A large number of macrophages and haematopoietic progenitor cells accumulate in pre-metastatic lungs in which chemoattractants, such as S100A8 and S100A9, are produced by distant primary tumours serving as metastatic soil. The exact mechanism by which these chemoattractants elicit cell accumulation is not known. Here, we show that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion. We also show that in lung endothelial cells and macrophages, Toll-like receptor (TLR) 4 acts as a functional receptor for SAA3 in the pre-metastatic phase. In our study, SAA3 stimulated NF-kappaB signalling in a TLR4-dependent manner and facilitated metastasis. This inflammation-like state accelerated the migration of primary tumour cells to lung tissues, but this was suppressed by the inhibition of either TLR4 or SAA3. Thus, blocking SAA3-TLR4 function in the pre-metastatic phase could prove to be an effective strategy for the prevention of pulmonary metastasis.

In mouse models of lung metastasis, before the appearance of significant metastases, localized changes in vascular permeability have been observed, which appear to set the stage for tumour growth. However, it is unclear whether this is also true in human patients. Here, we show that MD-2, a coreceptor for Toll-like receptor 4 that has a key role in the innate immune response, triggers the formation of regions of hyperpermeability in mice by upregulating C-C chemokine receptor type 2 (CCR2) expression. The CCR2–CCL2 system induces the abundant secretion of permeability factors such as serum amyloid A3 and S100A8. Disruption of MD-2 or CCR2 abrogates the formation of hyperpermeable regions, resulting in reduced tumour cell homing. Furthermore, fibrinogen, which is processed during permeability-mediated coagulation, is also localized in areas of elevated CCR2 expression in tumour-bearing human lungs. Our findings raise the possibility that CCR2 upregulation might represent a marker for regions of increased susceptibility to metastatic homing in lung cancer.

We have previously shown that tumor necrosis factor (TNF)a produced from primary tumor-induced expression of two endogenous Toll-like receptor 4 (TLR4) ligands, S100A8 and serum amyloid A3 (SAA3), in pre-metastatic lungs. However, mechanistic details of the signaling network and relevance to pulmonary physiology are poorly understood. Here, we identify Clara cells as a control tower of the network. Clara cell ablation by naphthalene suppressed pulmonary recruitment of CD11b þ TLR4 þ cells and spontaneous lung metastasis. Clara cells turned out to express TLR4 through which SAA3 was auto-amplified. Reciprocal bone marrow transplantation between wild-type and TLR4 knockout mice demonstrated that pulmonary TLR4 þ Clara cells could be derived from bone marrow. SAA3-induced TNFa expression in both alveolar type II cells and macrophages. Primary co-cultures of alveolar type II cells and Clara cells revealed that the induction of TNFa in alveolar type II cells was dependent on the Clara cell-mediated amplification of SAA3. SAA3 induction by bacterial endotoxin also required both Clara cells and TLR4. Thus, pulmonary metastatic soil may feature deregulation of homeostatic inflammatory responses to constant assaults of microbes with endotoxin.

Primary tumours establish metastases by interfering with distinct organs. In pre‐metastatic organs, a tumour‐friendly microenvironment supports metastatic cells and is prepared by many factors including tissue resident cells, bone marrow‐derived cells and abundant fibrinogen depositions. However, other components are unclear. Here, we show that a third organ, originally regarded as a bystander, plays an important role in metastasis by directly affecting the pre‐metastatic soil. In our model system, the liver participated in lung metastasis as a leucocyte supplier. These liver‐derived leucocytes displayed liver‐like characteristics and, thus, were designated hepato‐entrained leucocytes (HepELs). HepELs had high expression levels of coagulation factor X (FX) and vitronectin (Vtn) and relocated to fibrinogen‐rich hyperpermeable regions in pre‐metastatic lungs; the cells then switched their expression from Vtn to thrombospondin, both of which were fibrinogen‐binding proteins. Cell surface marker analysis revealed that HepELs contained B220+ CD11c+ NK1.1+ cells. In addition, an injection of B220+ CD11c+ NK1.1+ cells successfully eliminated fibrinogen depositions in pre‐metastatic lungs via FX. Moreover, B220+ CD11c+ NK1.1+ cells demonstrated anti‐metastatic tumour ability with IFNγ induction. These findings indicate that liver‐primed B220+ CD11c+ NK1.1+ cells suppress lung metastasis.