||Studies of the Functions of Tumoricidal Factors on MS-K Tumor Formation and Angiogenesis
74 , 2016-03-23 , 新潟大学
学位の種類: 博士（理学）. 報告番号: 甲第4180号. 学位記番号: 新大院博（理）甲第409号. 学位授与年月日: 平成28年3月23日
Inhibition of blood vessel formation in tumors by IL-18-polarized M1 macrophages. Genes to Cells. 2016, 21(3), 287-295.
Tumor growth depends on angiogenesis, which transports the oxygen and nutrients to the tumor cells and provides an essential microenvironment for the tumor growth and even metastasis. The tumor-associated angiogenesis, together with the tumor cells, stromal cells and some immune cells, forms the tumor niche. In the tumor niche, various protein factors, such as vascular endothelial growth factors (VEGFs), platelet-derived growth factors (PDGFs), fibroblast growth factors (FGFs), interleukins and chemokines, transduce the signals via their receptors and influence the tumor-associated angiogenesis. A lot of reports demonstrated that the immune cells, resident in the tumor niche, and the protein factors related for the tumor formation and angiogenesis. Nevertheless, the precise anti-tumoral functions of the immune cells remain to be explored further. Our group previously demonstrated that Interleukin-18 (IL-18) and C-C type chemokine ligand, CCL11 were highly expressed in a NFSA tumor cell line than the MS-K tumor cell line. The NFSA tumor showed limited angiogenesis and severe necrosis. On the other hand, the MS-K tumor showed enriched well-developed blood vessel network. It was thought that IL-18 produced by NFSA cells induced the M1 type of macrophages in NFSA tumors, and it may have resulted in the severe necrosis of NFSA tumors by enhancing macrophage phagocytosis and cytotoxicity. Really, the activated macrophages caused the destruction of endothelial cells in vitro. However, the effect of IL-18 on blood vessel formation in vivo and the origin of the infiltrated immune cells, into tumor have not been elucidated. Therefore, in this study, I aimed to elucidate the roles of the IL-18 and CCL11 on angiogenesis and accumulation of the immune cells in tumor, using these cell lines. Firstly, I established IL-18-overexpressing MS-K cell clones (MS-K-IL-18) to address the roles of IL-18 in angiogenesis. The overexpression of IL-18 in MS-K cells inhibited the proliferation rate of the MS-K-IL-18 cells in vitro and blood vessel formation in the MS-K-IL-18 tumors. Interestingly, CD14-positive cells from the MS-K-IL-18 tumor had upregulated expression of the M1 type macrophage marker il-6. Furthermore, FACS analysis revealed more accumulation of CD11b+CD80+ M1 macrophages in the MS-K-IL-18 tumors than in the parental MS-K tumor. Moreover, an in vitro co-culture assay demonstrated that MS-K-IL-18-conditioned medium (CM) stimulated macrophages to induce the apoptosis of endothelial cells. However, there was no significant difference in the number of the accumulated immune cells in the tumor between the parental MS-K-tumor and MS-K-IL-18-tumor. Thus, it revealed that the IL-18 was not responsible for the immune cell accumulation in the NFSA cells.
Next, I attempted to clarify the relevance of CCL11-recruited immune cells in angiogenesis and tumor formation. Then I established CCL11-overexpressing MS-K cell clones (MS-K-CCL11) to clarify the role of CCL11 on immune cell accumulation and angiogenesis. The proliferation rate and colony formation ability in vitro were almost the same between MS-K-CCL11 and parental MS-K cells. However, interestingly, the MS-K-CCL11 cells could not form any tumors in mice. The MS-K-CCL11-conditioned medium and recombinant CCL11 induced macrophage and eosinophil differentiation from bone marrow cells. The MS-K-CCL11-CM effectively attracted eosinophils, which were differentiated from bone marrow cells by the MS-K-CCL11-CM. Furthermore, the eosinophils damaged the MS-K, NFSA and endothelial cells in a dose dependent manner by co-culture experiment. Administration of CCL11-receptor CCR3 specific antagonist SB328437 into NFSA cells-transplanted mice restored the blood vessel formation and blockaded of eosinophils infiltration into NFSA tumor. These data indicated that the CCL11 was responsible for differentiation of eosinophils, and also recruitment of them into tumor. Cumulatively, these data demonstrated that IL-18 and CCL11 both have tumoricidal effects on MS-K tumors: Excess IL-18 produced by MS-K-IL-18 induced M1 macrophages and inhibited blood vessel formation by pro-apoptotic capability exerted by macrophages to endothelial cells. Whereas, excess CCL11 produced by MS-K-CCL11 cells induced and recruited eosinophils to tumor; which would damage not only the MS-K-CCL11 cells but also the endothelial cells and would block MS-K tumor formation. These results indicate that CCL11 and IL-18 are the key factors to modulate the tumor immune cells infiltration and angiogenesis; and also indicates that they would be potential targets of tumor treatment strategy.