Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Tokyo Women's Medical University

Shinshu University

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.

Membrane fixation of vascular endothelial growth factor receptor 1 ligand-binding domain is important for vasculogenesis and angiogenesis in mice.

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.

Vascular endothelial growth factor A (VEGF-A) is involved in guidance of VEGF receptor-positive cells to the anterior portion of early embryos.

VEGF receptor 1 (VEGFR-1/Flt-1) is a high-affinity tyrosine kinase (TK) receptor for VEGF and regulates angiogenesis as well as monocyte/macrophage functions. We previously showed that the osteoclast deficiency in osteopetrotic Csf1op/Csf1op (op/op) mice is gradually restored in an endogenous, VEGF-dependent manner. However, the molecular basis of the recovery is still not clear. To examine which VEGFR is important and to clarify how colony-stimulating factor 1 (CSF-1) and VEGF signals interact in osteoclastogenesis, we introduced a VEGFR-1 signaling deficiency (Flt1(TK)-/-) into op/op mice. The original Flt1(TK)-/- mice showed mild osteoclast reduction without bone marrow suppression. The double mutant (op/opFlt1(TK)-/-) mice, however, exhibited very severe osteoclast deficiency and did not have numbers of osteoclasts sufficient to form the bone marrow cavity. The narrow bone marrow cavity in the op/opFlt1(TK)-/- mice was gradually replaced with fibrous tissue, resulting in severe marrow hypoplasia and extramedullary hematopoiesis. In addition to osteoclasts, osteoblasts also decreased in number in the op/opFlt1(TK)-/- mice. These results strongly suggest that the interaction of signals by means of VEGFR-1 and the CSF-1 receptor plays a predominant role not only in osteoclastogenesis but also in the maintenance of bone marrow functions.

VEGF receptor 1 signaling is essential for osteoclast development and bone marrow formation in colony-stimulating factor 1-deficient mice.

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 alpha (TNFalpha) and transforming growth factor beta (TGFbeta) 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.

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

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.

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

The circulatory system comprises a tubular network of blood vessels including arterioles, capillaries, venules, and lymphatic vessels. This circulatory system is essential for the embryonic development and maintenance of all tissues, which requires the transportation of oxygen, carbon dioxide, and nutrition. The system regulates the movement of fluid into and out of organs with high level of efficiency. "Tumor angiogenesis" describes the rapid growth of certain components of the circulatory system in an abnormal fashion that is both heterogeneous and dysregulated. The aberrant flow between abnormal tumor vessels and normal vessels poses a high risk for seeding of potentially metastatic cancer cells. Moreover, it has also been reported that premetastatic distant organ vessels already undergo specific changes due to the presence of a remote primary tumor. Therapeutic strategies aimed at targeting tumor vessels have the potential to suppress tumor growth, and also influence the effects of tumor-derived cytokines and circulating tumor cells. Furthermore, focusing on vessels in a premetastatic organ that have responded to a primary tumor may be one possibility for reducing metastatic risk.

Vasculogenensis, angiogenesis and special features of tumor blood vessels.

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.

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

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.

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

It is well established that organs of future metastasis are not passive receivers of circulating tumour cells, but are instead selectively and actively modified by the primary tumour before metastatic spread has even occurred. Sowing the 'seeds' of metastasis requires the action of tumour-secreted factors and tumour-shed extracellular vesicles that enable the 'soil' at distant metastatic sites to encourage the outgrowth of incoming cancer cells. In this Review, we summarize the main processes and new mechanisms involved in the formation of the pre-metastatic niche.

Pre-metastatic niches: organ-specific homes for metastases.

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 B220CD11cNK1.1 cells. In addition, an injection of B220CD11cNK1.1 cells successfully eliminated fibrinogen depositions in pre-metastatic lungs via FX Moreover, B220CD11cNK1.1 cells demonstrated anti-metastatic tumour ability with IFNγ induction. These findings indicate that liver-primed B220CD11cNK1.1 cells suppress lung metastasis.

Article	Year
Membrane fixation of vascular endothelial growth factor receptor 1 ligand-binding domain is important for vasculogenesis and angiogenesis in mice. Molecular and cellular biology\| PubMed ID: 15601855	2005
Vascular endothelial growth factor A (VEGF-A) is involved in guidance of VEGF receptor-positive cells to the anterior portion of early embryos. Molecular and cellular biology\| PubMed ID: 15601856	2005
VEGF receptor 1 signaling is essential for osteoclast development and bone marrow formation in colony-stimulating factor 1-deficient mice. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 16172397	2005
Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis. Nature cell biology\| PubMed ID: 17128264	2006
The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase. Nature cell biology\| PubMed ID: 18820689	2008
Vasculogenensis, angiogenesis and special features of tumor blood vessels. Frontiers in bioscience (Landmark edition)\| PubMed ID: 21196239	2011
Endothelial focal adhesion kinase mediates cancer cell homing to discrete regions of the lungs via E-selectin up-regulation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21321210	2011
Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci. Nature communications\| PubMed ID: 23673638	2013
Pre-metastatic niches: organ-specific homes for metastases. Nature reviews. Cancer\| PubMed ID: 28303905	2017
Hepato-entrained B220CD11cNK1.1 cells regulate pre-metastatic niche formation in the lung. EMBO molecular medicine\| PubMed ID: 29930175	2018

Sachie Hiratsuka

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