Hepato‐entrained B220 + CD 11c + NK 1.1 + cells regulate pre‐metastatic niche formation in the lung
2018; Springer Nature; Volume: 10; Issue: 7 Linguagem: Inglês
10.15252/emmm.201708643
ISSN1757-4684
AutoresSachie Hiratsuka, Takeshi Tomita, Taishi Mishima, Yuta Matsunaga, Tsutomu Omori, Sachie Ishibashi, Satoshi Yamaguchi, Tsuyoshi Hosogane, Hiroshi Watarai, Miyuki Omori‐Miyake, Tomoko Yamamoto, Noriyuki Shibata, Akira Watanabe, Hiroyuki Aburatani, Michio Tomura, Katherine A. High, Yoshiro Maru,
Tópico(s)Immunotherapy and Immune Responses
ResumoResearch Article21 June 2018Open Access Source DataTransparent process Hepato-entrained B220+CD11c+NK1.1+ cells regulate pre-metastatic niche formation in the lung Sachie Hiratsuka Corresponding Author Sachie Hiratsuka [email protected] orcid.org/0000-0002-6507-7362 Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Japan Search for more papers by this author Takeshi Tomita Takeshi Tomita Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Taishi Mishima Taishi Mishima Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Yuta Matsunaga Yuta Matsunaga Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Tsutomu Omori Tsutomu Omori Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Sachie Ishibashi Sachie Ishibashi Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Satoshi Yamaguchi Satoshi Yamaguchi Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Search for more papers by this author Tsuyoshi Hosogane Tsuyoshi Hosogane Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroshi Watarai Hiroshi Watarai Division of Stem Cell Cellomics, The Institute of Medical Science of the University of Tokyo, Tokyo, Japan Search for more papers by this author Miyuki Omori-Miyake Miyuki Omori-Miyake Department of Microbiology and Immunology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Tomoko Yamamoto Tomoko Yamamoto Department of Pathology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Noriyuki Shibata Noriyuki Shibata Department of Pathology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Akira Watanabe Akira Watanabe Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroyuki Aburatani Hiroyuki Aburatani Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Search for more papers by this author Michio Tomura Michio Tomura Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan Search for more papers by this author Katherine A High Katherine A High Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA Search for more papers by this author Yoshiro Maru Corresponding Author Yoshiro Maru [email protected] orcid.org/0000-0002-9477-7974 Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Sachie Hiratsuka Corresponding Author Sachie Hiratsuka [email protected] orcid.org/0000-0002-6507-7362 Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Japan Search for more papers by this author Takeshi Tomita Takeshi Tomita Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Taishi Mishima Taishi Mishima Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Yuta Matsunaga Yuta Matsunaga Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Tsutomu Omori Tsutomu Omori Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Sachie Ishibashi Sachie Ishibashi Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Satoshi Yamaguchi Satoshi Yamaguchi Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Search for more papers by this author Tsuyoshi Hosogane Tsuyoshi Hosogane Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroshi Watarai Hiroshi Watarai Division of Stem Cell Cellomics, The Institute of Medical Science of the University of Tokyo, Tokyo, Japan Search for more papers by this author Miyuki Omori-Miyake Miyuki Omori-Miyake Department of Microbiology and Immunology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Tomoko Yamamoto Tomoko Yamamoto Department of Pathology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Noriyuki Shibata Noriyuki Shibata Department of Pathology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan Search for more papers by this author Akira Watanabe Akira Watanabe Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroyuki Aburatani Hiroyuki Aburatani Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Search for more papers by this author Michio Tomura Michio Tomura Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan Search for more papers by this author Katherine A High Katherine A High Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA Search for more papers by this author Yoshiro Maru Corresponding Author Yoshiro Maru [email protected] orcid.org/0000-0002-9477-7974 Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan Search for more papers by this author Author Information Sachie Hiratsuka *,1,2,10, Takeshi Tomita1, Taishi Mishima1, Yuta Matsunaga1, Tsutomu Omori1, Sachie Ishibashi1, Satoshi Yamaguchi3, Tsuyoshi Hosogane3, Hiroshi Watarai4, Miyuki Omori-Miyake5, Tomoko Yamamoto6, Noriyuki Shibata6, Akira Watanabe7,11, Hiroyuki Aburatani7, Michio Tomura8, Katherine A High9 and Yoshiro Maru *,1 1Department of Pharmacology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo, Japan 2PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Japan 3Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan 4Division of Stem Cell Cellomics, The Institute of Medical Science of the University of Tokyo, Tokyo, Japan 5Department of Microbiology and Immunology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan 6Department of Pathology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan 7Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan 8Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani University, Osaka, Japan 9Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA 10Present address: Department of Biochemistry and Molecular Biology, Shinshu University School of Medicine, Nagano, Japan 11Present address: Genome/Epigenome Analysis Core Facility, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan *Corresponding author. Tel: +81 3 5269 7417; E-mail: [email protected] *Corresponding author. Tel: +81 3 5269 7417; E-mail: [email protected] EMBO Mol Med (2018)10:e8643https://doi.org/10.15252/emmm.201708643 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis During tumour progression, the pre-metastatic and post-metastatic organs contain inflammatory immune cells with pro-metastatic characteristics rather than anti-metastatic. This newly found liver-priming immune cell population is shown to be involved in metastatic suppression. Liver-educated B220+CD11c+NK1.1+ cells (HepELs) relocated to pre-metastatic lungs. HepELs eliminated pre-metastatic fibrinogen deposit to reduce the risk of metastasis via coagulation factor X (FX). Metastatic tumour cells were attacked by HepELs with increase of IFNγ. Lung metastasis was suppressed by HepELs in mice. Expression pattern of FX+ leukocytes in patients was similar to that of mouse FX+ HepELs, suggesting that an anti-metastatic cell population exists in human. Introduction Cancer remains a leading cause of deaths globally, with the number expected to increase to 21 million by 2030 (Vinay et al, 2015). Tumour metastases account for approximately 90% of all cancer-related deaths, although recent medical advances have aided many patients (Spano et al, 2012; Vinay et al, 2015). Metastatic mobilization is generated by the triangular interplay among a primary tumour, a metastatic tissue and the bone marrow (Wels et al, 2008). Although the tumour-induced systemic environment likely affects metastases, the precise role of non-metastatic organs has yet to be elucidated. In some organs, the local microenvironment can be altered by a distant primary tumour resulting in conditions referred to as pre-metastatic soil, or niche (Hiratsuka et al, 2002, 2006, 2008; Kaplan et al, 2005; Erler et al, 2009; Huang et al, 2009; Jung et al, 2009; Kim et al, 2009; Hood et al, 2011; Peinado et al, 2012, 2017; Sceneay et al, 2013; McAllister & Weinberg, 2014; Costa-Silva et al, 2015). We have reported that the pre-metastatic niche in the lung occurs within distinct regions composed of fibrinogen deposition, resident tissue cells and infiltrating immune cells (Hiratsuka et al, 2013). Pre-metastatic organs contain a variety of resident tissue cells, as well as infiltrating bone marrow-derived cells (BMDCs). Importantly, the pre-metastatic soil contains a complex mixture of factors that either inhibit or promote metastasis (Qian & Pollard, 2010; Granot et al, 2011). In addition, post-metastatic regions contain more inflammatory immune cells in which pro-metastatic cells were activated rather than anti-metastatic cells (McAllister & Weinberg, 2014). Taken together, a distinct fibrinogen area may be a scaffold that attracts various immune cells as well as circulating tumour cells. However, it is not known whether any cell population can eliminate metastatic niche or combat cancer cells. Thus, we tried to find out anti-metastatic cells in pre- and post-metastatic niche. In this paper, we found a specific immune cell population that was educated in liver, accumulated in lung niche via circulation from liver and capable of eliminating pre-metastatic fibrinogen deposition and killing metastatic tumour cells. In addition, application of those liver-primed cells efficiently functions with anti-metastatic ability. Results FX+CD45+ cells accumulate in pre-metastatic lung niche To search for potential anti-metastatic factors, we sought functional cell populations related to fibrinogen clearance because the fibrinogen deposition area is scaffold for immune cells in pre-metastatic lungs (Hiratsuka et al, 2013). Fibrinogen is primarily produced in the liver and associated with a coagulation cascade; thus, we screened molecules related to liver-specific and coagulation system genes in circulating leucocytes of tumour-bearing mice. We generated tumour-bearing mice using E0771 breast cancer, LLC lung carcinoma and B16 melanoma cells. We used 6- to 8-week male and female mice for LLC and B16 and only female for E0771 tumours. In this study, a key point of our pre-metastatic model system is that spontaneous metastasis from the primary site was observed only after the primary tumour resection, although an intravenous injection of these cells easily attained lung metastasis (Hiratsuka et al, 2002, 2006, 2008) (see Materials and Methods). In addition, it should be noted that these tumour cells failed to metastasize to the liver. As shown in Appendix Table S1, tumour-bearing mice exhibited a threefold increase in coagulation factor X (FX, F10) expression in leucocytes compared with tumour-free controls. This trend was also apparent in CD45+ leucocytes derived from E0771 and LLC tumour-bearing mice (Fig 1A). We next demonstrated that livers derived from tumour-bearing mice more significantly induced FX expression in CD45+ leucocytes than those from no tumour-bearing mice (Fig 1B). Immunostaining results confirmed the results also at protein levels (Fig 1C). In addition, bone marrow transplantation (BMT) strategy using GFP+-BM revealed that CD45+ leucocytes, which were derived from BM showed strong FX expression in tumour-bearing mouse liver (tumour-bearing mouse liver meaning liver derived from tumour-bearing mice) (Appendix Fig S1). Primary tumours induced lung fibrinogen depositions (Fig 1D, Appendix Fig S2), and accumulation of FX+CD45+ cells was detected in fibrinogen deposition areas in pre-metastatic lungs (Fig 1E). These results led us to hypothesize that the pre-metastatic liver induces FX+ leucocytes in tumour-bearing mice. Figure 1. Appearance of coagulation factor X (FX) positive-hepato-entrained leucocytes (HepELs) in peripheral blood and lungs during the pre-metastatic phase A. Relative mRNA levels of FX in CD45+ leucocytes in the peripheral blood of E0771 (abbreviated to E) or LLC tumour-bearing mice. The mean sizes of E0771 and LLC tumours were 9.8 mm and 9.5 mm, respectively. Shown are averages (N = 8 mice/group) with SEM and one-way ANOVA. B. Relative mRNA levels of FX in CD45+ leucocytes in various organs such as lung (Lu), liver (Li), spleen (Sp), bone marrow (BM) and lymph nodes (Lymph; inguinal (Ing) and mesenteric (Mes)) derived from no tumour-bearing or E0771-bearing mice. "Tumour" stands for mRNA levels of FX in E0771 tumours. Shown are averages (N = 6: organs from no tumour-bearing mice, N = 6: organs from E0771-bearing mice) with SEM and one-way ANOVA. C. Immunohistochemical quantifications of FX expression in CD45+ leucocytes in the liver and lungs of no tumour-bearing or E0771-bearing mice. Shown are averages (N = 6) with SEM and Welch's t-test. D. Immunohistochemical quantifications of fibrinogen deposition in no tumour-bearing and tumour-bearing mouse lungs. Shown are averages (N = 7/group, 14 random fields) with SEM. Welch's t-test. E. Representative photographs of immunohistochemical co-localization of FX+CD45+ cells and fibrinogen deposition in tumour-bearing mouse lungs. Circles show high fibrinogen deposition areas (scale bar, 50 μm). Download figure Download PowerPoint FX+CD45+ cells contain B220+CD11c+NK1.1+ cells that relocate from liver to lung in tumour-bearing mouse To examine cellular trafficking between the liver and pre-metastatic lungs of tumour-bearing mice, we developed an in vivo cell-tracking system using KikGR mice (Tomura et al, 2014). KikGR is a marker protein with a violet light-induced green-to-red photoconvertible fluorescent group. We prepared tumour-bearing KikGR mice using E0771 and LLC tumours. In addition, we used a tumour-conditioned media (TCM) injection technique to generate mice in the pre-metastatic phase, with no possibility of micrometastasis. For this model, we first applied violet light to a liver lobe of tumour-bearing- or TCM-stimulated KikGR mice (see model in Fig 2A, with further details in Materials and Methods). This irradiation protocol was established so that there would be no evidence of inflammation as monitored by the accumulation of CD11b+ cells and inflammation-related gene expression in violet light-treated mice (see details in Materials and Methods). Briefly, a small frontal area of the liver (circle area in 10 mm diameter, 100 μm depth) was exposed to violet light twice for 2 min while supplying phosphate-buffered saline (PBS). We kept this protocol because when we expanded area and time for irradiation to obtain more photoconverted cells, CD11b+ cell mobilization and inflammation-related gene expression were markedly stimulated. Blood perfusion was carried out before tissue collection to reduce the effect of circulating blood cells. The signal of KikGR red protein was clearly observed in the lungs 72 h after photoconversion, but not in non-photoconverted lungs (Fig 2B). The photoconverted cells were also detected by flow cytometric analysis (Fig EV1A, B and D, and Appendix Figs S3 and S4). After 72 h, the photoconverted KikGR red cells continued to produce KikGR green resulting in shifting upper-left direction in the dot plot (see arrow in Fig EV1D). The ratio of photoconverted cells in the lungs of unstimulated mice was low; however, it became tangible in the case of TCM-stimulated KikGR mice (Appendix Fig S5A and B). We further analysed the ratios of photoconverted KikGR cells in the liver and lung by using TCM-stimulated KikGR mice. The ratios of photoconverted CD45+ cells per total CD45+ cells in liver were higher than those in lung (N = 5, liver, 0.52 ± 0.08%, lung, 0.12 ± 0.02% (mean ± SEM) in Fig 2C). Our results demonstrated that a part of liver CD45+ cells that underwent photoconversion were diverted to the lung (Fig 2C and Appendix Fig S5B). To determine the cell surface markers expressed in lung HepELs, we conducted flow cytometric analyses using antibodies for CD45, NK1.1, CD4, CD8, CD11b, CD11c and B220 using spleen and lungs (Appendix Figs S3 and S4). First, it should be emphasized that very few photoconverted CD45-negative cells were observed in the lungs (Fig EV1C and E). Second, TCM stimulation increased the populations of CD4+CD45+, CD8+CD45+, NK1.1+CD45+, CD11c+CD45+ and B220+CD45+ compared with conCM (control culture medium without tumour cells) stimulation (Figs 2D and EV1C and E). Since we confirmed that the primary tumours induced the relocation of CD4+CD45+, CD8+CD45+, NK1.1+CD45+, CD11c+CD45+ and B220+CD45+HepELs in the lungs, we examined whether these cells expressed FX. The FX signal was immunohistochemically detected in CD4+CD45+, NK1.1+CD45+, CD11c+CD45+ and B220+CD45+ cells, indicating that FX+ HepELs are a mixed population of mononuclear cells (MNCs) (Fig 2E). To clarify which type of cell relocated to the pre-metastatic lung, we further examined photoconverted HepELs using candidate cell markers such as CD4, NK1.1, CD11c and B220. We found that about 1% of B220+CD11c+NK1.1+ leucocytes emerged in TCM-stimulated lungs (Fig 3A and B). On the other hand, control CM (conCM) did not induce B220+CD11c+NK1.1+ cells in the lung (Fig 3A). As shown in Figs 3B and EV2, the relocated photoconverted HepELs in TCM-stimulated lungs were confirmed to be B220+CD11c+NK1.1+ leucocytes. In examination of the relocated HepELs, TCRβ+NK1.1− T cell mobilization was not observed in TCM-stimulated lungs (Appendix Fig S6). We could not clarify whether B220+CD11c+NK1.1+ cells were also NK1.1+TCRβdim NKT cells in this assay system (Appendix Fig S6). B220+CD11c+NK1.1+ cells in various organs such as lung, liver, peripheral blood, bone marrow, lymph node and the primary tumour were investigated. We collected samples 2, 7 and 14 days after the tumour cell implantation; their approximate tumour sizes were 0 mm (2 days), 3 mm (7 days) and 10 mm (14 days) in diameter, respectively. Among them, the FX expression levels in B220+CD11c+NK1.1+ cells isolated from the liver of 3 mm tumour-bearing mice were remarkably high (Appendix Fig S7, upper panel). We would like to note that the FX expressions in the cells derived from the lung and tumour tissues in 10 mm tumour-bearing mice were also observed (Appendix Fig S7, upper panel). Figure 2. In vivo tracking of HepELs in a primary tumour-stimulated mouse A. An experimental tracing model of CD45+ leucocytes in "KikGR" mice using a photoconversion system. Colour conversion from KikGR green-to-KikGR red occurred in liver cells upon violet light irradiation. In the tumour-bearing- or tumour-conditioned media (TCM)-stimulated KikGR mice, and the cells later moved into the lungs. The KikGR red cells, obtained from TCM-stimulated liver and lungs, were isolated by a cell sorter and CD45 microbeads, and these purified cells were used for microarray screening. B. KikGR red cells were detected in the TCM-stimulated liver and lungs after liver exposure to violet light (arrow). Images taken from animals with no light exposure were also shown (scale bar, 100 μm). C. Flow cytometric quantifications of photoconverted HepELs in TCM (three times)-stimulated KikGR mouse liver and lungs. Cells were isolated 72 h after photoconversion. Ratio was calculated as the number of photoconverted cells (KikGR red) observed in the region (gated in Fig EV1) in comparison with the number of liver or lung cells pre-sorted with CD45-beads. Shown are averages (N = 5) with SEM and Welch's t-test. D. Surface marker analyses of photoconverted KikGR cells. Vertical axes represent ratio of each marker+ cell/photoconverted KikGR cell. Shown are averages (conCM, control: N = 4, TCM: N = 9) with SEM and Welch's t-test. E. Representative immunostaining images of FX expression in NK1.1+, CD11c+, B220+ and CD4+ cells in tumour-bearing lungs (scale bar, 10 μm). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Flow cytometric analyses of moving HepELs in a KikGR mouse lungs A–E. Representative flow cytometric analysis in lungs 72 h after liver exposure to violet light. Cells obtained from mouse with no light exposure gave KikGR red versus KikGR green dot plot (A). Those with light exposure (B, D) exhibited photoconverted cells in a red polygonal region. Cells in the gated region were further analysed by using CD4, CD8, CD45, CD11c, CD11b and B220 antibodies (C, E). Percent is shown for total cells in (A, B and D) and for gated cells in (C and E). conCM (control) and TCM were injected (3 times, every 2 days). Comparison of isotype control-Ab and specific-Ab are also shown (Appendix Figs S3 and S4). Download figure Download PowerPoint Figure 3. Relocation of B220+CD11c+NK1.1+HepELs in pre-metastatic lungs A. Flow cytometric analysis of B220+CD11c+NK1.1+ cells in the lung of TCM-stimulated mice. Values in the dot plots present ratios for total cells gated KikGR red and KikGR green cell. B. Representative flow cytometric analyses of the relocation of B220+CD11c+NK1.1+ cells in lungs that were photoconverted in TCM-stimulated KikGR mouse liver (gated in red polygonal region). Percent is shown for total cells (upper panel) and for gated KikGR red or KikGR green cells (middle and lower panels). Region where the photoconverted cells locate is magnified in the right. Yellow arrow shows the photoconversion direction. Three independent experiments. LTCM stands for TCM derived from LLC. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Representative three independent flow cytometric analyses related to Fig 3BThe movement of B220+CD11c+NK1.1+ cells in lungs that were photoconverted in liver of a E0771-TCM (ETCM)-stimulated KikGR mouse. Percent is shown for total cells in the upper panel (total cells gated KikGR red and KikGR green) and for gated cells in the lower panels. Download figure Download PowerPoint B220+CD11c+NK1.1+ HepELs attack tumour cells via IFNγ It has been reported that B220+CD11c+NK1.1+ cells were identified as a subset of natural killer (NK) cells in lymphoid organs (Blasius et al, 2007). NK cells are prototypic innate lymphoid cells endowed with potent cytolytic function, which provides a host defence against tumours and microbial infections (Robinette et al, 2015; Morvan & Lanier, 2016). In addition, NK cell-type cytotoxic capacities of CD3−NK1.1+ cells were reduced by hypoxic primary tumour-derived factors in the pre-metastatic niche (Sceneay et al, 2012). To investigate the nature of B220+CD11c+NK1.1+ cells against metastatic tumour cells, we examined the IFNγ secretion capacity and NK cell cytotoxic activity. For IFNγ secretion, we compared organ-specific microenvironments such as liver and lungs. The IFNγ in liver B220+CD11c+NK1.1+ cells was kept at a moderate level, but was markedly induced by conditioned media (CM) cultured with LLC-bearing lungs (Fig 4A and B left). In contrast, IFNγ induction in spleen with B220+ CD11c+ NK1.1+ cells was prominent by both liver-CM and lung-CM (Fig 4B, right). To determine whether NK cell-mediating cytotoxic activity is dependent on tumour-stimulating organ education, we co-cultured LLC cells with B220+CD11c+NK1.1+ cells derived from liver or lung in LLC-bearing mice. In accordance with IFNγ induction, lung-CM-primed B220+CD11c+NK1.1+ cells clearly attacked co-existing tumour cells (Fig 4C–E). Figure 4. IFNγ induction and anti-tumour activity of B220+CD11c+NK1.1+HepELs A. Representative photographs of IFNγ expression in B220+CD11c+NK1.1+ cells derived from TCM-stimulating mouse liver. These cells were cultured with LiCM or LuCM that had been prepared by culture with tumour-bearing- liver or lungs, respectively. NoCM stands for CM without tissues, and it was used as control (scale bar, 10 μm). B. Immunohistochemical quantifications of IFNγ in B220+CD11c+NK1.1+ cells. Cells derived from liver and spleen were primed with LiCM or LuCM. Shown are averages (N = 6/group) with SEM and one-way ANOVA. C. Flow cytometric analysis of living Zombie− tumour cells with B220+CD11c+NK1.1+ cells that were primed by NoCM or LuCM. NoCM: N = 3, LuCM: N = 4. Shown are averages with SEM. Welch's t-test. Rhodamine+ tumour cells were counted as viable cells. D. Representative immunohistochemical stainings of rhodamine+ (red) Zombie+ (green) tumour cells (scale bar, 10 μm). E. Ratios of living tumour cells after co-culture with B220+CD11c+NK1.1+ cells that had been primed with lung- or liver-CM. Shown are averages (N = 6/group) with SEM and one-way ANOVA. Download figure Download PowerPoint Differential expression patterns of FX+CD45+ cells between liver and lungs of tumour-bearing mice To better understand the molecular mechanisms mediating HepEL relocalization from the liver to the lungs in response to tumour progression, we isolated photoconverted HepELs from the liver and lungs of TCM-treated mice using a cell sorter (see Materials and Methods, "Isolation of CD45+ cells and B220+CD11c+NK1.1+ cells"). Gene expression between the two cell populations was assessed by microarray analysis (Appendix Table S2). Interestingly, HepELs remaining in the liver displayed increased Vtn (Seiffert et al, 1994) expression compared with those that migrated to the lungs (Appendix Table S2, right). In contrast, lung HepELs showed a higher expression of thrombospondin (TSP) (THBS1) (Good et al, 1990; Watnick et al, 2014) (Appendix Table S2, left). Notably, both Vtn and TSP have been reported to bind fibrinogen (Panetti et al, 1999; Podor et al, 2002), which is prevalent in the focal areas of pre-metastatic lungs. We attempted to visualize Vtn/TSP expression pattern in autopsy samples from cancer patients. First, we pathologically examined the liver and lung tissue to identify regions devoid of tumour metastasis, atelectasis or inflammation and then performed immunohistochemistry to quantify the presence of Vtn+FX+CD45+ and TSP+FX+CD45+ cells in the liver and lungs of non-cancer and cancer patients. We found that Vtn expression in CD45+ cells was higher in the liver than in the lung tissue from the same patient (Fig EV3A). In contrast, TSP expression was upregulated in CD45+ cells in the lung, but not in the liver (Fig EV3A). A unique pattern of Vtn-TSP signal
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