scholarly journals Integrated metabolomics and transcriptomics analysis of monolayer and neurospheres from glioblastoma cells

2020 ◽  
Author(s):  
Joana Peixoto ◽  
Sudha Janaki-Raman ◽  
Lisa Schlicker ◽  
Werner Schmitz ◽  
Susanne Walz ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Metabolic Pathways ◽  
Culture Conditions ◽  
Cellular Systems ◽  
Metabolic Reprogramming ◽  
Arginine Biosynthesis ◽  
Science Data ◽  
Data Set ◽  
Metabolome Data

Abstract Background: Altered metabolism is a hallmark of cancer and metabolic reprogramming can regulate several malignant properties to drive tumourigenesis. Metabolic processes contribute to carcinogenesis by modulating proliferation, survival and differentiation. Here, we studied how metabolic pathways are deregulated in cancer stem-like cells, a specific pool of cells that is thought to be responsible for cancer growth and recurrence. Cancer stem-like cells are particularly relevant in glioblastoma (GBM), the most lethal form of primary brain tumours.Methods: We have analysed the transcriptome and metabolome of an established GBM cell line (U87) and a patient-derived GBM stem-like cell line (NCH644) exposed to neurosphere or monolayer culture conditions. By integrating transcriptome and metabolome data, we identified key metabolic pathways and gene signatures that are associated with stem-like and differentiated states in GBM cells.Results: By principal component analysis and hierarchical clustering, we demonstrated that neurospheres and monolayer cells differ substantially in their metabolism and gene regulation. Furthermore, by performing a joint pathway analysis of transcriptome and metabolome data, we found that neurosphere culture conditions induce a similar metabolic rewiring in the two cellular systems and significantly regulate the same metabolic pathways. Finally, arginine biosynthesis was identified as the most significantly regulated pathway in neurospheres from both cell lines, although individual nodes of this pathway were distinctly regulated in the two cellular systems.Conclusions: Neurosphere conditions, as opposed to monolayer conditions, cause a transcriptomic and metabolic rewiring that may be crucial for the regulation of stem-like features. Arginine biosynthesis may be a key metabolic pathway in stemness regulation, by supporting the specific needs of the different cell populations. Different GBM cell lines show distinct regulation of these metabolic pathways, which are crucial to direct metabolites towards nucleotide or arginine synthesis, respectively. Finally, as part of open science data, the data set generated is of great value as a resource for the scientific community.

scholarly journals Integrated Metabolomics and Transcriptomics Analysis of Monolayer and Neurospheres from Established Glioblastoma Cell Lines

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1327
Author(s):  
Joana Peixoto ◽  
Sudha Janaki-Raman ◽  
Lisa Schlicker ◽  
Werner Schmitz ◽  
Susanne Walz ◽  
...  
Keyword(s):  
Cell Line ◽  
Metabolic Pathway ◽  
Metabolic Pathways ◽  
Monolayer Culture ◽  
Culture Conditions ◽  
Cellular Systems ◽  
Arginine Biosynthesis ◽  
Primary Brain Tumours ◽  
Relevant Role ◽  
Metabolome Data

Altered metabolic processes contribute to carcinogenesis by modulating proliferation, survival and differentiation. Tumours are composed of different cell populations, with cancer stem-like cells being one of the most prominent examples. This specific pool of cells is thought to be responsible for cancer growth and recurrence and plays a particularly relevant role in glioblastoma (GBM), the most lethal form of primary brain tumours. Here, we have analysed the transcriptome and metabolome of an established GBM cell line (U87) and a patient-derived GBM stem-like cell line (NCH644) exposed to neurosphere or monolayer culture conditions. By integrating transcriptome and metabolome data, we identified key metabolic pathways and gene signatures that are associated with stem-like and differentiated states in GBM cells, and demonstrated that neurospheres and monolayer cells differ substantially in their metabolism and gene regulation. Furthermore, arginine biosynthesis was identified as the most significantly regulated pathway in neurospheres, although individual nodes of this pathway were distinctly regulated in the two cellular systems. Neurosphere conditions, as opposed to monolayer conditions, cause a transcriptomic and metabolic rewiring that may be crucial for the regulation of stem-like features, where arginine biosynthesis may be a key metabolic pathway. Additionally, TCGA data from GBM patients showed significant regulation of specific components of the arginine biosynthesis pathway, providing further evidence for the importance of this metabolic pathway in GBM.

scholarly journals Growth properties and alloantigenic expression of murine lymphoblastoid cell lines.

1975 ◽  
Vol 142 (2) ◽  
pp. 378-390 ◽  
Author(s):  
R K Zwerner ◽  
R T Acton
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Fetal Bovine Serum ◽  
Horse Serum ◽  
Culture Conditions ◽  
Lymphoblastoid Cell Lines ◽  
Stationary Growth ◽  
Growth Properties ◽  
Fetal Bovine ◽  
Logarithmic Growth

Murine lymphoblastoid cell lines were evaluated for their expression of Thy-1 and thymus leukemia (TL) differentiation alloantigens. Two culture conditions were shown to affect this expression. Cells grown in fetal bovine serum (FBS)-enriched medium expressed up to 15 times the amount of TL as cells grown in horse serum (HS)-enriched medium. Thy-1 expression was less affected by the type of serum used for culture. The phase of growth when the cells were harvested, was demonstrated to affect the expression of Thy-1. The expression of Thy-1.2 for one cell line examined, L-251A, during logarithmic growth was threefold greater than cells collected during either lag or stationary growth. When culture conditions were standardized a ranking of the amount of Thy-1 and TL expressed by several cell lines was made. All cell lines, except one, L-1210, expressed Thy-1. There was a 450-fold difference in the expression of Thy-1 between the cell lines evaluated. Seven cell lines expressed TL-1,2,3 with a ninefold difference in the amount of expression. The L-251A cell line was cultured in a 14 liter fermentor for a 26 day period. During this time TL and Thy-1 expression did not vary significantly, demonstrating that lymphoblastoid cell lines can be cultured on a continuous basis and will continue to express their surface alloantigens.

scholarly journals Ranking small molecules by how much they preferentially inhibit the growth of cancer cell lines with either BRAF or KRAS oncogene mutations

2014 ◽  
Author(s):  
James Langham
Keyword(s):  
Growth Inhibition ◽  
Small Molecules ◽  
Cell Line ◽  
Cell Lines ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Braf Inhibitor ◽  
Data Set ◽  
Cytidine Analogs ◽  
Knowledge Of Cancer

We were interested in the question of whether it might be possible to use knowledge of cancer-related mutations in the cell lines of the NCI60 screening data set to identify small molecules that preferentially inhibit the growth of cell lines containing either BRAF or KRAS oncogene mutations. Our hypothesis was that this cell line mutation knowledge could help to identify small molecules that were more likely to preferentially inhibit growth of cell lines with a particular mutation. It seems that any such molecules might be further investigated to try to better understand the molecular mechanisms of growth inhibition. We defined a quantity, \(\text{Diff}_{\text{mut}}\), that estimates how much more a given small molecule inhibits cell lines with a mutation of interest than cell lines without that mutation. We ranked the small molecules in descending order of \(\text{Diff}_{\text{mut}}\) and then tried to explain whether the ranking of the highest ranked molecules made sense in terms of independent facts about these molecules. This method showed the BRAF inhibitor vemurafenib to be highly ranked in the BRAF ranking. The cytidine analog cytarabine was found to be highly ranked in the KRAS ranking. Other cytidine analogs were also found to be highly ranked with respect to KRAS.

scholarly journals Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

2020 ◽  
Author(s):  
Thailín Lao-González ◽  
Alexi Bueno Soler ◽  
Arnelys Duran Hernandez ◽  
Katya Sosa Aguiar ◽  
Luis Eduardo Hinojosa Puerta ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Culture Media ◽  
Screening Strategy ◽  
Culture Conditions ◽  
Host Cells ◽  
Selection Strategy ◽  
Cell Line Development ◽  
Early Screening ◽  
Gene Optimization

Abstract The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

scholarly journals Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thailin Lao-Gonzalez ◽  
Alexi Bueno-Soler ◽  
Arnelys Duran-Hernandez ◽  
Katya Sosa-Aguiar ◽  
Luis Eduardo Hinojosa-Puerta ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Culture Media ◽  
Screening Strategy ◽  
Culture Conditions ◽  
Host Cells ◽  
Selection Strategy ◽  
Cell Line Development ◽  
Early Screening ◽  
Gene Optimization

AbstractThe high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

scholarly journals Propagation of Arthropod-Borne Rickettsia spp. in Two Mosquito Cell Lines

2007 ◽  
Vol 73 (20) ◽  
pp. 6637-6643 ◽  
Author(s):  
Joyce M. Sakamoto ◽  
Abdu F. Azad
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Spotted Fever ◽  
Culture Conditions ◽  
Spotted Fever Group ◽  
Rickettsia Felis ◽  
Transitional Group ◽  
Mosquito Cell Lines

ABSTRACT Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.

scholarly journals Potentially Novel Candidate Biomarkers for Head and Neck Squamous Cell Carcinoma Identified Using an Integrated Cell Line-based Discovery Strategy

2012 ◽  
Vol 11 (11) ◽  
pp. 1404-1415 ◽  
Author(s):  
Lusia Sepiashvili ◽  
Angela Hui ◽  
Vladimir Ignatchenko ◽  
Willa Shi ◽  
Susie Su ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Head And Neck ◽  
Cell Line ◽  
Cell Lines ◽  
Western Blotting ◽  
Squamous Cell ◽  
Control Cell ◽  
Data Set ◽  
Qrt Pcr ◽  
Increased Risk

Head and neck squamous cell carcinomas (HNSCC) can arise from the oral cavity, oropharynx, larynx or hypopharynx, and is the sixth leading cancer by incidence worldwide. The 5-year survival rate of HNSCC patients remains static at 40–60%. Hence, biomarkers which can improve detection of HNSCC or early recurrences should improve clinical outcome. Mass spectrometry-based proteomics methods have emerged as promising approaches for biomarker discovery. As one approach, mass-spectrometric identification of proteins shed or secreted from cancer cells can contribute to the identification of potential biomarkers for HNSCC and our understanding of tumor behavior. In the current study, mass spectrometry-based proteomic profiling was performed on the conditioned media (i.e. secretome) of head and neck cancer (HNC) cell lines (FaDu, UTSCC8 and UTSCC42a) in addition to gene expression microarrays to identify over-expressed transcripts in the HNSCC cells in comparison to a normal control cell line. This integrated data set was systematically mined using publicly available resources (Human Protein Atlas and published proteomic/transcriptomic data) to prioritize putative candidates for validation. Subsequently, quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and ELISAs were performed to verify selected markers. Our integrated analyses identified 90 putative protein biomarkers that were secreted or shed to the extracellular space and over-expressed in HNSCC cell lines, relative to controls. Subsequently, the over-expression of five markers was verified in vitro at the transcriptional and translational levels using qRT-PCR and Western blotting, respectively. IHC-based validation conducted in two independent cohorts comprising of 40 and 39 HNSCC biopsies revealed that high tumor expression of PLAU, IGFBP7, MMP14 and THBS1 were associated with inferior disease-free survival, and increased risk of disease progression or relapse. Furthermore, as demonstrated using ELISAs, circulating levels of PLAU and IGFBP7 were significantly higher in the plasma of HNSCC patients compared with healthy individuals.

scholarly journals Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

2015 ◽  
Vol 35 (1) ◽  
pp. 305-314 ◽  
Author(s):  
Tobias Hannes ◽  
Marie Wolff ◽  
Michael Xavier Doss ◽  
Kurt Pfannkuche ◽  
Moritz Haustein ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Cardiac Development ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Cardiac Differentiation ◽  
Multielectrode Arrays ◽  
Electrophysiological Properties

Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs) requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs) of different murine ESC lines. Methods: Two wild-type (D3 and R1) and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7) were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP) and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC) promoter. Action potentials (APs) were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD) as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

scholarly journals Meta-Analysis of Microarray Expression Studies on Metformin in Cancer Cell Lines

2019 ◽  
Vol 20 (13) ◽  
pp. 3173 ◽  
Author(s):  
Hans-Juergen Schulten ◽  
Sherin Bakhashab
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Nucleic Acid ◽  
Cell Lines ◽  
Cancer Cell ◽  
Metabolic Pathways ◽  
P Value ◽  
Nucleic Acid Binding ◽  
Data Set ◽  
Microarray Expression

Several studies have demonstrated that metformin (MTF) acts with variable efficiency as an anticancer agent. The pleiotropic anticancer effects of MTF on cancer cells have not been fully explored yet. By interrogating the Gene Expression Omnibus (GEO) for microarray expression data, we identified eight eligible submissions, representing five different studies, that employed various conditions including different cell lines, MTF concentrations, treatment durations, and cellular components. A compilation of the data sets of 13 different conditions contained 443 repeatedly up- and 387 repeatedly down-regulated genes; the majority of these 830 differentially expressed genes (DEGs) were associated with higher MTF concentrations and longer MTF treatment. The most frequently upregulated genes include DNA damage inducible transcript 4 (DDIT4), chromodomain helicase DNA binding protein 2 (CHD2), endoplasmic reticulum to nucleus signaling 1 (ERN1), and growth differentiation factor 15 (GDF15). The most commonly downregulated genes include arrestin domain containing 4 (ARRDC4), and thioredoxin interacting protein (TXNIP). The most significantly (p-value < 0.05, Fisher’s exact test) overrepresented protein class was entitled, nucleic acid binding. Cholesterol biosynthesis and other metabolic pathways were specifically affected by downregulated pathway molecules. In addition, cell cycle pathways were significantly related to the data set. Generated networks were significantly related to, e.g., carbohydrate and lipid metabolism, cancer, cell cycle, and DNA replication, recombination, and repair. A second compilation comprised genes that were at least under one condition up- and in at least another condition down-regulated. Herein, the most frequently deregulated genes include nuclear paraspeckle assembly transcript 1 (NEAT1) and insulin induced gene 1 (INSIG1). The most significantly overrepresented protein classes in this compilation were entitled, nucleic acid binding, ubiquitin-protein ligase, and mRNA processing factor. In conclusion, this study provides a comprehensive list of deregulated genes and biofunctions related to in vitro MTF application and individual responses to different conditions. Biofunctions affected by MTF include, e.g., cholesterol synthesis and other metabolic pathways, cell cycle, and DNA replication, recombination, and repair. These findings can assist in defining the conditions in which MTF exerts additive or synergistic effects in cancer treatment.

scholarly journals CBMT-39. TARGETING METABOLIC PATHWAYS IN HYPOXIA FOR GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi41-vi41
Author(s):  
Aleksandra Gruslova ◽  
Alessia Lodi ◽  
Mengxing Li ◽  
Mei Zhou ◽  
Michael Garcia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Overall Survival ◽  
Cell Line ◽  
Cell Lines ◽  
Metabolic Pathways ◽  
Progression Free Survival ◽  
Metabolic Adaptation ◽  
Metabolic Resistance ◽  
Free Survival ◽  
Hypoxic Volume

Abstract BACKGROUND Metabolic adaptation to hypoxia is a crucial consideration in combating anti-angiogenic resistance. We previously explored the potential of targeting the peroxisomal fatty acid oxidation (FAO) pathway and observed higher potency to peroxisomal FAO inhibitor thioridazine in hypoxia in vitro. In this study we further examine the effects of peroxisomal FAO enzyme ACOX1, looking at differences in mitochondrial and peroxisomal FAO in hypoxia between subtypes, and explore additional metabolic pathways that can be potentially further studied. METHODS We utilized a CRISPR doxycycline-inducible U251 knockout cell line for the peroxisomal FAO gene ACOX1 and examined growth and viability in 2 weeks hypoxia (2% O2). We analyzed the gene expression of 13 peroxisomal and mitochondrial FAO enzymes in six different patient-derived cell lines. Finally, we characterized serum metabolites that are associated with tumor hypoxic volume, progression-free survival, and overall survival in patients undergoing clinical trial for TH302 and bevacizumab with bevacizumab refractory tumors. RESULTS We observed decreased cell growth and viability (p < .03) specifically in hypoxia but not normoxia with ACOX1 knock out. We saw some changes in gene expression (|ΔΔCt| >1) in hypoxia for all genes, which differed between cell lines. ACOX1 and CPT1A expression were strongly decreased (|ΔΔCt| >2) while ACADSB expression was strongly increased for the proneural cell line. ACOX1 expression was strongly decreased in our most thioridazine-sensitive cell line. ACOX2, ACADVL, CPT1A, CPT1C, and DECR2 expression were strongly increased in one of the proneural cell lines. Twelve polar metabolites were positively or negatively correlated (|r| >.4) with both hypoxic volume and either overall survival or progression-free survival. CONCLUSION Peroxisomal FAO and other metabolic pathways may be essential to target in order combat metabolic resistance during anti-angiogenic therapy. Better understanding differences in metabolism in different tumor environments will help determine which targets will be most therapeutically useful.

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