Immuno-Thrombotic Regulation of Pulmonary Pre-Metastatic Niche by Liver

Introduction

We have previously shown that primary tumor-derived cytokines such as TNFα up-regulate focal expression of CCL2 and S100A8 in the lungs, which causes increased pulmonary vascular permeability with fibrinogen deposition and subsequent entry of metastatic tumor cells (Nat Cell Biol., 10, 1349, 2008; Nat Commun., 4, 1853, 2013). Mechanistically, S100A8 serves as an endogenous ligand for TLR4, stimulating mobilization of TLR4-expressing myeloid cells from bone marrow that participate in the formation of pulmonary pre-metastatic niche and TLR4-expressing tumor cells from the primary tumor. A TLR4 inhibitor eritoran can abrogate those processes (Oncogene, 35, 1445, 2016). Given that TLR4 is a sensor for endotoxin that induces systemic coagulopathy, we wished to connect fibrinogen deposition in the vascular hyper-permeability areas and innate immune response against tumor.

Materials and method

Our lung pre-metastasis models have been described (Nat. Cell Biol., 8, 1369, 2006). cDNA microarray analysis was performed with circulating leukocytes between tumor-bearing and non-bearing mice. KikGR mice (Sci. Rep., 4, 6030, 2014) were utilized for in vivo cell tracking from liver to lungs by observing photo-conversion. Bone marrow transplantation strategy with GFP+ bone marrow was used to determine the origin of leukocytes. Sorted cells were administered to tumor-bearing mice during pre-metastatic phase to evaluate pulmonary fibrinogen deposition levels by immunostaining.

Results and discussion

Significantly augmented expression of coagulation factor X was found in CD45+ leukocytes in tumor-bearing mice, and CD45+X+ cells were originated from bone marrow and observed in the fibrinogen deposition areas in the lungs. This prompted us to elucidate the roles of X+ leukocytes in pre-metastatic niche formation. A subpopulation of CD45+X+ cells was B220+CD11c+NK1.1+, and those triple+ NK cells relocated from liver to lungs as demonstrated by the KikGR system. Those cells expressed thrombospondin, a known fibrinogen-binding molecule, and binding of those triple+ cells and fibrinogen in vitro was abolished by antibody against thrombospondin. They showed IFNγ secretion and cytotoxic activity against tumor cells. An injection of the triple+ cells derived from tumor-bearing wild-type mice eliminated fibrinogen deposition in pre-metastatic lungs and reduced metastatic tumor cell homing more efficiently than those from X-knockout mice. Fibrinogen deposition may not be a passive event in the hyper-permeability regions in pre-metastatic lungs but actively contribute to host defense against metastasis.

Conclusion

Primary tumor induces not only pro-metastatic S100A8 expression but also mobilization of liver-educated anti-metastatic triple+ NK cells in pre-metastatic lungs.