September 12, 2023 | The recent discovery that the metastatic spread of breast cancer accelerates during sleep sparked a lively conversation at last month’s Next Generation Dx Summit about new paths of exploration for thwarting a deadly pan-cancer process claiming 12 lives worldwide every minute. Due to the dynamics of circadian- rhythm-related hormones, the proliferation of circulating tumor cells (CTCs) in primary tumors is higher during the rest phase compared to the active phase and these are the aggressive CTCs that grow into metastatic lesions, according to a presentation by Zoi Diamantopoulou, Ph.D., a postdoctoral fellow in the molecular oncology lab at the Institute of Molecular Health Sciences at ETH Zurich (Switzerland).
These results, validated through a series of experiments in mice, are as fascinating as they are perplexing. For example, Diamantopoulou and her research team succeeded in inhibiting the number of CTCs in the rest phase after treating the animals with insulin for seven days during the opposite (active) phase of their circadian rhythm when hormone levels are known to reach their daily peak. But in doing so, they increased proliferation of breast cancer cells during the active phase.
While glucose transporters are known to be abnormally upregulated in cancer, the treatment was given many weeks after the tumor had an established phenotype, she later explained. In general, study findings indicate that CTC proliferation and intravasation—a key event initiating the escape of cancerous cells from their primary site—are controlled by daily oscillations in insulin as well as glucocorticoid and testosterone.
A single treatment with a well-known glucocorticoid (dexamethasone), and implantation of a testosterone pellet, each resulted in a marked reduction in single CTCs, CTC clusters and CTC–white blood cell (WBC) clusters when mice were sampled during the rest phase. While tumor size was unaffected, a reduction in the metastatic burden was observed in the testosterone-treated animals, Diamantopoulou reported.
CTC clusters are more aggressive than single CTCs and so this is the population scientists are targeting to block metastasis, Diamantopoulou began. They represent an active field of study, with more than 3,000 clinical trials underway attempting to identify novel biomarkers.
Her recent quest was to answer the basic but fundamental question with potentially significant impact in the clinic: “Is the generation of CTCs constant throughout the day?” The project began with an analysis of the number of CTCs in hospitalized breast cancer patients in both early and late stages of the disease.
One blood sample was collected from each patient at 4 a.m. and a second one a few hours later. In all the samples analyzed, a dramatic increase was seen in the number of CTCs during the rest phase of the circadian rhythm, Diamantopoulou says.
These results were confirmed by Diamantopoulou and her colleagues by analyzing the intravasation rate of CTCs at different time points in both immune-competent and immune-compromised mice. First, they shifted the animals’ exposure to light by eight hours every three days, mimicking the jetlag stress experienced by people traveling internationally, before collecting their blood in the rest phase. Compared to the control animals whose CTCs were increasing, she says, those of the jetlagged mice were “dramatically reduced.”
To verify that these results were real, the research team gave the jetlagged mice the sleep hormone melatonin, either alone or in combination with the melatonin-specific inhibitor luzindole. Those administered only melatonin showed a marked increase in the production of single CTCs, CTC clusters, and CTC-WBC, while those receiving melatonin along with the luzindole were effectively rescued from those effects, she says.
Next, investigators looked for the presence of CTCs in blood collected at different time points from either wild-type BL/6 (inbred) mice or Bmal1-homozygous-knockout mice—the only arrhythmic single-gene-knockout model—after the animals developed symptoms, Diamantopoulou continues. The same pattern again emerged, where levels were highest during the rest phase of the circadian rhythm in the control animals but in the Bmal1 mice that oscillation in CTC counts was lost.
The possibility remained that the fewer number of CTCs seen in the active phase were the ones to worry about, or perhaps all CTCs have the same metastatic-forming potential throughout the day, Diamantopoulou says. To rule that out, she and her team collected CTCs that were labelled with green fluorescent protein during the rest phase of the circadian rhythm and red fluorescent protein during the active phase in tumor-free mice injected with CTCs at different time points of the circadian rhythm. More than 97% of metastatic lesions formed in the animals who were injected during their rest phase.
To confirm this apparent time-dependency in the development of metastatic lesions, they repeated the experiment to analyze the metastatic potential of each cell type in parallel, she says. CTC clusters were observed to be more aggressive than the single CTCs and the phenotype was seen in cancers that metastasize to the bone and liver as well as the lung.
The same collection procedure was followed yet again when identifying the expression profile of CTCs in the active and rest phases via genetic analysis, shares Diamantopoulou. As it turns out, most of the genes expressed during the rest phase correlate with cell division whereas more in the active phase are associated with a high level of ribosome biogenesis activity.
Confirmation of these results was accomplished by staining primary tumors and CTCs with Ki67, a well-known proliferation marker that is broadly used in the clinic, she adds. With the primary tumors, a proliferation pattern was seen with the highest CTC intravasation observed during the rest phase.
Diamantopoulou says her first assumption was that the tumor has a functional circadian clock, and this is why CTCs proliferate at a specific time. With that in mind, investigators collected samples from the tumor, liver, and adrenal gland of the mice at different time points and analyzed the expression levels of Bmal1.
The Bmal1 had a “very nice intravasation pattern in the case of the liver and adrenal glands but not in the case of the tumor,” she says. That led to the thought that only a few cells had the circadian clock, rather than the entire tumor, and they went on to become CTCs and intravasate at a specific time.
To test that hypothesis, the research team next investigated whether the expression of circadian clock genes in cancer cells changed between the rest and active phases. “Surprisingly, we didn’t see any difference,” says Diamantopoulou, “so evidently the breast cancer does not have a functional circadian clock.” But this begged the question, “Why then do these cells intravasate at a specific time of the day?”
The theory was now that “people who don’t have a functional circadian clock probably can’t respond to circadian-rhythm-regulated hormones,” she continues. So, the team went back and analyzed results from the human tumor samples to identify three receptors that were secreting hormones and they corresponded to the receptors for glucocorticoids, insulin and testosterone.
This is where the idea came up to administer the hormones during the rest phase of the circadian rhythm to see if they could inhibit generation of the CTCs.
As pointed out during the Q&A following Diamantopoulou’s presentation, blood draws on cancer patients are routinely done in the morning but would perhaps turn up different results if they instead happened in the evening. Since she was unable to collect blood samples more than twice from the same patient, and the study was conducted during COVID when it was harder in general to get into the hospital environment, that question remains unanswered.
The development of anti-metastatic therapies will undoubtedly require understanding more than the hormones involved and the role of the circadian clock, which is itself under the control of the Bmal1 master clock gene, she says. “There are [other] clock-independent functions and molecules that may affect circadian rhythmicity... [and] contribute to the final phenotype.”
One additional surprise coming out of the recent series of experiments is that immunosurveillance remains stable at different time points of the circadian rhythm, says Diamantopoulou. No differences were seen in the immune cell populations during the active and rest phases of the circadian rhythm when analyzing both the peripheral blood from cancer patients and the tumor-infiltrated immune cells. Perhaps something changes systematically in all these immune cells during early stages of disease, she muses.
In terms of the findings around testosterone, Diamantopoulou references a study where the hormone was administered for a long time to more than 1,000 women undergoing a sex change procedure. Among those that went on to develop metastatic cancer, researchers found “very clear differences in progression-free survival” where testosterone was thought to confer the protective effect.
Top questions on the mind of Diamantopoulou currently are whether it is sleep or the lack of activity that contributes to metastatic disease, as well as the impact of anesthesia on the phenotype. She and her team are now trying to get ethical approval to examine the latter question in patients with breast, prostate, and colorectal cancer by taking blood samples from them before they’re anesthetized, during the anesthesia, and one day afterwards, she reports.