In vivo validation of metastasis-regulating microRNA-766 in human triple-negative breast cancer cells / 한국실험동물학회지

Breast cancer is the second most common cancer and the most frequent cancer in women worldwide. Recent improvements in early detection and effective adjuvant chemotherapies have improved the survival of breast cancer patients. Even with initial disease remission, one-third of all breast cancer patients will relapse with distant metastasis. Breast cancer metastasis is largely an incurable disease and the main cause of death among breast cancer patients. Cancer metastasis is comprised of complex processes that are usually not controllable by intervention of a single molecular target. As a single microRNA (miRNA) can affect the aggressiveness of breast cancer cells by concurrently modulating multiple pathway effectors, a metastasis-regulating miRNA would represent a good disease target candidate. In this study, we evaluated the functional capacity of a newly defined human metastasis-related miRNA, miR-766, which was previously identified by comparing a patient-derived xenograft primary tumor model and a metastasis model. Compared to vector-transfected control cells, miR-766-overexpressed triple-negative breast cancer cells exhibited similar primary tumor growth in the orthotopic xenograft model. In contrast, tumor sphere formation and Matrigel invasion were significantly decreased in miR-766-overexpressed breast cancer cells compared with control cancer cells. In addition, lung metastasis was dramatically reduced in miR-766-overexpressed breast cancer cells compared with control cells. Thus, miR-766 affected the distant metastasis process to a greater extent than cancer cell proliferation and primary tumor growth, and may represent a future therapeutic target to effectively control fatal breast cancer metastasis.

Osteopontin Potentiates Pulmonary Inflammation and Fibrosis by Modulating IL-17/IFN-gamma-secreting T-cell Ratios in Bleomycin-treated Mice

Lung fibrosis is a life-threatening disease caused by overt or insidious inflammatory responses. However, the mechanism of tissue injury-induced inflammation and subsequent fibrogenesis remains unclear. Recently, we and other groups reported that Th17 responses play a role in amplification of the inflammatory phase in a murine model induced by bleomycin (BLM). Osteopontin (OPN) is a cytokine and extracellular-matrix-associated signaling molecule. However, whether tissue injury causes inflammation and consequent fibrosis through OPN should be determined. In this study, we observed that BLM-induced lung inflammation and subsequent fibrosis was ameliorated in OPN-deficient mice. OPN was expressed ubiquitously in the lung parenchymal and bone-marrow-derived components and OPN from both components contributed to pathogenesis following BLM intratracheal instillation. Th17 differentiation of CD4+ alphabeta T cells and IL-17-producing gammadelta T cells was significantly reduced in OPN-deficient mice compared to WT mice. In addition, Th1 differentiation of CD4+ alphabeta T cells and the percentage of IFN-gamma-producing gammadelta T cells increased. T helper cell differentiation in vitro revealed that OPN was preferentially upregulated in CD4+ T cells under Th17 differentiation conditions. OPN expressed in both parenchymal and bone marrow cell components and contributed to BLM-induced lung inflammation and fibrosis by affecting the ratio of pathogenic IL-17/protective IFN-gamma T cells.

Murine Sca1+Lin- bone marrow contains an endodermal precursor population that differentiates into hepatocytes

The direct differentiation of hepatocytes from bone marrow cells remains controversial. Several mechanisms, including transdifferentiation and cell fusion, have been proposed for this phenomenon, although direct visualization of the process and the underlying mechanisms have not been reported. In this study, we established an efficient in vitro culture method for differentiation of functioning hepatocytes from murine lineage-negative bone marrow cells. These cells reduced liver damage and incorporated into hepatic parenchyma in two independent hepatic injury models. Our simple and efficient in vitro protocol for endodermal precursor cell survival and expansion enabled us to identify these cells as existing in Sca1+ subpopulations of lineage-negative bone marrow cells. The endodermal precursor cells followed a sequential developmental pathway that included endodermal cells and hepatocyte precursor cells, which indicates that lineage-negative bone marrow cells contain more diverse multipotent stem cells than considered previously. The presence of equivalent endodermal precursor populations in human bone marrow would facilitate the development of these cells into an effective treatment modality for chronic liver diseases.

Tissue transglutaminase-interleukin-6 axis facilitates peritoneal tumor spreading and metastasis of human ovarian cancer cells / 한국실험동물학회지

Inflammation has recently been implicated in cancer formation and progression. As tissue transglutaminase (TG2) has been associated with both inflammatory signaling and tumor cell behavior, we propose that TG2 may be an important link inducing interleukin-6 (IL-6)-mediated cancer cell aggressiveness, including cancer stem cell-like characteristics and distant hematogenous metastasis. We evaluated the effect of differential TG2 and IL-6 expression on in vivo distant metastasis of human ovarian cancer cells. IL-6 production in human ovarian cancer cells was dependent on their TG2 expression levels. The size and efficiency of tumor sphere formation were correlated with TG2 expression levels and were dependent on TG2-mediated IL-6 secretion in human ovarian cancer cells. Primary tumor growth and propagation in the peritoneum and distant hematogenous metastasis into the liver and lung were also dependent on TG2 and downstream IL-6 expression levels in human ovarian cancer cells. In this report, we provide evidence that TG2 is an important link in IL-6-mediated tumor cell aggressiveness, and that TG2 and downstream IL-6 could be important mediators of distant hematogenous metastasis of human ovarian cancer cells. Intervention specific to TG2 and/or downstream IL-6 in ovarian cancer cells could provide a promising means to control tumor metastasis.

Kinetics of IFN-gamma and IL-17 Production by CD4 and CD8 T Cells during Acute Graft-versus-Host Disease

Graft-versus-host disease (GVHD) is a fatal complication that occurs after allogeneic hematopoietic stem cell transplantation. To understand the dynamics of CD4 and CD8 T cell production of IFN-gamma and IL-17 during GVHD progression, we established a GVHD model by transplanting T cell-depleted bone marrow (TCD-BM) and purified T cells from B6 mice into irradiated BALB.B, creating an MHC-matched but minor histocompatibility (H) antigen-mismatched transplantation (B6 --> BALB.B GVHD). Transplantation-induced GVHD was confirmed by the presence of the appropriate compositional changes in the T cell compartments and innate immune cells in the blood and the systemic secretion of inflammatory cytokines. Using this B6 --> BALB.B GVHD model, we showed that the production of IFN-gamma and IL-17 by CD4 T cells preceded that by CD8 T cells in the spleen, mesenteric lymph node, liver, and lung in the BALB.B GVHD host, and Th1 differentiation predated Th17 differentiation in all organs during GVHD progression. Such changes in cytokine production were based on changes in cytokine gene expression by the T cells at different time points during GVHD development. These results demonstrate that both IFN-gamma and IL-17 are produced by CD4 and CD8 T cells but with different kinetics during GVHD progression.

CP-690550 Treatment Ameliorates Established Disease and Provides Long-Term Therapeutic Effects in an SKG Arthritis Model

Although pathogenesis of human rheumatoid arthritis (RA) remains unclear, arthritogenic T cells and downstream signaling mediators have been shown to play critical roles. An increasing numbers of therapeutic options have been added for the effective control of RA. Nevertheless, there is still a category of patients that fails treatment and suffers from progressive disease. The recently developed immunosuppressant CP-690550, a small molecule JAK kinase inhibitor, has been implicated as an important candidate treatment modality for autoimmune arthritis. In this study, we evaluated the therapeutic effect of CP-690550 on established arthritis using an SKG arthritis model, a pathophysiologically relevant animal model for human RA. CP-690550 treatment revealed remarkable long-term suppressive effects on SKG arthritis when administered to the well-advanced disease (clinical score 3.5~4.0). The treatment effect lasted at least 3 more weeks after cessation of drug infusion, and suppression of disease was correlated with the reduced pro-inflammatory cytokines, including IL-17, IFN-gamma, and IL-6 and increased level of immunoregulatory IL-10.

Establishment of High Throughput Screening System Using Human Umbilical Cord-derived Mesenchymal Stem Cells

The use of high throughput screening (HTS) in drug development is principally for the selection new drug candidates or screening of chemical toxicants. This system minimizes the experimental environment and allows for the screening of candidates at the same time. Umbilical cordderived stem cells have some of the characteristics of fetal stem cell and have several advantages such as the ease with which they can be obtained and lack of ethical issues. To establish a HTS system, optimized conditions that mimic typical cell culture conditions in a minimal space such as 96 well plates are needed for stem cell growth. We have thus established a novel HTS system using human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). To determine the optimal cell number, hUC-MSCs were serially diluted and seeded at 750, 500, 200 and 100 cells per well on 96 well plates. The maintenance efficiencies of these dilutions were compared for 3, 7, 9, and 14 days. The fetal bovine serum (FBS) concentration (20, 10, 5 and 1%) and the cell numbers (750, 500 and 200 cells/well) were compared for 3, 5 and 7 days. In addition, we evaluated the optimal conditions for cell cycle block. These four independent optimization experiments were conducted using an MTT assay. In the results, the optimal conditions for a HTS system using hUC-MSCs were determined to be 300 cell/well cultured for 8 days with 1 or 5% FBS. In addition, we demonstrated that the optimal conditions for a cell cycle block in this culture system are 48 hours in the absence of FBS. In addition, we selected four types of novel small molecule candidates using our HTS system which demonstrates the feasibility if using hUC-MSCs for this type of screen. Moreover, the four candidate compounds can be tested for stem cell research application.

Maturation-Resistant Dendritic Cells Ameliorate Experimental Autoimmune Uveoretinitis

BACKGROUND: Endogenous uveitis is a chronic inflammatory eye disease of human, which frequently leads to blindness. Experimental autoimmune uveoretinitis (EAU) is an animal disease model of human endogenous uveitis and can be induced in susceptible animals by immunization with retinal antigens. EAU resembles the key immunological characteristics of human disease in that both are CD4+ T-cell mediated diseases. Dendritic cells (DCs) are specialized antigen-presenting cells that are uniquely capable of activating naive T cells. Regulation of immune responses through modulation of DCs has thus been tried extensively. Recently our group reported that donor strain-derived immature DC pretreatment successfully controlled the adverse immune response during allogeneic transplantation. METHODS: EAU was induced by immunization with human interphotoreceptor retinoid-binding protein (IRBP) peptide(1-20). Dendritic cells were differentiated from bone marrow in the presence of recombinant GM-CSF. RESULTS: In this study, we used paraformaldehyde-fixed bone marrow-derived DCs to maintain them in an immature state. Pretreatment with fixed immature DCs, but not fixed mature DCs, ameliorated the disease progression of EAU by inhibiting uveitogenic CD4+ T cell activation and differentiation. CONCLUSION: Application of iBMDC prepared according to the protocol of this study would provide an important treatment modality for the autoimmune diseases and transplantation rejection.

Intratumoral Administration of Secondary Lymphoid Chemokine and Unmethylated Cytosine-phosphorothioate-guanine Oligodeoxynucleotide Synergistically Inhibits Tumor Growth in Vivo

Secondary lymphoid tissue chemokine (SLC), which is expressed in T cell zones of secondary lymphoid organs, including the spleen and lymph nodes, strongly recruits both T lymphocytes and mature dendritic cells. As appropriate interaction of tumor-specific T cells and mature dendritic cells, equipped with tumor antigens, is a prerequisite for effective T cell immunity against established tumors, we mobilized lymphocytes and dendritic cells to tumor sites by intratumoral injection of secondary lymphoid tissue chemokine-Fc (SLC-Fc) fusion protein using the B16F10 murine melanoma model. Activation of dendritic cells, another prerequisite for the effective activation of naive tumor-specific T cells, was achieved by the addition of immunostimulatory cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG-ODN) into the tumor site. Intratumoral administration of SLC-Fc or CpG-ODN revealed antitumor effects against B16F10 murine melanoma grown in the subcutaneous space. Co-treatment of SLC-Fc and CpG-ODN displayed synergistic effects in reducing the tumor size. The synergistic antitumor effect in co-treatment group was correlated with the synergistic/additive increase in the infiltration of CD4+ T cells and CD11c+ dendritic cells in the tumor mass compared to the single treatment groups. These results suggest that the combined use of chemokines and adjuvant molecules may be a possible strategy in clinical tumor immunotherapy.

Antigen Nonspecific Death of Immature Thymocytes by Corticosteroids and TNF

BACKGROUND: In the thymus, developing thymocytes continually interact with thymic epithelial cell components. Self MHC restriction of mature T cells are imposed in the thymus through interaction of immature double positive thymocytes and thymic cortical epithelial cells. The site of negative selection, however, is a matter of debate. Through systemic injection of anti-TCR antibody or antigenic peptides, investigators suggested that most of the negative selection occurs in the thymic cortex. But the requirements for negative selection, i.e cellular counterparts and costimulatory molecules are more available in the medulla or cortico-medullary junction rather than in the thymic cortex. METHODS: The direct and indirect pathways of thymocyte death after systemic anti-TCR antibody injection were separated through several experimental systems. B6 mice were either adrenalectomized or sham-adrenalectomized to evaluate the role of endogenous glucocorticoids from adrenal gland. Role of TNF were evaluated through using TNF receptor double knockout mice. RESULTS: We found that without indirectly acting mediators such as TNF-alpha or corticosteroid, double positive thymocyte death were minimal by systemic injection of anti-TCR antibody in TNF receptor double knockout neonatal mice. Also by analyzing neonatal wild-type mice with adoptively transferred mature T cells, only peripheral activation of mature T cells could induce extensive double positive thymocyte death. CONCLUSION: Thus, systemically injected anti-TCR antibody mediated thymocyte death are mostly induced through indirect pathway.