Chil3+ immature neutrophils inhibit anti-tumor immunity and impede immune checkpoint blockade therapy in bone metastases [scRNA-seq]

Bone metastases remain incurable and respond poorly to immune checkpoint blockade (ICB) therapy. The impact of neutrophil maturation in cancer remains largely unknown, especially in refractory tumors including bone metastases. Here, we observed the predominance of immature neutrophils in the bone metastasis microenvironment of both mouse models and cancer patients. DKK1 induces the immature-like functional state in neutrophils, which exert robust immunosuppressive capabilities to inhibit anti-tumor response of CD8+ T cells. Mechanistically, driven by the DKK1-CKAP4-STAT6 signaling, Chil3 expression is identified and required for the immune suppression mediated by immature neutrophils in bone metastases. Moreover, using multiple syngeneic and human immune system (HIS) mouse models, we found that DKK1 blockade efficiently allows neutrophil maturation to improve the immune microenvironment, controls tumor progression, and enhances ICB therapy response in bone metastases. Thus, our findings uncover an essential role for immature neutrophils in cancer, especially in bone metastases, and propose a potential strategy modulating neutrophils to improve cancer immunotherapy. Overall design: 4T1 (TNBC model) cells were injected into the caudal artery (CA) of mice using syringe needles immediately after anesthetization to construct syngeneic bone metastasis models. After CA inoculation of cancer cells, mice were randomized in groups and treated i.p. with mDKN-01 or IgG2a (Isotype control for mDKN-01). Single-cell suspensions from mouse bone metastasis samples upon termination were isolated. CD45 positive cells were harvested from these tumors by CD45 MicroBeads and used for single cell sequencing analysis.