Siglec-9 is a sialic acid binding lectin predominantly expressed on myeloid cells. Aberrant glycosylation occurs in essentially all types of cancers resulting in increased sialylation. Thus when MUC1 is expressed on cancer cells it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). Here we show that this cancer-specific MUC1 glycoform could, through the engagement of Siglec-9, educate myeloid cells to release factors associated with tumor microenvironment determination and disease progression. Moreover MUC1-ST induced macrophages to display a TAM-like phenotype with increased expression of PD-L1. MUC1-ST binding to Siglec-9 did not activate SHP-1/2 but surprisingly induced calcium flux leading to MEK-ERK activation. This work defines a critical role for aberrantly glycosylated MUC1 and identifies an activating pathway following Siglec-9 engagement.

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Somatic neural and neural crest stem cells are promising sources for cellular therapy of several neurodegenerative diseases. However, because of practical considerations such as inadequate accessibility of the source material, the application of neural crest stem cells is strictly limited. The secondary palate is a highly regenerative and heavily innervated tissue, which develops embryonically under direct contribution of neural crest cells. Here, we describe for the first time the presence of nestin-positive neural crest-related stem cells within Meissner corpuscles and Merkel cell-neurite complexes located in the hard palate of adult Wistar rats. After isolation, palatal neural crest-related stem cells (pNC-SCs) were cultivated in the presence of epidermal growth factor and fibroblast growth factor under serum-free conditions, resulting in large amounts of neurospheres. We used immunocytochemical techniques and reverse transcriptase-polymerase chain reaction to assess the expression profile of pNC-SCs. In addition to the expression of neural crest stem cell markers such as Nestin, Sox2, and p75, we detected the expression of Klf4, Oct4, and c-Myc. pNC-SCs differentiated efficiently into neuronal and glial cells. Finally, we investigated the potential expression of stemness markers within the human palate. We identified expression of stem cell markers nestin and CD133 and the transcription factors needed for reprogramming of somatic cells into pluripotent cells: Sox2, Oct4, Klf4, and c-Myc. These data show that cells isolated from palatal rugae form neurospheres, are highly plastic, and express neural crest stem cell markers. In addition, pNC-SCs may have the ability to differentiate into functional neurons and glial cells, serving as a starting point for therapeutic studies. Stem Cells 2009;27:1899–1910

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We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galβ1-3GalNAc (core 1) and mono-and disialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1-and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.

For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines.Electronic supplementary materialThe online version of this article (doi:10.1007/s00449-010-0502-y) contains supplementary material, which is available to authorized users.

The response of endothelial energy metabolism to oxygen supply was studied in cultured coronary endothelial cells from the rat at defined PO2 levels between 0.1 and 100 Torr. In the presence of glucose (5 mM), endothelial respiration (4 nmol O2.min-1.mg protein-1) was independent of the exterior PO2 greater than 3 Torr; oxygen consumption was half maximal at 0.8 Torr. At 100 Torr, lactate production was 26 nmol.min-1.mg protein-1; the decrease of the PO2 to 0.1 Torr resulted in a 2.2-fold increase in lactate production. The contents of ATP, ADP, and AMP were 21, 4, and 2 nmol/mg protein, respectively; they remained constant for 2.5-h incubations at PO2 levels between 0.1 and 100 Torr. In the presence of palmitate (100 microM) plus glutamine (0.5 mM), oxygen consumption was 8 nmol.min-1.mg protein-1 at PO2 levels greater than 3 Torr, and the half-maximal rate was again observed at 0.8 Torr. Lactate production was negligible. At PO2 levels greater than 3 Torr, the cells remained well energized. Below 3 Torr, however, the adenine nucleotide contents rapidly declined. These results demonstrate that the oxygen demand of coronary endothelial cells is low compared with the beating myocardium. In the presence of glucose, aerobic glycolysis is pronounced and the Pasteur effect small. In severe hypoxia (PO2 less than 0.1 Torr) the energetic state remained stable. In the absence of glucose, the energetic state of coronary endothelial cells is sensitive to the exterior PO2 less than 3 Torr, declining concomitantly with the decrease in respiration.