythmicity in such genes at the cellular level, which might influence the observations reported in the new study.  

All researchers interviewed by The Scientist agree on the importance of testing these results in other cancers—Aceto, who is also a cofounder and a board member of PAGE Therapeutics AG and a consultant for other pharmaceutical companies, writes that he and his colleagues plan to investigate whether other cancers become more metastatic during rest as well.

Now that this paper has been published, Meng says, “a lot of labs probably will follow this up in their own [cancer] models,” manipulating clock genes or environmental factors to address whether this higher CTC generation during sleep “is a true and generalized [circadian] phenomena.” If it is, this will “open up new opportunities” for understanding cancer progression and perhaps allow clinicians to optimize treatment based on the time of day, Meng concludes. 

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