A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human -interferon (-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.Abbreviation HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers

Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (10⁷–2×10⁷ cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

High cell density perfusion culture of hybridoma cells recycling high molecular weight components

We have developed a high cell density and high product concentration culture system recycling high molecular weight components. The production of monoclonal antibodies in high concentration was performed by this culture system with mouse human hybridoma H2 and V6 cells in serum-free defined media.The concentration of IgG after 48 days culture of H2 cells in ITES-eRDF reached 2 mg/ml and the purity of IgG in culture fluid was 61%. In addition, high molecular weight components in serum-free media, such as transferrin or BSA, could be reduced to 5% of the original concentration.     

Egg yolk lipoprotein,a new supplement for the growth of mammalian cells in serum-free medium

Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity.     

Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors

A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.Abbreviations Ag8 Mouse myeloma cell line P3-X63-Ag8.653 - BME Basal Medium Eagle - BSA Bovine Serum Albumin - DMEM Dulbecco's Modified Eagle's Medium - EDTA Ethylenediaminete-traacetic Acid - e-PC Phosphatidyl choline from egg yolk - FCS Fetal Calf Serum - FGF Fibroblast Growth Factor - GHL Glycyl-histidyl-lysine - HDL High Density Lipoprotein - HPL Human Plasma Lipid - IF 1:1 mixture of IMDM and Ham's F12 - IMDM Iscove's Modified Dulbecco's medium - LDL Low Density Lipoprotein - NS1 Mouse myeloma cell line NSI-1-Ag4-1 - PBS Phosphate Buffered Saline - s-PC Phosphatidylcholine from soy beans - s-PE Phosphatidylethanolamine from soy beans - s-lecithin lecithin from soy beans