Systemic inflammation induced by calprotectin (S100A8/S100A9 complex) triggers reactivation of dormant cells in mouse models of breast cancer

Breast cancer is the most common cancer in women in the developed countries. Cancer stem cells play key roles in tumorigenesis, dormancy and recurrence in breast cancer, yet they are resistant to many treatments. Despite recent advances in the field in terms of imaging and adjuvant therapies, disseminated dormant tumor cells (DTCs) resistant to these therapies are major cause of late metastatic relapse. However, the mechanisms by which the dormancy maintenance and reactivation, particularly within bone and lung niches remain poorly understood, due in part to limited preclinical models. There is a need for better mouse models to study breast cancer dormancy to mitigate the risk of late metastatic recurrence. Systemic inflammation is increasingly recognized as a trigger of dormancy escape but its mechanistic role in awakening DTCs in vivo is not fully explored. To address this, we evaluated two murine breast cancer syngeneic models in mice to establish a tractable platform for studying tumor dormancy and its awakening. Using orthotopic implantation of EMT6-Luci cells followed by surgical resection in BALB/c mice and intracardiac injection of D2.0R-Luci cells, we examined whether systemic inflammatory cytokines, including IL-1b, G-CSF or Calprotectin (S100A8-S100A9 heterotetramer) promote escape from dormancy. Beginning 30 days post-resection of EMT6-Luci or post-injection of D2.0R-Luci cells, mice received intraperitoneal treatment of indicated cytokines. We monitored the dormancy awakening longitudinally using IVIS bioluminescence imaging as well as the peripheral immune profile assessed by flow cytometry. D2.0R-luci cells demonstrated a robust sensitivity to inflammatory cues. Treatment with Il-1b, G-CSF or calprotectin all markedly increased the frequency of mice exhibiting bioluminescent reactivation of dormant cells compared to the control vehicle mice. Notably, calprotectin induced reawakening in 100% of treated mice versus 33% in control animals highlighting its potent pro-inflammatory and pro-metastatic role. In contrast, disseminated EMT6-luci cells remined below the detection threshold in lung and bone marrow following resection, indicating differences in dormancy biology. Our findings reinforce emerging evidence that systemic inflammation is a critical driver of dormant tumor cell awakening. Our work provides a robust syngeneic dormancy platform for dissecting systemic and microenvironmental regulators of metastatic reactivation and establishes calprotectin as strong candidate mediating dormancy escape. Ultimately, these models will facilitate the development of therapeutic strategies to eliminate dormant cells and prevent late metastatic relapses in breast cancer patients.