Let Sleeping Cells Lie: How Immune Cells Influence Metastatic Dormancy

With around 2.3 million new diagnoses reported in 2022, breast cancer poses a significant global burden and is the most common cancer in women. With early diagnosis and appropriate treatment, the chances of survival are often high. For example, if the cancer is detected at stage 1, the 5-year survival rate is almost 100%.

Despite this, many individuals will still die as a result of breast cancer, often due to recurrence and metastasis (Varzaru et al. 2025). Recurrences generally occur within a few years of treatment completion; however, they can also appear decades later. In addition to the physical threat, this risk of recurrence can place considerable emotional strain on the patient.

The in-depth study of recurrence has been obstructed by the use of mouse models of metastatic dormancy—the state in which cancer cells persist in a quiescent state—that rely on injecting human cancer cells into immunocompromised mice. This significantly hinders the ability to investigate any immune cell contributions and thus limits clinical relevance.

In this blog, we discuss how Bushnell et al. (2024) used flow cytometry to examine metastatic dormancy in immunocompetent mice and found a new role for immune cells in regulating this process.

Who’s Keeping Dormant Cells in Check?

The researchers initially determined that the breast cancer cell lines PyMT, Met1, and D2.0R could exhibit metastatic dormancy when inoculated into the hearts of mice and decided to investigate the immune system's contribution to this phenotype.

To do this, they used mice with different levels of immune competency: fully immunocompetent mice, NOD scid mice, which lack T cells and B cells, or NSG mice, which lack T, B, and NK cells. They inoculated them with each cell line and examined their disease recurrence following a dormancy period.

Interestingly, they observed different results depending on the cell line used.

When PyMT cells were used, NOD scid mice had reduced survival compared with their immunocompetent counterparts, and this was further reduced in NSG mice, suggesting that, here, dormancy is in part controlled by the adaptive immune system, but also by contributions from NK cells.

The use of Met1 cells again led to a decrease in the survival of immunodeficient mice, but there was no difference between NOD scid and NSG groups, indicating that only the adaptive immune system plays a role in controlling dormant cells in this case.

Finally, the D2.0R cell transfer triggered no change in survival between immunocompetent and NOD scid mice, but a significantly reduced survival in NSG mice, pointing to a prominent role of NK cells in maintaining the dormancy phenotype.

These results support the idea that there are multiple routes to metastatic dormancy, with different immune cells playing various roles depending on the specific clinical situation. It’s important to understand the full picture of immune cell contribution to dormancy.

In the present study, however, the scientists sought to first shed some light on NK cell involvement.

Unraveling CSC Population with Flow

Cancer stem cells (CSCs) are highly resistant to many forms of cancer therapy; therefore, they are frequently the root of cancer recurrence (Lee et al. 2025).

So, understanding how NK cells and CSCs interact was the next step in Bushnell et al.’s plan.

To do this, they first cocultured D2.0R cells with NK cells and assessed the subpopulations compared to monocultures to determine their susceptibility or resistance to NK cell cytotoxicity.

Using flow cytometry (using Bio-Rad’s ZE5 Cell Analyzer), the cultures were stained for Sca-1 and CD90 to identify CSCs and markers of epithelial-mesenchymal transition (EMT), a process that enhances the motility and invasiveness of the cells.

They found that Sca1+ CD90- CSCs were susceptible to NK cell cytotoxicity, and that, when exposed to NK cell cultures, EpCAM-expressing cells decreased while vimentin-expressing cells increased, indicating that epithelial CSCs are more vulnerable to NK cells than their mesenchymal counterparts.

The researchers also used fluorescent label retention together with flow cytometry to assess the proliferation of the cells and observed that proliferative cells were more sensitive to NK cells, whereas quiescent cells were more resistant.

Could STING Be the Answer?

To delve deeper into the mechanisms that can make certain cell types more resistant to NK cell cytotoxicity, they performed RNA-seq analysis on the different populations to look at differentially expressed genes.

One difference they found was that the stimulator of interferon genes (STING), a pathway that activates the innate immune response, and STING target gene expression were increased in the cells that were susceptible to NK cells. Activation of the STING pathway has previously been shown to trigger the anti-tumor immune response (Wang et al. 2024).

This prompted the researchers to investigate the effect of the STING agonist MSA-2, which has recently been shown to prevent reactivation of metastatic cells from dormancy in lung adenocarcinoma (Hu et al. 2023).

They used fluorescence-activated cell sorting (FACS) to isolate the NK cell-sensitive and resistant populations, and cultured them with MSA-2 for one week before coculture with NK cells to see if any changes to their susceptibility would be observed.

Upon analysis, the scientists found that pretreatment with the agonist increased NK cell cytotoxicity in both populations through a tumor cell-intrinsic mechanism. Furthermore, treatment of mice with MSA-2 seven days after inoculation with the cancer cell line significantly increased survival compared to untreated mice.

Summary

This paper emphasizes the importance of using immunocompetent mouse models when investigating metastatic dormancy due to the prominent role of the immune system in regulating this phenomenon.

The study notes that differences in the mechanisms behind dormancy exist depending on the cell line used to induce cancer, which could reflect the intricacies and complexities of this process in humans. This would suggest that deeper research is necessary to pinpoint the key drivers of recurrence from dormancy under different clinical circumstances.

Here, however, the focus is on NK cells. Quiescent, mesenchymal CSCs displayed more resistance to NK cell cytotoxicity, which could explain their persistence in dormancy and implicate them as the root of disease recurrence. In support of this, treatment with STING agonists increased their susceptibility to NK cell killing and increased survival in mice, suggesting the possibility of targeting this mechanism to prevent recurrence in breast cancer.