Metastatic breast cancer causes most cancer-related deaths in women worldwide. Disseminated tumor cells (DTCs), which seed metastases in distant organs, can remain in a reversible dormant state before eventually initiating metastatic outgrowth. Predicting when and how DTCs exit dormancy remains a major challenge in oncology. Interactions between DTCs and the surrounding microenvironment, including immune populations, play a critical role in regulating metastatic outgrowth. However, how distinct organ-specific cellular and molecular mechanisms control the formation of breast cancer metastases is just beginning to be understood. Here we aimed at understanding the dynamics of immune cells during the transition from dormancy to metastasis in a common and deadly site of breast cancer metastasis, the liver.

We used syngeneic mouse models of targeted experimental breast cancer liver metastasis via intrasplenic injection of EO771 cells expressing a dual-reporter system that distinguishes proliferative from quiescent DTCs. We isolated immune cells from liver microenvironments corresponding to distinct stages of metastatic progression, specifically tumor-free, dormancy, and low and high burden metastases, and characterized them using single-cell RNA sequencing and multiplexed flow cytometry analysis.

We found significant changes in immune cell type proportions across disease stages, which paralleled distinct spatiotemporal immune transcriptomic profiles accompanying breast cancer metastatic progression. We have prioritized finer-grained analysis of the immune cell types and associated molecular targets that most significantly changed from dormancy to metastasis. For example, we observed a marked influx of monocytes and monocyte-derived cells with a remarkable plasticity, illustrated by distinct transcriptional changes that distributed differently across disease stages. We have developed panels to identify and isolate these monocyte subtypes by flow cytometry and we are currently conducting gain- and loss-of-function experiments to determine their functional relevance on the emergence of liver-specific metastases.

Collectively, our findings uncover the composition and dynamics of the immune microenvironment during the formation of liver metastases, and identify unique monocyte subtypes associated with metastatic breast cancer dormancy. Our data suggest that monocyte-driven cues may actively sustain DTC dormancy, holding potential to be therapeutically targeted for preventing liver metastatic relapse.