Biochemistry and Molecular Biology

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Summary of Activity

Our efforts are directed to reveal molecular mechanisms of tumorigenesis and tumor metastasis. By using tumor-bearing mouse model system, we have determined that primary tumor elicits an inflammatory reaction in a remote organ before metastasis, which is termed as "premetastatic soil". This inflammatory reaction enhances vascular permeability, and recruitment of immunosuppressor cells and/or circulating tumor cells, these factors increase the risk of tumor metastasis. Thus, we consider that controlling the premetastatic phase is an important step to prevent tumor metastasis.

Research Subject

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First, tumor-bearing mice were generated. Tumor cells (such as Lewis Lung Carcinoma, LLC) can be cultured in a standard mammalian cell culture condition. LLC cells, injected in the back of C57BL6 mouse, forms tumor. To find out an effect of the LLC tumor in the lung, potential metastatic target, we carried out Evans blue assay. After an injection of Evans blue dye in the tail vein, lungs from tumor- and non-tumor ?bearing mouse (but no metastasis in the lung) were taken out to check the leakage of the dye. In this assay, dense blue region indicates high vascular permeability, and such regions were observed in the tumor-bearing mice but not in the non-tumor bearing mice.

Next, to decipher the molecular mechanisms governing vascular permeability in the tumor-bearing mouse, we compared gene expression profiles between high and low permeability regions in the same tumor-bearing mouse. The gene expression analysis unveiled that in the high permeability region inflammation related genes, including TLR4/MD-2 and chemokine receptors, were remarkably upregulated. To confirm that these receptors play an important role in the process of tumor metastasis, we obtained a strand of knockout mice (TLR4/MD2-KO, etc.) to perform Evans blue assay and tumor metastasis assay. These knockout mice did not show high permeability region even in the tumor bearing state. Moreover, in tumor metastasis assay, (fluorescent labeled tumor cells were injected via tail vein and after a certain period lungs were excised to observe tumor cells under fluorescent microscopy) the lungs of knockout mice showed no focal points created by the labeled cells that were observed in the lungs of wild type tumor-bearing mice. Our interpretation is that blocking inflammation related receptor(s) may reduce vascular permeability in the lungs to prevent tumor metastasis. To apply the theory found in the mouse study into clinical studies, we are about to execute analyses of clinical samples. Our first round analysis revealed that cancer patients hold inflammatory regions in the lung that are very similar to that in the tumor?bearing mouse. Thus, we consider that our premetastatic phase theory can be applied to clinical cases, and firmly believe that studying this theory is necessary to explore a new way to diagnose or prevent tumor metastasis.

Major Publications

1. Tomita T, Kato M, Mishima T, Matsunaga Y, Sanjo H, Ito KI, Minagawa K, Matsui T, Oikawa H, Takahashi S, Takao T, Iwai N, Mino T, Takeuchi O, Maru Y, Hiratsuka S.(2021) Extracellular mRNA transported to the nucleus exerts translation-independent function Nat. Commun. 12(1):3655. doi: 10.1038/s41467-021-23969-1.

2. Hiratsuka S., Tomita T., Mishima T., Matsunaga Y., Omori T., Ishibashi S., Yamaguchi S., Hosogane T., Watarai H., Omori-Miyake M., Yamamoto T., Shibata N., Watanabe A., Aburatani H., Tomura M., High. KA., Maru Y. (2018) Hepato-entrained B220+CD11c+NK1.1+ cells regulate pre-metastatic niche formation in the lung. EMBO Mol. Med. e8643, doi: 10.15252/emmm.201708643

3. Hiratsuka S., Ishibashi S., Tomita T., Watanabe A., Akashi-Takamura S., Murakami M., Kijima H., Miyake K., Aburatani H., Maru Y. (2013) Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci. Nature Communications 4: Article number: 1853 doi:10.1038/ncomms2856.

4. Hiratsuka S, Goel S, Kamoun WS, Maru Y, Fukumura D, Duda DG, Jain RK. (2011) Endothelial focal adhesion kinase mediates cancer cell homing to discrete regions of the lungs via E-selectin up-regulation. Proc Natl Acad Sci U S A. 108: 3725-3730.

5. Hiratsuka S, Duda DG, Huang Y, Goel S, Sugiyama T, Nagasawa T, Fukumura D, Jain RK. (2011) C-X-C receptor type 4 promotes metastasis by activating p38 mitogen-activated protein kinase in myeloid differentiation antigen (Gr-1)-positive cells. Proc Natl Acad Sci U S A. 108: 302-307.

6. Hiratsuka S, Watanabe A, Sakurai Y, Akashi-Takamura S, Ishibashi S, Miyake K, Shibuya M, Akira S, Aburatani H, Maru Y. (2008) The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase. Nat. Cell Biol. 10: 1349-1355.

7. Hiratsuka S, Watanabe A, Aburatani H, Maru Y. (2006) Tumor-mediated upregulation of chemoattractants and recruitment of myeloid cells pre-determines lung metastasis. Nat. Cell Biol. 8: 1369-1375.

8. Hiratsuka S, Nakamura K, Iwai S, Murakami M, Itoh T, Kijima H, Shipley JM, Senior RM, Shibuya M. (2002) Involvement of MMP9 induction by Vascular Endothelial Growth Factor Receptor-1 in lung specific metastasis. Cancer Cell 2 : 289-300.
(The first paper in which pre-metastatic microenvironment in lung was prepared by a distant primary tumor.)