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The antitumor activity of LPS was first described by Dr. William Coley. However,its role in lung cancer remains unclear. The aim of our study was to elucidate the dose-dependent effects of LPS (0.1-10 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth increased at 3 and 7 d after the administration of low-dose LPS (0.1 μg/mouse) compared with control mice. This was associated with an influx of plasmacytoid dendritic cells (pDCs),regulatory T cells,myeloid-derived suppressor cells,and CD8(+) regulatory T cells. In contrast,high-dose LPS (10 μg/mouse) reduced lung tumor burden and was associated with a greater influx of pDCs,as well as a stronger Th1 and Th17 polarization. Depletion of pDCs during low-dose LPS administration resulted in a decreased lung tumor burden. Depletion of pDCs during high-dose LPS treatment resulted in an increased tumor burden. The dichotomy in LPS effects was due to the phenotype of pDCs,which were immunosuppressive after the low-dose LPS,and Th1- and T cytotoxic-polarizing cells after the high-dose LPS. Adoptive transfer of T cells into nude mice demonstrated that CD8(+) T cells were responsible for pDC recruitment following low-dose LPS administration,whereas CD4(+) T cells were required for pDC influx after the high-dose LPS. In conclusion,our data suggest differential effects of low-dose versus high-dose LPS on pDC phenotype and tumor progression or regression in the lungs of mice. View Publication

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1,a cell surface glycoprotein expressed on MM cells. In preclinical models,elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein,we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary,human MM cells. Taken together,these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone. View Publication

Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells,presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study,20 ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35-121.5); P=0.02),number of metastatic sites (hazard ratio 3.87 (1.66-9.03); P=0.02),tumor burden (hazard ratio 5.72 (1.57-20.9); P=0.01),and percentage of NES-expressing CMCs ≥ 35% (hazard ratio 5.73 (1.66-19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis. View Publication

The activating NK cell receptor DNAX accessory molecule-1 (DNAM-1) contributes to tumor immune surveillance and plays a crucial role in NK cell-mediated recognition of several types of human tumors,including ovarian carcinoma. Here,we have analyzed the receptor repertoire and functional integrity of NK cells in peritoneal effusions from patients with ovarian carcinoma. Relative to autologous peripheral blood NK cells,tumor-associated NK cells expressed reduced levels of the DNAM-1,2B4,and CD16 receptors and were hyporesponsive to HLA class I-deficient K562 cells and to coactivation via DNAM-1 and 2B4. Moreover,tumor-associated NK cells were also refractory to CD16 receptor stimulation,resulting in diminished Ab-dependent cellular cytotoxicity against autologous tumor cells. Coincubation of NK cells with ovarian carcinoma cells expressing the DNAM-1 ligand CD155 led to reduction of DNAM-1 expression. Therefore,NK cell-mediated rejection of ovarian carcinoma may be limited by perturbed DNAM-1 expression on tumor-associated NK cells induced by chronic ligand exposure. Thus,these data support the notion that tumor-induced alterations of activating NK cell receptor expression may hamper immune surveillance and promote tumor progression. View Publication

Protein kinase Ciota (PKCiota) is an oncogene required for maintenance of the transformed phenotype of non-small cell lung cancer cells. However,the role of PKCiota in lung tumor development has not been investigated. To address this question,we established a mouse model in which oncogenic Kras(G12D) is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCiota gene,Prkci. Genetic loss of Prkci dramatically inhibits Kras-initiated hyperplasia and subsequent lung tumor formation in vivo. This effect correlates with a defect in the ability of Prkci-deficient bronchioalveolar stem cells to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo. Furthermore,the small molecule PKCiota inhibitor aurothiomalate inhibits Kras-mediated bronchioalveolar stem cell expansion and lung tumor growth in vivo. Thus,Prkci is required for oncogene-induced expansion and transformation of tumor-initiating lung stem cells. Furthermore,aurothiomalate is an effective antitumor agent that targets the tumor-initiating stem cell niche in vivo. These data have important implications for PKCiota as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment. View Publication

Platelets have a crucial role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion,which underlie stroke and acute coronary syndromes. Anucleate platelets contain mRNAs and are capable of protein synthesis,raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs),which are known as key regulators of mRNA translation in other cell types. Further analyses revealed that platelets contain the Dicer and Argonaute 2 (Ago2) complexes,which function in the processing of exogenous miRNA precursors and the control of specific reporter transcripts,respectively. Detection of the receptor P2Y(12) mRNA in Ago2 immunoprecipitates suggests that P2Y(12) expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system. View Publication

The optimization of a purely negative depletion,enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling,and subsequent depletion,of CD45 positive cells. A number of relevant variables are quantified,or attempted to be quantified,which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross,fresh,peripheral blood from normal donors,and fresh peripheral blood from human cancer patients. After optimization,the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 x 10(9) to 8.04 x 10(3) cells/mL and still recover,on average,2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood,with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patient's blood is unknown,and most probably varies from patient to patient,the recovery of the CTC is unknown. However,spiking studies of a cancer cell line into normal blood,and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies,this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log(10)) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final,enrich product contains CTCs or other cell type relevant to the specific question (i.e.,does the CTC have predominantly epithelial or mesenchymal characteristics?). View Publication

Hereditary hemochromatosis (HH),an iron overload disease associated with mutations in the HFE gene,is characterized by increased intestinal iron absorption and consequent deposition of excess iron,primarily in the liver. Patients with HH and Hfe-deficient (Hfe-/-) mice manifest inappropriate expression of the iron absorption regulator hepcidin,a peptide hormone produced by the liver in response to iron loading. In this study,we investigated the contribution of Hfe expression in macrophages to the regulation of liver hepcidin levels and iron loading. We used bone marrow transplantation to generate wild-type (wt) and Hfe-/- mice chimeric for macrophage Hfe gene expression. Reconstitution of Hfe-deficient mice with wt bone marrow resulted in augmented capacity of the spleen to store iron and in significantly decreased liver iron loading,accompanied by a significant increase of hepatic hepcidin mRNA levels. Conversely,wt mice reconstituted with Hfe-deficient bone marrow had a diminished capacity to store iron in the spleen but no significant alterations of liver iron stores or hepcidin mRNA levels. Our results suggest that macrophage Hfe participates in the regulation of splenic and liver iron concentrations and liver hepcidin expression. View Publication