Sachie Hiratsuka

Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis

Primary tumours influence the environment in the lungs before metastasis. However, the mechanism of metastasis is not well understood. Here, we show that the inflammatory chemoattractants S100A8 and S100A9, whose expression is induced by distant primary tumours, attract Mac 1 (macrophage antigen 1)+-myeloid cells in the premetastatic lung. In addition, tumour cells use this mechanism, through activation of the mitogen-activated protein kinase (MAPK) p38, to acquire migration activity with pseudopodia for invasion (invadopodia). The expression of S100A8 and S100A9 was eliminated in lung Mac 1+-myeloid cells and endothelial cells deprived of soluble factors, such as vascular endothelial growth factor A (VEGF-A), tumour necrosis factor α (TNFα) and transforming growth factor β (TGFβ) both in vitro and in vivo. Neutralizing anti-S100A8 and anti-S100A9 antibodies blocked the morphological changes and migration of tumour cells and Mac 1+-myeloid cells. Thus, the S100A8 and S100A9 pathway may be common to both myeloid cell recruitment and tumour-cell invasion.

The S100A8–serum amyloid A3–TLR4 paracrine cascade establishes a pre-metastatic phase

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-κB 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.

Endothelial focal adhesion kinase mediates cancer cell homing to discrete regions of the lungs via E-selectin up-regulation

Primary tumors secrete factors that alter the microenvironment of distant organs, rendering those organs as fertile soil for subsequent metastatic cancer cell colonization. Although the lungs are exposed to these factors ubiquitously, lung metastases usually develop as a series of discrete lesions. The underlining molecular mechanisms of the formation of these discrete lesions are not understood. Here we show that primary tumors induce formation of discrete foci of vascular hyperpermeability in premetastatic lungs. This is mediated by endothelial cell-focal adhesion kinase (FAK), which up-regulates E-selectin, leading to preferential homing of metastatic cancer cells to these foci. Suppression of endothelial-FAK or E-selectin activity attenuates the number of cancer cells homing to these foci. Thus, localized activation of endothelial FAK and E-selectin in the lung vasculature mediates the initial homing of metastatic cancer cells to specific foci in the lungs.

Membrane Fixation of Vascular Endothelial Growth Factor Receptor 1 Ligand-Binding Domain Is Important for Vasculogenesis and Angiogenesis in Mice

ABSTRACT Vascular endothelial growth factor (VEGF) regulates vasculogenesis and angiogenesis by using two tyrosine kinase receptors, VEGFR1 and VEGFR2. VEGFR1 null mutant mice die on embryonic day 8.5 (E8.5) to E9.0 due to an overgrowth of endothelial cells and vascular disorganization, suggesting that VEGFR1 plays a negative role in angiogenesis. We previously showed that the tyrosine kinase (TK) domain of VEGFR1 is dispensable for embryogenesis, since VEGFR1 TK-deficient mice survived and were basically healthy. However, the molecular basis for this is not yet clearly understood. To test the hypothesis that the specific role of VEGFR1 during early embryogenesis is to recruit its ligand to the cell membrane, we deleted the transmembrane (TM) domain in TK-deficient VEGFR1 mice. Surprisingly, about half of the VEGFR1(TM-TK)-deficient mice succumbed to embryonic lethality due to a poor development of blood vessels, whereas other mice were healthy. In VEGFR1(TM-TK)−/− mice with growth arrest, membrane-targeted VEGF was reduced, resulting in the suppression of VEGFR2 phosphorylation. Furthermore, the embryonic lethality in VEGFR1(TM-TK)−/− mice was significantly increased to 80 to 90% when the genotype of VEGFR2 was changed from homozygous (+/+) to heterozygous (+/−) in 129/C57BL6 mice. These results strongly suggest that the membrane-fixed ligand-binding region of VEGFR1 traps VEGF for the appropriate regulation of VEGF signaling in vascular endothelial cells during early embryogenesis.

Vascular Endothelial Growth Factor A (VEGF-A) Is Involved in Guidance of VEGF Receptor-Positive Cells to the Anterior Portion of Early Embryos

ABSTRACT The hemangioblast in the mesoderm gives rise to both angioblasts and hematopoietic stem cells. The movement of hemangioblast precursor cells in the fetal trunk is a critical event in early embryogenesis. Vascular endothelial growth factor (VEGF) signaling is likely involved in this migration given the partial disturbance of VEGF receptor (VEGFR)-positive cell accumulation and migration in VEGFR2 null mice or mice with a truncated VEGFR1. However, it is not clear how the VEGF system regulates this migration or its direction. We show here that the expression of VEGF-A is dominant in the anterior portion of the embryo, whereas VEGFR1 and VEGFR2 are expressed in the posterior portion of the embryo. An inhibitor of VEGFR kinase blocked the migration of VEGFR-positive cells in a whole-embryo culture system. In addition, VEGFR-positive cells migrated toward a VEGFR1- or VEGFR2-specific ligand in vitro. Furthermore, VEGFR-positive cells derived from wild-type or VEGFR2+/− mice moved rapidly anteriorly, whereas cells derived from VEGFR2+/− mice carrying a truncated VEGFR1 [VEGFR1(TM-TK)−/−] migrated little when injected into wild-type mice. These results suggest that the VEGF-A protein concentrated in the anterior region plays an important role in the guidance of VEGFR-positive cells from the posterior portion to the head region by interacting with VEGFR in the mouse embryo.

C-X-C receptor type 4 promotes metastasis by activating p38 mitogen-activated protein kinase in myeloid differentiation antigen (Gr-1)-positive cells

Increasing evidence suggests that myeloid bone marrow-derived cells (BMDCs) play a critical role in lung metastasis. Blockade of VEGF receptor 1 (VEGFR1) has been proposed as a potential strategy to limit myeloid BMDC recruitment to tumors. However, preclinical evidence indicates that this strategy may not be effective in all tumors. Thus, establishing which molecular mechanisms are responsible for the “escape” of these BMDCs from VEGFR1 inhibition would facilitate development of strategies to control metastasis. Here, we report the complementary role of the chemokine (C-X-C motif) ligand 12/C-X-C chemokine receptor 4 (CXCR4) and VEGF/VEGFR1 pathways in promoting lung metastasis in mice via BMDC recruitment using chimeric mice with deficiency in CXCR4 and VEGFR1–tyrosine kinase in the BMDCs. We first demonstrate that CXCR4 activity is essential for recruitment of myeloid differentiation antigen (Gr-1)-positive BMDCs, whereas VEGFR1 activity is responsible for macrophage recruitment in established tumors. Inhibition of both VEGFR1 and CXCR4 signaling in myeloid BMDCs exerted greater effects on tumor vascular density, growth, and lung metastasis than inhibition of VEGFR1 alone. These effects were reproduced after pharmacologic inhibition of CXCR4 with AMD3100. VEGFR1 and CXCR4 independently exerted a promigratory effect in myeloid BMDCs by activating p38 mitogen-activating protein kinase. Thus, combining CXCR4 blockade with inhibition of VEGFR1 may induce greater tumor growth delay and prevent or inhibit metastasis.

Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci

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.

Hepato‐entrained B220+CD11c+NK1.1+ cells regulate pre‐metastatic niche formation in the lung

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.