Reduced deformability of stored red blood cells is associated with generation of extracellular vesicles.

Throughout storage, red blood cells (RBCs) undergo detrimental changes in viability and their ability to effectively transport oxygen. RBC storage lesions are mediated, in part, by a progressive loss of cell deformability, and associated with the release of extracellular vesicles (EVs). Accumulation of EVs during the storage of RBCs correlates with a decrease in RBC surface area to volume ratio. Similarly, the loss of RBC-deformability is associated with loss of RBC surface area to volume ratio. In this study we thus tested whether loss of RBC-deformability is associated with increased RBC-EV production during blood storage. EVs obtained by differential centrifugation of stored RBCs (non-leukoreduced non-irradiated or leukoreduced γ-irradiated RBCs stored 35 or 28 days respectively) were enumerated by high-sensitivity flow cytometry. RBC deformability was quantified, using a cell-flow-properties-analyzer, by measuring the median cell elongation ratio (MER) and percentage of low and high deformable cells in the population (%, LDFC, and HDFC, respectively). The number of EVs was inversely correlated with the MER and positively correlated with the %LDFC with both measures showing highly significant logarithmic dependence with EV levels in stored RBCs. Considering how highly deformable cells did not correlate with EV formation as compared with low deformable RBCs we propose that the formation of EVs is a key factor leading to increased RBC-rigidity.

Upregulation of RET induces perineurial invasion of pancreatic adenocarcinoma.

Tumor spread along nerves, a phenomenon known as perineurial invasion, is common in various cancers including pancreatic ductal adenocarcinoma (PDAC). Neural invasion is associated with poor outcome, yet its mechanism remains unclear. Using the transgenic Pdx-1-Cre/KrasG12D /p53R172H (KPC) mouse model, we investigated the mechanism of neural invasion in PDAC. To detect tissue-specific factors that influence neural invasion by cancer cells, we characterized the perineurial microenvironment using a series of bone marrow transplantation (BMT) experiments in transgenic mice expressing single mutations in the Cx3cr1, GDNF and CCR2 genes. Immunolabeling of tumors in KPC mice of different ages and analysis of human cancer specimens revealed that RET expression is upregulated during PDAC tumorigenesis. BMT experiments revealed that BM-derived macrophages expressing the RET ligand GDNF are highly abundant around nerves invaded by cancer. Inhibition of perineurial macrophage recruitment, using the CSF-1R antagonist GW2580 or BMT from CCR2-deficient donors, reduced perineurial invasion. Deletion of GDNF expression by perineurial macrophages, or inhibition of RET with shRNA or a small-molecule inhibitor, reduced perineurial invasion in KPC mice with PDAC. Taken together, our findings show that RET is upregulated during pancreas tumorigenesis and its activation induces cancer perineurial invasion. Trafficking of BM-derived macrophages to the perineurial microenvironment and secretion of GDNF are essential for pancreatic cancer neural spread.

Macrophages mediate gemcitabine resistance of pancreatic adenocarcinoma by upregulating cytidine deaminase.

Resistance to pharmacologic agents used in chemotherapy is common in most human carcinomas, including pancreatic ductal adenocarcinoma (PDA), which is resistant to almost all drugs, including gemcitabine, a nucleoside analog used as a first-line treatment. Poor survival rates of PDA patients have, therefore, not changed much over 4 decades. Recent data indicated that tumor-associated macrophages (TAMs), which are abundant in the microenvironment of several tumors, including PDA, secrete pro-tumorigenic factors that contribute to cancer progression and dissemination. In this study, we show for the first time that TAMs can also induce chemoresistance of PDA by reducing gemcitabine-induced apoptosis. Macrophages co-cultured with cancer cells or TAM-conditioned medium significantly reduced apoptosis and activation of the caspase-3 pathway during gemcitabine treatment. In vivo PDA models of mice, which have reduced macrophage recruitment and activation, demonstrated improved response to gemcitabine compared with controls. Similarly, inhibition of monocytes/macrophages trafficking by a CSF1-receptor antagonist GW2580 augmented the effect of gemcitabine in a transgenic mouse PDA model that was resistant to gemcitabine alone. Analysis of multiple proteins involved in gemcitabine delivery and metabolism revealed that TAMs induced upregulation of cytidine deaminase (CDA), the enzyme that metabolizes the drug following its transport into the cell. Decreasing CDA expression by PDA cells blocked the protective effect of TAMs against gemcitabine. These results provide the first evidence of a paracrine effect of TAMs, which mediates acquired resistance of cancer cells to chemotherapy. Modulation of macrophage trafficking or inhibition of CDA may offer a new strategy for augmenting the response of PDA to chemotherapy.

Utilizing glycogen synthase kinase-3ß as a marker for the diagnosis of graft-versus-host disease.

INTRODUCTION: Graft-versus-host disease (GVHD) is a deadly complication of allogeneic hematopoietic stem cell transplantation. Timely diagnosis is critical, because mortality rates for GVHD are high, increasing with disease severity. A diagnostic tool to predict GVHD before the onset of clinical symptoms could save many lives. On the cellular level, GVHD occurs when T cells from the transplant attack the tissues of the host, after perceiving them to be foreign. T-cell proliferation occurs even before clinical symptoms appear. Glycogen synthase kinase (GSK)-3ß is a protein which regulates proliferation in many cell types including T-cells. GSK-3ß has never been directly connected with GVHD and we applied GSK-3ß as a novel marker for GVHD prediction, seeking herein to determine whether GSK-3ß can be utilized as a marker for the early diagnosis of GVHD. METHODS: For the mouse model of acute GVHD, irradiated mice underwent allogeneic splenocyte transplantation and GSK-3ß expression levels and phosphorylation states were monitored in harvested spleens by western blot. FACS analysis was used to measure the number of T cells within the harvested spleens. RESULTS: Mice developed observable GVHD symptoms by day 5 post-transplantation, with severe symptoms on day 6 requiring mice to be killed for humane reasons. A significantly increased number of T cells in the allogeneic mice correlated with GVHD development. GSK-3ß protein expression levels and phosphorylation levels were significantly lower in allogeneic (GVHD) mice compared with negative (untreated) and positive (syngeneic transplant; non-GVHD) controls over time. CONCLUSION: GSK-3ß was directly connected with the onset and progression of GVHD. Therefore, it can be utilized as a marker for GVHD diagnosis in animals and potentially in humans.