Characterization of a SILAC method for proteomic analysis of primary rat microglia

Microglia play important and dynamic roles in mediating a variety of physiological and pathological processes during the development, normal function and degeneration of the central nervous system. Application of SILAC‐based proteomic analysis would greatly facilitate the identification of cellular pathways regulating the multifaceted phenotypes of microglia. We and others have successfully SILAC‐labeled immortalized murine microglial cell lines in previous studies. In this study, we report the development and evaluation of a SILAC‐labeled primary rat microglia model. Although the isotope labeling scheme for primary microglia is drastically different from that of immortalized cell lines, our de novo and uninterrupted primary culture labeling protocol (DUP‐SILAC) resulted in sufficient incorporation of SILAC labels for mass spectrometry‐based proteomic profiling. In addition, label incorporation did not alter their morphology and response to endotoxin stimulation. Proteomic analysis of the endotoxin‐stimulated SILAC‐labeled primary microglia identified expected as well as potentially novel activation markers and pro‐inflammatory pathways that could be quantified in a more physiologically relevant cellular model system compared to immortalized cell lines. The establishment of primary microglia SILAC model will further expand our capacity for global scale proteomic profiling of pathways under various physiological and pathological conditions. Proteomic MS data are available via ProteomeXchange with identifier PXD002759.     

A proteomics approach to identifying fish cell lines

Fish cell lines are relatively easy to culture and most have simple growth requirements that make cross contamination a potential problem. Cell line contamination is not an uncommon incident in laboratories handling more than one cell line and many reports have been made on cross contamination of mammalian cell lines. Although problems of misidentification and cross-contamination of fish cell lines have rarely been reported, these are issues of concern for cell culturists that can make scientific results and their reproducibility unreliable. Proper identification of cell lines is thus crucial and protocols for routine and rapid screening are preferred. Cytogenetic evaluation, DNA fingerprinting, microsatellite analysis and PCR methods have been published for inter-species identification of many cell lines, but discerning intra-species contamination has been challenging. More complex DNA fingerprinting and hybridization techniques coupled with isoenzyme analysis have been developed to discriminate intra-species contamination, however, these require complex and time consuming procedures to enable cell identification thus are difficult to apply for routine use. A simple proteomic approach has been made to identify several fish cell lines derived from tissues of the same or differing species. Protein expression signatures (PES) of the evaluated fish cell lines have been developed using 2-DE and image analysis. A higher degree of concordance was seen among cell lines derived from rainbow trout, than from other fish species. Similar concordance was seen in cells derived from the same tissues than from other tissues within the same species. These profiles have been saved in an electronic databank and could be made available to be used for discerning the origins of the various cell lines evaluated. This proteomic approach could thus serve as an additional, valuable and reliable technique for the identification of fish cell lines.     

Characterization and comparison of the properties of sarcoma cell lines in vitro and in vivo

To expand the available tools for investigating human sarcomas, we characterized the primary properties of 22 common, uncommon, and newly characterized sarcoma cell lines representing eight different histological subtypes. Throughout the characterization process we noticed that in vitro markers and assays are poor indicators of tumorigenicity and that generated xenografts often bear little resemblance to the original histopathology. In vitro properties examined included morphology, proliferation rate, cell cycle characteristics, invasiveness, and immunohistochemical expression of p53 and phospho-AKT. In vivo properties examined included days to tumor formation in NOD/SCID mice, xenograft morphology in several locations and immunohistochemical expression of Ki67, p53 and phospho-AKT. We believe that such an in depth comparison of a large cohort of sarcoma cell lines will be useful in both designing and interpreting experiments aimed at elucidating both the molecular biology and efficacy of therapeutic agents in sarcomas. However, that data generated also suggests a small set of sarcoma cell lines may be inappropriate for generalizations regarding biological behavior of specific sarcoma subtypes. Integration of functional genomics or other more sophisticated assays of cell lines may help bridge the differences in vitro and in vivo .     

Proteomic signature of human cancer cells

We assessed proteomic profiles as biomarkers for monitoring cell phenotypes. Protein expression profiles were obtained by fluorescence two-dimensional difference gel electrophoresis (2-D-DIGE), in which quantitative ability is improved by labeling proteins with fluorescent dyes prior to electrophoresis. Integrated protein spot intensities were analyzed by a statistical approach. The proteomic data of two groups of cell lines: (1) adenocarcinoma (AC) cell lines derived from lung, pancreas and colon tissues and (2) lung cancer cell lines with different histological backgrounds, including AC, squamous cell carcinoma and small cell carcinoma, were assessed on the basis of prior biological information. Hierarchical clustering analysis and principal component analysis were used to divide the cell lines into subgroups on the basis of similarities between their protein expression profiles. The majority of cell lines were grouped according to their organ of origin or histological background. A machine-learning algorithm selected 32 protein spots that were responsible for the classification. The results indicate that proteomic data generated by 2-D-DIGE can provide a signature of essential cell phenotypes, suggesting that it might be possible to apply this technique to developing tumor markers that could identify the organ of origin of metastatic tumors and contribute to the differential diagnosis of lung cancer.     

T‐cell‐derived Hodgkin lymphoma has motility characteristics intermediate between Hodgkin and anaplastic large cell lymphoma

Classic Hodgkin lymphoma (cHL) is usually characterized by a low tumour cell content, derived from crippled germinal centre B cells. Rare cases have been described in which the tumour cells show clonal T‐cell receptor rearrangements. From a clinicopathological perspective, it is unclear if these cases should be classified as cHL or anaplastic large T‐cell lymphoma (ALCL). Since we recently observed differences in the motility of ALCL and cHL tumour cells, here, we aimed to obtain a better understanding of T‐cell‐derived cHL by investigating their global proteomic profiles and their motility. In a proteomics analysis, when only motility‐associated proteins were regarded, T‐cell‐derived cHL cell lines showed the highest similarity to ALK⁻ ALCL cell lines. In contrast, T‐cell‐derived cHL cell lines presented a very low overall motility, similar to that observed in conventional cHL. Whereas all ALCL cell lines, as well as T‐cell‐derived cHL, predominantly presented an amoeboid migration pattern with uropod at the rear, conventional cHL never presented with uropods. The migration of ALCL cell lines was strongly impaired upon application of different inhibitors. This effect was less pronounced in cHL cell lines and almost invisible in T‐cell‐derived cHL. In summary, our cell line‐derived data suggest that based on proteomics and migration behaviour, T‐cell‐derived cHL is a neoplasm that shares features with both cHL and ALCL and is not an ALCL with low tumour cell content. Complementary clinical studies on this lymphoma are warranted.     

动物细胞无血清培养基的研究与设计方法

随着哺乳动物细胞培养规模的扩大和生物药物需求的增长,基于细胞及产品特性的无血清培养基的研制已成为细胞工程领域的重要课题。运用统计学实验方法,可科学有效地考察细胞培养基中多因素、多水平间的交互作用。应用新型蛋白质组分析技术及生物芯片技术定位胞浆信号通路相关蛋白、膜表面的生长因子受体、激素受体、细胞因子受体、粘附分子等,用于确定细胞培养基中调控分子的添加组合。本文系统概述了目前无血清培养基几种新型及常用的研究和设计方法,并对其应用特点做了分析,希望为动物细胞无血清培养基的研制提供可借鉴的思路。     

Analysis of surface membrane antigens, cytoskeletal proteins and N-myc oncogene in pediatric solid malignant tumors, their diagnostic usefulness and relevant problems]

Neuroblastoma (NB), primitive neuroectodermal tumor (PNET), Ewing's sarcoma and rhabdomyosarcoma (RMS) are solid malignant tumors in childhood. Microscopically these tumors are grouped as small-round-cell tumors, and a different diagnosis is sometimes difficult. Cell surface membrane antigen, cytoskeletal protein and N-myc amplification and over-expression were analyzed in these cell lines and tumor tissues for the accurate diagnosis. NB and PNET could be distinguished from Ewing's sarcoma and RMS by the panel of monoclonal antibodies against cell surface membrane antigens. The cytoskeletal protein analysis is useful for the diagnosis of RMS and leiomyosarcoma. Alpha-smooth muscle actin and/or desmin were demonstrated in the S-type (epithelial-like) cells in 3 NB cell lines, suggesting the differentiation pathway of NB into smooth muscle cells. N-myc amplification and over-expression were observed in NB cell lines as well as one RMS cell line. The occurrence of N-myc amplification and over-expression in the RMS cell line cautions us against using N-myc as a distinguishable marker for NB.     

Effect of Glutamate Analogues on Brain Tumor Cell Lines

Glutamate analogues have been used in many different experimental approaches in neurobiology. A small number of these analogues have been classified as gliotoxic. We have examined the effect of seven glutamate analogues (five gliotoxic and two neurotoxic) on the growth and viability of four human glioma cell lines, one human medulloblastoma cell line, and one human sarcoma cell line. Aminoadipic acid and homocysteic acid predominantly affected the growth of two glioma cell lines in the presence of 4 mM glutamine. Phosphonobutyric acid predominantly affected the other two glioma cell lines and the medulloblastoma cell line in the presence of 4 mM glutamine. In medium containing no glutamine, all three analogues had marked effects on all the cell lines except the sarcoma cell line. These effects were dose dependent. We postulate that these results can in part be explained on the basis of metabolic compartmentalization.     

食管鳞癌的蛋白质组学

大部分食管鳞癌（esophageal squamous cell carcinoma, ESCC）确诊时已发展至中晚期,临床治疗效果差,是导致我国华北地区ESCC死亡率居高不下的主要原因之一.因此,亟需筛查ESCC特异性、敏感性的生物标志物,以期用于ESCC早期诊断、个体化分子靶向治疗和预后评价. 与相对稳定、携带遗传信息的基因组不同,蛋白质组具有时空变化特性,由此构成生命活动复杂性的物质基础.在病理情况下,蛋白质组能够精确反映患病组织器官的功能状态,因此为疾病的监测提供了窗口.本文总结了ESCC蛋白质组研究现状及差异表达的蛋白质谱,并探讨了ESCC候选分子标志物的潜在临床应用价值.     

Establishment and proteomic characterization of a novel cell line,NCC-UPS2-C1, derived from a patient with undifferentiated pleomorphic sarcoma

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal malignancy requiring novel therapeutic approaches to improve clinical outcome. Patient-derived cancer cell lines are an essential tool for investigating molecular mechanisms underlying cancer initiation and development; however, there is a lack of patient-derived cell lines of UPS available for research. The objective of this study was to develop a patient-derived cell model of UPS. A cell line designated NCC-UPS2-C1 was established from the primary tumor tissue of an 84-yr-old female patient with UPS. The short tandem repeat pattern of NCC-UPS2-C1 cells was identical to that of the original tumor and distinct from that of any other cell lines deposited in public cell banks. NCC-UPS2-C1 cells were maintained as a monolayer culture for over 80 passages during 30 mo and exhibited spindle-like morphology, continuous growth, and ability for spheroid formation and invasion. Proteomic profiling using mass spectrometry and functional treemap analysis revealed that the original tumor and the derived NCC-UPS2-C1 cells had similar but distinct protein expression patterns. Our results indicate that a novel UPS cell line was successfully established and could be used to study UPS development and effects of anti-cancer drugs. However, the revealed difference between proteomes of the original tumor and NCC-UPS2-C1 cells should be further investigated to determine the appropriate applications of this cell line in UPS research.     

Review: Production, characterization, and testing of banked mammalian cell substrates used to produce biological products

Summary A critical component in controlling the production of biological products derived from human and animal cell lines in the characterization and testing of banked cell substrates. The objective is to confirm the identity, purity, and suitability of these cells for manufacturing use. Quality concerns for biological products derived from cell lines originate from the presence of cellular and adventitious contaminants. Well-characterized cell banks not only permit a consistent source of production cells throughout the life of a product but also decrease the likelihood of contamination by other cell lines and adventitious agents. An important part of qualifying a cell line is choosing the appropriate testing for the presence of adventitious contaminants. The qualification of cell banks includes tests for cell identity and endogenous and adventitious microbial contaminants (bacteria, fungi, mycoplasmas, and viruses). For cells producing recombinant deoxyribonucleic acid-derived products, analysis of the expression construct at the nucleic acid level (genetic stability) is also a primary concern. The strategy for designing a safety-testing program for banked cells should be based on sound scientific principles and current regulatory guidance.     

Isolation and proteomic analysis of exosomes secreted by human cancer cells in vitro

Exosomes are 20-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro and in vivo. Since exosomes contain both RNA (mRNA and microRNA) and proteins, which can be transferred to another cell, and be functional in that new environment, these vesicles may be involved in the communication between cells. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo suggest their participation in pathological situations. Our purpose here is to describe methods for the production, purification, and proteomic characterization of exosomes derived from human cancer cells in vitro. Based on exosomes' unique lipidic composition, we have developed the new approach to increase production of exosomes by cells in vitro. Secondly, we have developed quality control by laser correlation spectroscopy for exosomal assays based on the amount of MHC class I and CD63 molecules on their surface. At last, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was used after 2D electrophoresis for the proteomic analysis of exosomes derived from cancer cell lines. This study describes the protein composition of brain tumor cell-derived exosomes in more detail.     

Role of the embryology laboratory in the human embryonic stem cell line derivation process

With the introduction of regenerative medicine and cell therapy programmes by means of human embryonic stem cells (hESC), several research centres have begun projects of derivation of hESC lines. In some stem cell banks, such as the Andalusian Stem Cell Bank, the law also permits the creation of these cell lines. Therefore, the recovery of cryopreserved embryos, their culture and the subsequent derivation to hESC lines requires a suitable embryology laboratory and specialized and highly qualified staff. Moreover, new techniques, from therapeutic nuclear transfer, need this type of laboratory and staff, too. Several International Associations have drawn up some guidelines for laboratories where embryos are manipulated and they reflect the physical space, the staff and the equipment needed in these kinds of laboratories. Nevertheless, we can see that these guidelines do not distinguish between IVF laboratories and other laboratories that obtain hESC lines, so it would be convenient to make a distinction. Following these guidelines, we have tried to draw up concurrent aspects applicable to areas of embryology within stem cell banks. So, the design and the specific implementation programmes for these areas and other research centres with this area but which do not use IVF techniques is vital to develop embryonic cell lines in optimum conditions for future therapeutic applications, although maybe it is rather premature to standardize this type of research.     

Morphology of the surface of normal and virus transformed cells in vitro]

The cell surface morphology of two cell lines--from the mink lung (Mv1Lu) and from the Kirsten sarcoma virus transformed derivate (Ki-Mv1Lu)--was studied by scanning electron microscopy. Marked differences are seen in cell morphology of these two lines at high cell densities. Mv1Lu cells at high densities had uniform flat polygonal shape with microvilli at their surface. A marked diversity in cell morphology was characteristic of Ki-Mv1Lu cells at comparable cell densities: variation in shape, in thickness degrees, and in the expression of cell surface ultrastructure (microvilli, blebs, filopodia). No dependence of Ki-Mv1Lu cell morphology from cell densities was observed. At low cell densities of Mv1Lu cells, cells with the morphology differing from the typical pattern of confluent Mv1Lu cells were seen. Morphological diversity of these cells was comparable with that of Ki-Mv1Lu cells. Nothing has been found in cell surface morphology that could be absolutely specific for transformed cells only.     

Establishment and proteomic characterization of a novel synovial sarcoma cell line,NCC-SS2-C1

Synovial sarcoma is an aggressive mesenchymal tumor, characterized by the presence of unique transfusion gene, SS18–SSX. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of SS18–SSX in relevant cellular contexts. We report the establishment and proteomic characterization of a novel synovial sarcoma cell line. Primary tissue culture was performed using tumor tissue of synovial sarcoma. The established cell line was authenticated by assessing its DNA microsatellite short tandem repeat analysis and characterized by in vitro assay. Proteomic study was achieved by mass spectrometry, and the results were analyzed by treemap. The cell line NCC-SS2-C1 was established from a primary tumor tissue of a synovial sarcoma patient. The cell line has grown well for 11 mo and has been subcultured more than 15 times. The established cells were authenticated by assessing their short tandem repeat pattern comparing with that of original tumor tissue. The cells showed polygonal in shape and formed spheroid when seeded on the low-attachment dish. Proteomic analysis revealed the molecular pathways which are unique to the original tumor tissue or the established cell line. In conclusion, a novel synovial sarcoma cell line NCC-SS2-C1 was successfully established from the primary tumor tissue. The cell line has characteristic transfusion SS18–SSX and poses aggressive in vitro growth and capability of spheroid formation. Thus, NCC-SS2-C1 cell line will be a useful tool for investigation of the mechanisms of disease and the biological role of fusion gene.     

Sensitivity of Ewing's sarcoma to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill transformed cells. We have studied the expression and functionality of the TRAIL apoptotic pathway in Ewing's sarcoma. We demonstrate that tumors from patients with Ewing's sarcoma express receptors TRAIL-R1 and -R2. Using a panel of nine Ewing's sarcoma cell lines TRAIL could induce apoptosis in seven cell lines. Preincubation with interferon-gamma rendered the two resistant cell lines sensitive. TRAIL was the most potent inducer of apoptosis when compared to Fas ligand or TNF. TRAIL-mediated apoptosis could be inhibited by various caspase-inhibitors. No difference in the surface expression of TRAIL-receptors was observed between sensitive and resistant cell lines. Also, all cell lines had similar levels of expression of Flice-like inhibitory protein (FLIP) on immunoblot. However, the two resistant cell lines had only very low level expression of caspase 8 on RNA and protein level. In summary, we show that Ewing's sarcoma expresses receptors for TRAIL, and that cells are exquisitely sensitive to TRAIL-mediated apoptosis. These results may warrant clinical trials with TRAIL in Ewing's sarcoma once the safety of TRAIL for humans has been established.     

Human Proteinpedia as a resource for clinical proteomics

Clinical proteomics is an emerging field that deals with the use of proteomic technologies for medical applications. With a major objective of identifying proteins involved in pathological processes and as potential biomarkers, this field is already gaining momentum. Consequently, clinical proteomics data are being generated at a rapid pace, although mechanisms of sharing such data with the biomedical community lag far behind. Most of these data are either provided as supplementary information through journal web sites or directly made available by the authors through their own web resources. Integration of these data within a single resource that displays information in the context of individual proteins is likely to enhance the use of proteomic data in biomedical research. Human Proteinpedia is one such portal that unifies human proteomic data under a single banner. The goal of this resource is to ultimately capture and integrate all proteomic data obtained from individual studies on normal and diseased tissues. We anticipate that harnessing of these data will help prioritize experiments related to protein targets and also permit meta-analysis to uncover molecular signatures of disease. Finally, we encourage all biomedical investigators to maximize dissemination of their valuable proteomic data to rest of the community by active participation in existing repositories such as Human Proteinpedia.     

Isobaric Tag‐Based Protein Profiling across Eight Human Cell Lines Using High‐Field Asymmetric Ion Mobility Spectrometry and Real‐Time Database Searching

A vast number of human cell lines are available for cell culture model‐based studies, and as such the potential exists for discrepancies in findings due to cell line selection. To investigate this concept, the authors determine the relative protein abundance profiles of a panel of eight diverse, but commonly studied human cell lines. This panel includes HAP1, HEK293T, HeLa, HepG2, Jurkat, Panc1, SH‐SY5Y, and SVGp12. A mass spectrometry‐based proteomics workflow designed to enhance quantitative accuracy while maintaining analytical depth is used. To this end, this strategy leverages TMTpro16‐based sample multiplexing, high‐field asymmetric ion mobility spectrometry, and real‐time database searching. The data show that the differences in the relative protein abundance profiles reflect cell line diversity. The authors also determine several hundred proteins to be highly enriched for a given cell line, and perform gene ontology and pathway analysis on these cell line‐enriched proteins. An R Shiny application is designed to query protein abundance profiles and retrieve proteins with similar patterns. The workflows used herein can be applied to additional cell lines to aid cell line selection for addressing a given scientific inquiry or for improving an experimental design.     

Single muscle fiber proteomics reveals unexpected mitochondrial specialization

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high‐sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype‐specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type‐resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.