CCR2- and CCR5-mediated macrophage infiltration contributes to glomerular endocapillary hypercellularity in antibody-induced lupus nephritis.

OBJECTIVES: LN comprises various glomerular lesions, including endocapillary hypercellularity with macrophage infiltration. In this study, we aimed to clarify the involvement of macrophage-tropic chemokine receptors in the pathogenesis of these glomerular lesions. METHODS: MRL/lpr mouse-derived monoclonal IgG3 antibody-producing hybridomas, 2B11.3 and B1, were injected intraperitoneally into BALB/c mice [wild type (WT)] to induce endocapillary hypercellularity and wire-loop lesions, respectively. The expression of chemokine and chemokine receptors was analysed by quantitative real-time PCR and IF. The roles of chemokine receptors in these lesions were evaluated using chemokine receptor-deficient mice or a selective CCR5 antagonist, maraviroc. RESULTS: 2B11.3 caused glomerular endocapillary hypercellularity with a significant number of glomerular CD68-positive macrophages. Further, enhanced expression of CCL2, CCL3, CCR2, CCR5 and CX3CR1 was observed in the renal cortex, compared with B1 injection, which induced wire-loop lesions. In 2B11.3-induced glomerular lesions, CD68 -positive glomerular macrophages expressed CCL2, CCL3, CCR2, CCR5 and CX3CR1, while glomerular endothelial cells expressed CCL2, CCL3, CX3CL1 and CCR2. When 2B11.3 was injected, CCR2-/- and CCR5-/-, but not CX3CR1-/-, mice exhibited reduced endocapillary hypercellularity, attenuated glomerular macrophage infiltration and improved serum blood urea nitrogen levels. Only CCR2-/- mice developed wire-loop lesions. B1 injection caused wire-loop lesions in these chemokine receptor-deficient mice to a similar extent as WT. Maraviroc treatment reduced 2B11.3-induced endocapillary hypercellularity and improved serum blood urea nitrogen levels. CONCLUSION: CCR2 and CCR5 regulate glomerular macrophage infiltration and contribute to the development of glomerular endocapillary hypercellularity in LN. CCR5 inhibition can be a specific therapy for endocapillary hypercellularity without inducing wire-loop lesions.

Crucial contribution of GPR56/ADGRG1, expressed by breast cancer cells, to bone metastasis formation.

From a mouse triple-negative breast cancer cell line, 4T1, we previously established 4T1.3 clone with a high capacity to metastasize to bone after its orthotopic injection into mammary fat pad of immunocompetent mice. Subsequent analysis demonstrated that the interaction between cancer cells and fibroblasts in a bone cavity was crucial for bone metastasis focus formation arising from orthotopic injection of 4T1.3 cells. Here, we demonstrated that a member of the adhesion G-protein-coupled receptor (ADGR) family, G-protein-coupled receptor 56 (GPR56)/adhesion G-protein-coupled receptor G1 (ADGRG1), was expressed selectively in 4T1.3 grown in a bone cavity but not under in vitro conditions. Moreover, fibroblasts present in bone metastasis sites expressed type III collagen, a ligand for GPR56/ADGRG1. Consistently, GPR56/ADGRG1 proteins were detected in tumor cells in bone metastasis foci of human breast cancer patients. Deletion of GPR56/ADGRG1 from 4T1.3 cells reduced markedly intraosseous tumor formation upon their intraosseous injection. Conversely, intraosseous injection of GPR56/ADGRG1-transduced 4T1, TS/A (mouse breast cancer cell line), or MDA-MB-231 (human breast cancer cell line) exhibited enhanced intraosseous tumor formation. Furthermore, we proved that the cleavage at the extracellular region was indispensable for GPR56/ADGRG1-induced increase in breast cancer cell growth upon its intraosseous injection. Finally, inducible suppression of Gpr56/Adgrg1 gene expression in 4T1.3 cells attenuated bone metastasis formation with few effects on primary tumor formation in the spontaneous breast cancer bone metastasis model. Altogether, GPR56/ADGRG1 can be a novel target molecule to develop a strategy to prevent and/or treat breast cancer metastasis to bone.

Alveolar Macrophages Drive Hepatocellular Carcinoma Lung Metastasis by Generating Leukotriene B4.

Macrophages in lungs can be classified into two subpopulations, alveolar macrophages (AMs) and interstitial macrophages (IMs), which reside in the alveolar and interstitial spaces, respectively. Accumulating evidence indicates the involvement of IMs in lung metastasis, but the roles of AMs in lung metastasis still remain elusive. An i.v. injection of a mouse hepatocellular carcinoma (HCC) cell line, BNL, caused lung metastasis foci with infiltration of AMs and IMs. Comprehensive determination of arachidonic acid metabolite levels revealed increases in leukotrienes and PGs in lungs in this metastasis model. A 5-lipoxygenase (LOX) inhibitor but not a cyclooxygenase inhibitor reduced the numbers of metastatic foci, particularly those of a larger size. A major 5-LOX metabolite, LTB4, augmented in vitro cell proliferation of human HCC cell lines as well as BNL cells. Moreover, in this lung metastasis course, AMs exhibited higher expression levels of the 5-LOX and LTB4 than IMs. Consistently, 5-LOX-expressing AMs increased in the lungs of human HCC patients with lung metastasis, compared with those without lung metastasis. Furthermore, intratracheal clodronate liposome injection selectively depleted AMs but not IMs, together with reduced LTB4 content and metastatic foci numbers in this lung metastasis process. Finally, IMs in mouse metastatic foci produced CCL2, thereby recruiting blood-borne, CCR2-expressing AMs into lungs. Thus, AMs can be recruited under the guidance of IM-derived CCL2 into metastatic lungs and can eventually contribute to the progression of lung metastasis by providing a potent arachidonic acid-derived tumor growth promoting mediator, LTB4.

MIP-1α/CCL3-expressing basophil-lineage cells drive the leukemic hematopoiesis of chronic myeloid leukemia in mice.

Basophilia is a frequently observed hematological abnormality in chronic myeloid leukemia (CML), but its pathophysiological roles are undefined. We previously demonstrated that an inflammatory chemokine, CCL3, preferentially acts on normal hematopoietic stem/progenitor cells and crucially contributes to the maintenance of leukemia initiating cells (LICs) in bone marrow (BM) during the initiation process of CML. However, the major cellular source of CCL3 in BM and the precise mechanism of CCL3-mediated maintenance of LICs remain to be investigated. To delineate the cellular process facilitating this CCL3-mediated crosstalk between normal and leukemic hematopoiesis, we precisely examined CCL3-expressing cells and their functions in both normal hematopoiesis and CML leukemogenesis. Herein, we demonstrate that basophils can constitutively express CCL3 to negatively regulate the normal hematopoietic process, especially hematopoietic reconstitution after BM transplantation. Moreover, CCL3-expressing basophil-like leukemia cells were found to accumulate in CML BM and supported the predominant expansion of LICs therein. These observations suggest that intra-BM basophil expansion can favor leukemia-tropic hematopoiesis in CML by providing CCL3, a potent inhibitor of normal hematopoiesis and that basophil-derived CCL3 may be a novel target molecule for the treatment of CML.

Chemokines as a Conductor of Bone Marrow Microenvironment in Chronic Myeloid Leukemia.

All blood lineage cells are generated from hematopoietic stem cells (HSCs), which reside in bone marrow after birth. HSCs self-renew, proliferate, and differentiate into mature progeny under the control of local microenvironments including hematopoietic niche, which can deliver regulatory signals in the form of bound or secreted molecules and from physical cues such as oxygen tension and shear stress. Among these mediators, accumulating evidence indicates the potential involvement of several chemokines, particularly CXCL12, in the interaction between HSCs and bone marrow microenvironments. Fusion between breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog (ABL)-1 gene gives rise to BCR-ABL protein with a constitutive tyrosine kinase activity and transforms HSCs and/or hematopoietic progenitor cells (HPCs) into disease-propagating leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). LSCs can self-renew, proliferate, and differentiate under the influence of the signals delivered by bone marrow microenvironments including niche, as HSCs can. Thus, the interaction with bone marrow microenvironments is indispensable for the initiation, maintenance, and progression of CML. Moreover, the crosstalk between LSCs and bone marrow microenvironments can contribute to some instances of therapeutic resistance. Furthermore, evidence is accumulating to indicate the important roles of bone marrow microenvironment-derived chemokines. Hence, we will herein discuss the roles of chemokines in CML with a focus on bone marrow microenvironments.

Gemcitabine-induced CXCL8 expression counteracts its actions by inducing tumor neovascularization.

Patients with pancreatic ductal adenocarcinoma (PDAC) are frequently complicated with metastatic disease or locally advanced tumors, and consequently need chemotherapy. Gemcitabine is commonly used for PDAC treatment, but with limited efficacy. The capacity of gemcitabine to generate reactive oxygen species (ROS) in human pancreatic cancer cells, prompted us to examine its effects on the expression of pro-inflammatory cytokines and chemokines. We observed that gemcitabine enhanced selectively the expression of CXCL8 in human pancreatic cancer cells through ROS generation and NF-κB activation. In vitro blocking of CXCL8 failed to modulate gemcitabine-mediated inhibition of cell proliferation in human pancreatic cancer cells. Gemcitabine also enhanced CXCL8 expression in pancreatic cancer cells in xenografted tumor tissues. Moreover, anti-CXCL8 antibody treatment in vivo attenuated tumor formation as well as intra-tumoral vascularity in nude mice, which were transplanted with Miapaca-2 cells and treated with gemcitabine. Thus, gemcitabine-induced CXCL8 may counteract the drug through inducing neovascularization.

Leukocyte cell-derived chemotaxin 2 is an antiviral regulator acting through the proto-oncogene MET.

Retinoic acid-inducible gene (RIG)-I is an essential innate immune sensor that recognises pathogen RNAs and induces interferon (IFN) production. However, little is known about how host proteins regulate RIG-I activation. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine and ligand of the MET receptor tyrosine kinase is an antiviral regulator that promotes the RIG-I-mediated innate immune response. Upon binding to MET, LECT2 induces the recruitment of the phosphatase PTP4A1 to MET and facilitates the dissociation and dephosphorylation of phosphorylated SHP2 from MET, thereby protecting RIG-I from SHP2/c-Cbl-mediated degradation. In vivo, LECT2 overexpression enhances RIG-I-dependent IFN production and inhibits lymphocytic choriomeningitis virus (LCMV) replication in the liver, whereas these changes are reversed in LECT2 knockout mice. Forced suppression of MET abolishes IFN production and antiviral activity in vitro and in vivo. Interestingly, hepatocyte growth factor (HGF), an original MET ligand, inhibits LECT2-mediated anti-viral signalling; conversely, LECT2-MET signalling competes with HGF-MET signalling. Our findings reveal previously unrecognized crosstalk between MET-mediated proliferation and innate immunity and suggest that targeting LECT2 may have therapeutic value in infectious diseases and cancer.

Cancer non-stem cells as a potent regulator of tumor microenvironment: a lesson from chronic myeloid leukemia.

A limited subset of human leukemia cells has a self-renewal capacity and can propagate leukemia upon their transplantation into animals, and therefore, are named as leukemia stem cells, in the early 1990's. Subsequently, cell subpopulations with similar characteristics were detected in various kinds of solid cancers and were denoted as cancer stem cells. Cancer stem cells are presently presumed to be crucially involved in malignant progression of solid cancer: chemoresitance, radioresistance, immune evasion, and metastasis. On the contrary, less attention has been paid to cancer non-stem cell population, which comprise most cancer cells in cancer tissues, due to the lack of suitable markers to discriminate cancer non-stem cells from cancer stem cells. Chronic myeloid leukemia stem cells generate a larger number of morphologically distinct non-stem cells. Moreover, accumulating evidence indicates that poor prognosis is associated with the increases in these non-stem cells including basophils and megakaryocytes. We will discuss the potential roles of cancer non-stem cells in fostering tumor microenvironment, by illustrating the roles of chronic myeloid leukemia non-stem cells including basophils and megakaryocytes in the pathogenesis of chronic myeloid leukemia, a typical malignant disorder arising from leukemic stem cells.

Cytoplasmic DNA accumulation preferentially triggers cell death of myeloid leukemia cells by interacting with intracellular DNA sensing pathway.

Accumulating evidence indicates the presence of cytoplasmic DNAs in various types of malignant cells, and its involvement in anti-cancer drug- or radiotherapy-mediated DNA damage response and replication stress. However, the pathophysiological roles of cytoplasmic DNAs in leukemias remain largely unknown. We observed that during hematopoietic stem cell transplantation (HSCT) in mouse myeloid leukemia models, double-stranded (ds)DNAs were constitutively secreted in the form of extracellular vesicles (EVs) from myeloid leukemia cells and were transferred to the donor cells to dampen their hematopoietic capabilities. Subsequent analysis of cytoplasmic DNA dynamics in leukemia cells revealed that autophagy regulated cytoplasmic dsDNA accumulation and subsequent redistribution into EVs. Moreover, accumulated cytoplasmic dsDNAs activated STING pathway, thereby reducing leukemia cell viability through reactive oxygen species (ROS) generation. Pharmaceutical inhibition of autophagosome formation induced cytoplasmic DNA accumulation, eventually triggering cytoplasmic DNA sensing pathways to exert cytotoxicity, preferentially in leukemia cells. Thus, manipulation of cytoplasmic dsDNA dynamics can be a novel and potent therapeutic strategy for myeloid leukemias.

Expansion of senescent megakaryocyte-lineage cells maintains CML cell leukemogenesis.

BCR-ABL, an oncogenic fusion gene, plays a central role in the pathogenesis of chronic myeloid leukemia (CML). Oncogenic signaling induces oncogene-induced senescence and senescence-associated secretory phenotype (SASP), which is characterized by enhanced production of various cytokines. BCR-ABL gene transduction confers senescent phenotype in vitro; however, the in vivo relevance of senescence has not been explored in this context. Transplantation of BCR-ABL-expressing hematopoietic stem/progenitor cells caused CML in mice with an increase in bone marrow BCR-ABL+CD41+CD150+ leukemic megakaryocyte-lineage (MgkL) cells, which exhibited enhanced senescence-associated ß-galactosidase staining and increased expression of p16 and p21, key molecules that are crucially involved in senescence. Moreover, knockout of p16 and p21 genes reduced both BCR-ABL-induced abnormal megakaryopoiesis and the maintenance of CML cell leukemogenic capacity, as evidenced by attenuated leukemogenic capacity at secondary transplantation. The expression of transforming growth factor-ß1 (TGF-ß1), a representative SASP molecule, was enhanced in the leukemic MgkL cells, and TGF-ß1 inhibition attenuated CML cell leukemogenic capacity both in vitro and in vivo. Furthermore, BCR-ABL-expressing MgkL cells displayed enhanced autophagic activity, and autophagy inhibition reduced bone marrow MgkL cell number and prolonged the survival of CML mice, which had transiently received the tyrosine kinase inhibitor, imatinib, earlier. Thus, BCR-ABL induced the expansion of senescent leukemic MgkL cells, which supported CML leukemogenesis by providing TGF-ß1.

Involvement of Prokineticin 2-expressing Neutrophil Infiltration in 5-Fluorouracil-induced Aggravation of Breast Cancer Metastasis to Lung.

Adjuvant chemotherapy is used for human breast cancer patients, even after curative surgery of primary tumor, to prevent tumor recurrence primarily as a form of metastasis. However, anticancer drugs can accelerate metastasis in several mouse metastasis models. Hence, we examined the effects of postsurgical administration with 5-fluorouracil (5-FU), doxorubicin, and cyclophosphamide, on lung metastasis process, which developed after the resection of the primary tumor arising from the orthotopic injection of a mouse triple-negative breast cancer cell line, 4T1. Only 5-FU markedly increased the numbers and sizes of lung metastasis foci, with enhanced tumor cell proliferation and angiogenesis as evidenced by increases in Ki67-positive cell numbers and CD31-positive areas, respectively. 5-FU-mediated augmented lung metastasis was associated with increases in intrapulmonary neutrophil numbers and expression of neutrophilic chemokines, Cxcl1 and Cxcl2 in tumor cells, with few effects on intrapulmonary T-cell or macrophage numbers. 5-FU enhanced Cxcl1 and Cxcl2 expression in 4T1 cells in a NFκB-dependent manner. Moreover, the administration of a neutrophil-depleting antibody or a Cxcr2 antagonist, SB225002, significantly attenuated 5-FU-mediated enhanced lung metastasis with depressed neutrophil infiltration. Furthermore, infiltrating neutrophils and 4T1 cells abundantly expressed prokineticin-2 (Prok2) and its receptor, Prokr1, respectively. Finally, the administration of 5-FU after the resection of the primary tumor failed to augment lung metastasis in the mice receiving Prokr1-deleted 4T1 cells. Collectively, 5-FU can enhance lung metastasis by inducing tumor cells to produce Cxcl1 and Cxcl2, which induced the migration of neutrophils expressing Prok2 with a capacity to enhance 4T1 cell proliferation. Mol Cancer Ther; 17(7); 1515-25. ©2018 AACR.

Blockade of the chemokine receptor, CCR5, reduces the growth of orthotopically injected colon cancer cells via limiting cancer-associated fibroblast accumulation.

We previously demonstrated that cancer-associated fibroblasts (CAFs) accumulate at tumor sites through the interaction between a chemokine, CCL3, and its receptor, CCR5, in the late phase of colitis-associated colon carcinogenesis. Here we examined the effect of a CCR5 antagonist, maraviroc, on tumor growth arising from the orthotopic injection of mouse or human colon cancer cell lines into the cecal wall by focusing on CAFs. Orthotopic injection of either cell line caused tumor formation together with leukocyte infiltration and fibroblast accumulation. Concomitant oral administration of maraviroc reduced tumor formation with few effects on leukocyte infiltration. In contrast, maraviroc reduced the intratumor number of α-smooth muscle actin-positive fibroblasts, which express epidermal growth factor, a crucial growth factor for colon cancer cell growth. These observations suggest that maraviroc or other CCR5 antagonists might act as novel anti-CRC drugs to dampen CAFs, an essential cell component for tumor progression.

Targeted and ultrasound-triggered cancer cell injury using perfluorocarbon emulsion-loaded liposomes endowed with cancer cell-targeting and fusogenic capabilities.

This study investigated the targeting and ultrasound-triggered injury of cancer cells using anticancer drug-free liposomes that contained an emulsion of perfluoropentane (ePFC5) and were co-modified with avidin as a targeting ligand for cancer cells and the hemagglutinating virus of Japan (HVJ) envelope to promote liposome fusion with the cells. These liposomes are designated as ePFC5-loaded avidin/HVJ liposomes. ePFC5-loaded liposomes were sensitized to ultrasound irradiation. Liposomes modified with avidin alone (avidin liposomes) showed binding to MCF-7 human breast cancer cells, and liposomes modified with HVJ envelope alone (HVJ liposomes) were found to fuse with MCF-7 cells. The irradiation of MCF-7 cells with 1 MHz ultrasound (30s, 1.2 W/cm(2), duty ratio 30%) combined with ePFC5-loaded avidin/HVJ liposomes resulted in a decrease in cell viability at 1h after irradiation to 43% of that of controls without ultrasound irradiation or liposomes. The cell viability was lower than that of cells treated with ultrasound irradiation with ePFC5-loaded avidin liposomes or ePFC5-loaded HVJ liposomes. This indicates that co-modification of liposome with avidin and HVJ envelope could enhance ultrasound-induced cell injury in the presence of ePFC5-loaded liposomes.