Chiyoko Nishime

Critical Role for CXC Chemokine Ligand 16 (SR-PSOX) in Th1 Response Mediated by NKT Cells

The transmembrane chemokine CXCL 16 (CXCL16), which is the same molecule as the scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX), has been shown to mediate chemotaxis and adhesion of CXC chemokine receptor 6-expressing cells such as NKT and activated Th1 cells. We generated SR-PSOX/CXCL16-deficient mice and examined the role of this chemokine in vivo. The mutant mice showed a reduced number of liver NKT cells, and decreased production of IFN-γ and IL-4 by administration of α-galactosylceramide (αGalCer). Of note, the αGalCer-induced production of IFN-γ was more severely impaired than the production of IL-4 in SR-PSOX-deficient mice. In this context, SR-PSOX-deficient mice showed impaired sensitivity to αGalCer-induced anti-tumor effect mediated by IFN-γ from NKT cells. NKT cells from wild-type mice showed impaired production of IFN-γ, but not IL-4, after their culture with αGalCer and APCs from mutant mice. Moreover, Propionibacterium acnes-induced in vivo Th1 responses were severely impaired in SR-PSOX-deficient as well as NKT KO mice. Taken together, SR-PSOX/CXCL16 plays an important role in not only the production of IFN-γ by NKT cells, but also promotion of Th1-inclined immune responses mediated by NKT cells.

Phosphorescence-Assisted Microvascular O2 Measurements Reveal Alterations of Oxygen Demand in Human Metastatic Colon Cancer in the Liver of Superimmunodeficient NOG Mice

We aimed to examine metabolism of human cancer in vivo and utilized superimmunodeficient NOG mice as an experimental model of hepatic metastasis, where human colon cancer cell line HCT116 transfected with Venus, the mutant GFP was injected intrasplenically. The mice were pretreated with Pd-porphyrin to quantify local O(2) tension through intravital phosphorescence assay. In this model, a majority of metastatic foci occurred in periportal regions but not in central regions. At 1 week after the transplantation, a PO(2) drop in periportal regions was minimal without any notable decrease in microvascular blood flow. Under these conditions, there was a negative correlation between the size of metastatic foci and the lobular O(2) consumption, suggesting that the tumor O(2) consumption is smaller than that in the residual liver. At 2 weeks, portal PO(2) was significantly smaller than controls, while the central PO(2) was not comparably decreased, indicating that metastatic foci increased the O(2) consumption, while the residual liver decreased it. These results suggest metastatic tumors derived from human colon cancer exhibit notable aerobic metabolism during their developmental process, compromising respiration of the rest of the tissue regenerated during tumor development.

Long-term maintenance of peripheral blood derived human NK cells in a novel human IL-15- transgenic NOG mouse

We generated a novel mouse strain expressing transgenic human interleukin-15 (IL-15) using the severe immunodeficient NOD/Shi-scid-IL-2Rγnull (NOG) mouse genetic background (NOG-IL-15 Tg). Human natural killer (NK) cells, purified from the peripheral blood (hu-PB-NK) of normal healthy donors, proliferated when transferred into NOG-IL-15 Tg mice. In addition, the cell number increased, and the hu-PB-NK cells persisted for 3 months without signs of xenogeneic graft versus host diseases (xGVHD). These in vivo-expanded hu-PB-NK cells maintained the original expression patterns of various surface antigens, including NK receptors and killer cell immunoglobulin-like receptor (KIR) molecules. They also contained significant amounts of granzyme A and perforin. Inoculation of K562 leukemia cells into hu-PB-NK-transplanted NOG-IL-15 Tg mice resulted in significant suppression of tumor growth compared with non-transplanted mice. Furthermore, NOG-IL-15 Tg mice allowed for engraftment of in vitro-expanded NK cells prepared for clinical cell therapy. These cells exerted antibody-dependent cell-mediated cytotoxicity (ADCC) on Her2-positive gastric cancer cells in the presence of therapeutic anti-Her2 antibody, and subsequently suppressed tumor growth. Our results collectively suggest that the NOG-IL-15 Tg mice are a useful model for studying human NK biology and evaluating human NK cell-mediated in vivo cytotoxicity.

Innate Response to Human Cancer Cells with or without IL-2 Receptor Common γ-Chain Function in NOD Background Mice Lacking Adaptive Immunity

Immunodeficient hosts exhibit high acceptance of xenogeneic or neoplastic cells mainly due to lack of adaptive immunity, although it still remains to be elucidated how innate response affects the engraftment. IL-2R common γ-chain (IL-2Rγc) signaling is required for development of NK cells and a subset of dendritic cells producing IFN-γ. To better understand innate response in the absence of adaptive immunity, we examined amounts of metastatic foci in the livers after intrasplenic transfer of human colon cancer HCT116 cells into NOD/SCID versus NOD/SCID/IL-2Rγcnull (NOG) hosts. The intravital microscopic imaging of livers in the hosts depleted of NK cells and/or macrophages revealed that IL-2Rγc function critically contributes to elimination of cancer cells without the need for NK cells and macrophages. In the absence of IL-2Rγc, macrophages play a role in the defense against tumors despite the NOD Sirpa allele, which allows human CD47 to bind to the encoded signal regulatory protein α to inhibit macrophage phagocytosis of human cells. Analogous experiments using human pancreas cancer MIA PaCa-2 cells provided findings roughly similar to those from the experiments using HCT116 cells except for lack of suppression of metastases by macrophages in NOG hosts. Administration of mouse IFN-γ to NOG hosts appeared to partially compensate lack of IL-2Rγc–dependent elimination of transferred HCT116 cells. These results provide insights into the nature of innate response in the absence of adaptive immunity, aiding in developing tumor xenograft models in experimental oncology.

Preclinical evaluation of heat-denatured [18F]FDG-labeled red blood cells for detecting splenic tissues with PET in rats

INTRODUCTION Heat-denatured 99mTc-labeled red blood cells (RBCs) are used for detecting splenic tissues with scintigraphy. The present study aimed to evaluate the feasibility of using heat-denatured [18F]fluorodeoxyglucose ([18F]FDG)-labeled RBCs in detecting splenic tissues using positron emission tomography (PET) in rats. METHODS RBCs were washed with phosphate buffered saline, labeled with [18F]FDG at 38°C, and heat-denatured at 50°C for 15 min. In vitro stability was assessed by measuring extracellular radioactivity during the 0-180 min incubation at 37°C. Thin layer chromatography (TLC) of the extracellular fluid was performed. The autologous RBCs were intravenously injected in four rats and PET scanning was simultaneously performed for 30 min. Time-activity curves of several organs, including the spleen, were analyzed on the PET images. RESULTS Labeling efficiency was 92%. Low levels of radioactivity were released from the labeled RBCs for 180 min. TLC revealed that 80% of the released radioactivity was due to [18F]FDG-6-phosphate. Whole body images showed strong uptake of heat-denatured [18F]FDG-labeled RBCs in the spleen soon after injection in all four rats. Time-activity curves revealed that the splenic uptake continued to increase for 30 min and the amount of radioactivity in the other organs, except the urinary bladder, decreased after the initial surge. CONCLUSIONS Heat-denatured [18F]FDG-labeled RBCs are suitable spleen-specific agents for PET. This method is clinically relevant as an alternative for heat-denatured 99mTc-labeled RBC scintigraphy.

Abstract 456: Adaptation of cancer cells to hypoxic environments for tumor progression

There have been a number of reports mentioning the significance of adaptation of cancer cells to hypoxic environments in tumor growth, invasion, and metastases. However, understanding of molecular aspects based on in vivo evidence still remains insufficient. We established an in vivo tumor progression model by transfer of human colorectal cancer HCT116 cells cultured under hypoxia to NOD/Shi-scid, IL-2Rγnull (NOG) mouse recipients. The hypoxic culture resulted in invasive proliferation of subcutaneous tumors. Immnohistochemical staining for molecular markers, such as E-cadherin, vimentin and BCRP1, demonstrated that adaptation of HCT116 cells to hypoxia was associated with increased frequencies of epithelial-mesenchymal transition and cancer stem cell-like appearance. To investigate the molecular basis of such effects of hypoxia on tumor progression, biological and molecular profiles of HCT116 cultured under hypoxia were analyzed. The hypoxic culture resulted in slowing cell cycling and increase in the side population cell frequency, suggesting induction of stemness. Capillary electrophoresis-mass spectrometric analysis revealed altered metabolism in hypoxic HCT116 cells, which appeared to be associated with adaptation to hypoxia. Gene expression profiling of the cultured cells under pharmacological inhibition of p53, which is not mutated in HCT116 cells, presented evidence for dependence of the adaptation to chronic mild but not acute severe hypoxia on p53 function. Our results provided insights into molecular basis of tumor progression critically directed by hypoxia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 456.

Abstract 2366: Reduced ecto-5′-nucleotidase CD73 expression and altered purine nucleotide metabolism in colorectal cancer cells robustly causing liver metastases

Liver metastases are frequently inoperative and severely impact the prognoses of patients with cancers. Colorectal cancers as well as pancreatic cancers most commonly metastasize to the liver, although there are some patients with colorectal cancers presenting liver metastases operative contrary to the cases of pancreatic cancers. Through liver metastasis models using NOD/SCID/IL-2Rγc null (NOG) mice, we have previously established and examined a highly liver-metastatic human pancreatic cancer cell subline, which was derived from the poorly liver-metastatic parental line, identifying a key molecular regulator of liver metastasis. In the present study, we established a highly liver-metastatic human colorectal cancer cell subline SW48LM2 through the serial intrasplenic transfer of cells derived from poor but visible hepatic tumor foci formed by parental SW48 cells transferred to NOG mice. The growth both under monolayer-culture conditions and during formation of the subcutaneous tumors was similar between the two cell lines, although there were morphological differences in the in vitro spheroid formation. Of 41 molecules reportedly associated positively or negatively with tumor progression, four were overexpressed and four were underexpressed in SW48LM2 cells. Most strikingly, this liver-metastatic cell subline exhibited strongly reduced expression of the ecto-5′ -nucleotidase CD73 as well as altered metabolism of purine nucleotides. Contrary to previous studies showing a positive correlation between CD73 expression and metastatic cancer phenotypes, reduced CD73 expression on tumor cells may contribute to the potential of liver metastases. The present study highlights the need for interpretative caution when investigating metastasis-associated molecules for the diagnostic and therapeutic applications, providing insights into the mechanisms of liver metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2366.