◎What is purpose of research?
We are trying to establish of diagnosis and therapy for early metastasis, especially focusing on the pre-metastatic phase.

◎What is pre-metastatic phase?
This phase is recognized the stage before metastasis with a primary tumor (Publication List 15).

◎What mechanism elicits the pre-metastatic microenvironment in the organ tissue that is stimulated by a distant primary tumor?
We found that this microenvironment was distinct soil with hyper-permeability in the tumor-bearing mouse lungs (Publication List 15 &6). This pre-metastatic soil is pre-metastatic niche, which was defined by Dr. David Lyden (Publication List 21). This pre-metastatic soil attracts circulating tumor cells resulting in metastasis.

◎Is there pre-metastatic microenvironment in cancer patients?
We found that cancer patients have the estimated area, which was very similar to the pre-metastatic soil in mouse (Publication List 6).

◎Is it possible to eliminate the pre-metastatic soil ? What strategy could suppress metastasis accompanied with decrease the soil?
Yes. We recently discovered effective anti-metastatic immune cells (Publication List 5). We try to establish it in cancer patients. If you will be interested in the project, please join us!

◎More detail
We further describe the pre-metastatic project in detail as shown below. Or please see our review (Publication List 23).

◎Summary of our findings (Fig.1)
Primary tumors secrete a lot of factors including proteins such as CCL2, VEGF, TNFα, and TGF-β. In the course of primary tumor growth, these factors stimulate bone marrow cells and tissue-resident cells in the distant host organ tissues. Despite ubiquitously spreading in blood vessels, the secreted factors from primary tumors mediate discrete hyperpermeability foci by several overlapping cascades such as S100A8-SAA3-TLR4/MD-2 and VEGF-FAK-E-selectin. The former is that these secreted factors stimulate CCR2 in potentially hyperpermeable areas, further inducing the secretion of SAA3 and S100A8, which increase vascular permeability via TLR4/MD-2. The latter is that tumor-derived VEGF generates the permeable foci with E-selectin upregulation through endothelial FAK in the premetastatic lungs. The metastatic tumor cells prefer to migrate the foci. Thus, it is an advantage to target premetastatic phase.

◎1) Detection of hyperpermeable foci
For our in vivo assay system, we implanted three murine tumor types with metastatic potential (LLC, Lewis lung carcinoma; E0771, mammary carcinoma; B16, melanoma), in immunocompetent C57BL/6 mice. Once tumors reached approximately 6-7mm in diameter, we injected Blue dye, Evans Blue (EB), intravenously and then excised the lungs 3 hours later. We found focal regions of increased EB leakage (Fig.2).

◎2) Circulating tumor cells accumulate the foci
To examine if hyperpermeable foci served as sites for tumor cell homing, we developed a three-step assay system. First, we applied a permeability inducer (tumor implantation or intravenous injection of tumor conditioned media). Second, we infused EB systemically to detect vascular leakage. Finally, we infused fluorescence-labeled metastatic tumor cells and measured the number of cells that homed to the lungs. We found increased numbers of fluorescent tumor cells homing in the lungs of tumor-stimulating mice(Fig.3). Importantly, the tumor cells preferentially homed to areas of high vascular permeability.

◎3) Blocking vascular permeability reduces tumor cell homing- KO of endothelial FAK
Src kinase-FAK complex activation mediates vascular permeability via TCM. To study the specific contribution of FAK in endothelial cells towards the formation of hyperpermeable foci, we developed an inducible transgenic mouse model. This model allowed in vivo regulation of endothelial cell-mediated vascular permeability. Doxycycline (Dox)-mediated endothelial-specific FRNK (the dominant negative form of FAK) overexpression significantly reduced TCM-induced formation of the hyperpermeability foci in the lungs. Moreover, this reduction in endothelial cell-mediated hyperpermeability led to a significant reduction in tumor cell homing to the lungs after metastatic tumor cell infusion.

◎4) Molecular-based inflammation in the foci (please see also Fig.1)
Because the inflammatory mediators all lie downstream of signaling factor NF-κB, the master regulator of inflammation, We found that TCM activate the NF-κB pathway in the lungs, and this NF-κB activation was largely restricted to the EB-high areas and was specific to the lungs rather than liver.

◎VEGF-mediating permeability
We found that TCM contained high concentrations of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Infusion of recombinant (r)VEGF or rPlGF also induced discrete foci of hyperpermeability with focal EB leakage in the lungs. Treatment of tumor-bearing mice with an anti-VEGF monoclonal blocking antibody inhibited the lung vascular hyperpermeability. This result supports that VEGF mediates in part this effect. (Fig.4)

◎S100A8-SAA3-mediating permeability
We further compared the gene expression levels of the receptors between hyperpermeable and poorly permeable regions in the tumor-bearing mouse lungs. C-C chemokine receptor type 2 (CCR2) showed the largest increase in the gene expression between the hyperpermeable area and the low permeable area in the tumor-bearing mice. To clarify the functional role of this system, we used CCL2 and CCR2 knockout mice. In association with lack of hyperpermeable foci in the tumor-bearing those knockout mice, tumor cell homing was suppressed. We found that S100A8 and SAA3, which are downstream molecules of the CCL2-cascade, produced strong permeability responses. (Fig.5)

◎5) The possibility of hyperpermeable foci in patients
To search whether distant primary tumors stimulate the co-localized induction of fibrinogen and CCR2 expression in tumor-bearing human lungs, we examined the expression of both molecules in healthy lung lobes from patients who carried tumors in their extrapulmonary organs. It should be noted that we detected relatively low levels of fibrinogen and CCR2 in lungs from non-cancer patients. In contrast, the co-localization of upregulated fibrinogen and CCR2 expression was detected in the lungs of patients with tumors. We observed that the tumor-bearing lung exhibited significantly increased fibrinogen and S100A8 expression levels compared with the non-tumor-bearing lungs (Fig.6).