Skarphedinn Halldorsson

Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices

Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture.

Ion mobility derived collision cross sections to support metabolomics applications.

Metabolomics is a rapidly evolving analytical approach in life and health sciences. The structural elucidation of the metabolites of interest remains a major analytical challenge in the metabolomics workflow. Here, we investigate the use of ion mobility as a tool to aid metabolite identification. Ion mobility allows for the measurement of the rotationally averaged collision cross-section (CCS), which gives information about the ionic shape of a molecule in the gas phase. We measured the CCSs of 125 common metabolites using traveling-wave ion mobility-mass spectrometry (TW-IM-MS). CCS measurements were highly reproducible on instruments located in three independent laboratories (RSD < 5% for 99%). We also determined the reproducibility of CCS measurements in various biological matrixes including urine, plasma, platelets, and red blood cells using ultra performance liquid chromatography (UPLC) coupled with TW-IM-MS. The mean RSD was < 2% for 97% of the CCS values, compared to 80% of retention times. Finally, as proof of concept, we used UPLC–TW-IM-MS to compare the cellular metabolome of epithelial and mesenchymal cells, an in vitro model used to study cancer development. Experimentally determined and computationally derived CCS values were used as orthogonal analytical parameters in combination with retention time and accurate mass information to confirm the identity of key metabolites potentially involved in cancer. Thus, our results indicate that adding CCS data to searchable databases and to routine metabolomics workflows will increase the identification confidence compared to traditional analytical approaches.

Azithromycin Maintains Airway Epithelial Integrity during Pseudomonas aeruginosa Infection

Tight junctions (TJs) play a key role in maintaining bronchial epithelial integrity, including apical-basolateral polarity and paracellular trafficking. Patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) often suffer from chronic infections by the opportunistic Gram-negative bacterium Pseudomonas aeruginosa, which produces multiple virulence factors, including rhamnolipids. The macrolide antibiotic azithromycin (azm) has been shown to improve lung function in patients with CF without reducing the bacterial count within the lung. However, the mechanism of this effect is still debated. It has previously been shown that azm increased transepithelial electrical resistance (TER) in a bronchial epithelial cell line. In this study we used an air-liquid interface model of human airway epithelia and measured TER, changes in TJ expression and architecture after exposure to live P. aeruginosa PAO1, and PAO1-Deltarhl which is a PAO1 mutant lacking rhlA and rhlB, which encode key enzymes for rhamnolipid production. In addition, the cells were challenged with bacterial culture medium conditioned by these strains, purified rhamnolipids, or synthetic 3O-C(12)-HSL. Virulence factors secreted by P. aeruginosa reduced TER and caused TJ rearrangement in the bronchial epithelium, exposing the epithelium to further bacterial infiltration. Pretreatment of the bronchial epithelium with azm attenuated this effect and facilitated epithelial recovery. These data suggest that azm protects the bronchial epithelium during P. aeruginosa infection independent of antimicrobial activity, and could explain in part the beneficial results seen in clinical trials of patients with CF.

deltaNp63 Has a Role in Maintaining Epithelial Integrity in Airway Epithelium

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

Real-time optical pH measurement in a standard microfluidic cell culture system

The rapid growth of microfluidic cell culturing in biological and biomedical research and industry calls for fast, non-invasive and reliable methods of evaluating conditions such as pH inside a microfluidic system. We show that by careful calibration it is possible to measure pH within microfluidic chambers with high accuracy and precision, using a direct single-pass measurement of light absorption in a commercially available phenol-red-containing cell culture medium. The measurement is carried out using a standard laboratory microscope and, contrary to previously reported methods, requires no modification of the microfluidic device design. We demonstrate the validity of this method by measuring absorption of light transmitted through 30-micrometer thick microfluidic chambers, using an inverted microscope fitted with a scientific-grade digital camera and two bandpass filters. In the pH range of 7-8, our measurements have a standard deviation and absolute error below 0.05 for a measurement volume smaller than 4 nL.

EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT.

Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend.

Metabolic re-wiring of isogenic breast epithelial cell lines following epithelial to mesenchymal transition

Epithelial to mesenchymal transition (EMT) has implications in tumor progression and metastasis. Metabolic alterations have been described in cancer development but studies focused on the metabolic re-wiring that takes place during EMT are still limited. We performed metabolomics profiling of a breast epithelial cell line and its EMT derived mesenchymal phenotype to create genome-scale metabolic models descriptive of both cell lines. Glycolysis and OXPHOS were higher in the epithelial phenotype while amino acid anaplerosis and fatty acid oxidation fueled the mesenchymal phenotype. Through comparative bioinformatics analysis, PPAR-γ1, PPAR- γ2 and AP-1 were found to be the most influential transcription factors associated with metabolic re-wiring. In silico gene essentiality analysis predicts that the LAT1 neutral amino acid transporter is essential for mesenchymal cell survival. Our results define metabolic traits that distinguish an EMT derived mesenchymal cell line from its epithelial progenitor and may have implications in cancer progression and metastasis. Furthermore, the tools presented here can aid in identifying critical metabolic nodes that may serve as therapeutic targets aiming to prevent EMT and inhibit metastatic dissemination.

Altered plasmalogen content and fatty acid saturation following epithelial to mesenchymal transition in breast epithelial cell lines

Epithelial to mesenchymal transition (EMT) is a developmental event characterized by phenotypic switching from a polarized epithelial phenotype to an unpolarized mesenchymal phenotype. Changes to plasma membrane function accompany EMT yet the differences in lipid composition of cells that have undergone EMT are relatively unexplored. To address this the lipidome of two cell models of EMT in breast epithelial tissue, D492 and HMLE, were analyzed by untargeted LC-MS. Detected masses were identified and their abundance was compared through multivariate statistical analysis. Considerable concordance was observed in eight lipid components between epithelial and mesenchymal cells in both cell models. Specifically, an increase in phosphatidylcholine and triacylglycerol were found to accompany EMT while phosphatidylcholine- and phosphatidylethanolamine plasmalogens, as well as diacylglycerols decreased. The most abundant fatty acid lengths were C16 and C18 but mesenchymal cells had on average shorter and more unsaturated fatty acids. The results are consistent with enhanced cell mobility post EMT and reflect a consequence of oxidative stress pre- and post EMT in breast epithelial tissue.

Abstract 4412: Metabolic reprogramming in EMT - targeting regulatory nodes in mesenchymal cells

To combat cancer we have to avoid development of resistant and metastatic disease. Breast cancer cells can switch from an epithelial to mesenchymal phenotype through a process called epithelial to mesenchymal transition/EMT. Emerging evidence suggests that this process is vital to avoid treatment pressure and to gain metastatic capacity. Furthermore, recent literature shows that metabolic reprogramming is an essential attribute of cellular plasticity. Metabolic targeting could therefore be an attractive possibility to prevent development of resistance and metastatic dissemination. Here we tried to understand the metabolic phenotype of EMT and the mechanisms linking the metabolic programs to cellular plasticity. We also aimed to unravel compensatory metabolic pathways and use the metabolic inhibitors to sensitize breast cancer cells to conventional therapy. To that end we have investigated the metabolic signature of the D492 EMT cell model. The D492 cell line, established from human breast epithelial progenitor cells, has retained stem cell characteristics and has the ability to undergo EMT upon stromal (endothelial) influence, forming the mesenchymal D492M cells. Thus, D492 cell system has preserved the natural flexibility of breast epithelial progenitor cells, and constitutes a unique platform to unravel the factors responsible for stromal cell-induced cellular plasticity. We show that metabolic reprogramming is essential for induction of the mesenchymal phenotype using metabolomic profiling. Using Ultra performance liquid chromatography Mass Spectrometry and gene expression profiling we have created genome-scale metabolic models of D492 and D492M. Our data show that glycolytic flux and oxidative phosphorylation is higher in D492, however, D492M cells rely more on amino acid anaplerosis and fatty acid oxidation to fuel the TCA cycle. Glutamine and glucose tracing using NMR will give further insight into the difference in metabolism between the two cell lines. We have used these data to find metabolic targets that lock the cells in the epithelial state or identify the means to induce lethality in the mesenchymal cells. Using the metabolic rewiring of EMT in the D492 cell model we can understand the mechanisms responsible for treatment resistance, identify compensatory metabolic pathways during treatment and find metabolic inhibitors that will sensitize BC cells to conventional therapy. Citation Format: Bylgja Hilmarsdottir, Skarphedinn Halldorsson, Maria T. Grinde, Anna Barkovskaya, Solveig Pettersen, Thorarinn Gudjonsson, Siver A. Moestue, Ottar Rolfsson, Gunhild M. Maelandsmo. Metabolic reprogramming in EMT - targeting regulatory nodes in mesenchymal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4412. doi:10.1158/1538-7445.AM2017-4412

Abstract 1021: Lipidomic analysis: a powerful tool for evaluating lipid metabolism in cultured cancer cells

Lipids have numerous functions in biological processes, structural as well as regulatory. Cancer cells show differences from healthy normal cells in their metabolism, including lipid metabolism, which contribute to their survival and growth. Overexpression of fatty acid synthase (FASN) is commonly observed in cancer cells and there is also evidence of other lipid pathways being changed in tumor cells. The general aim of this project is to establish a lipidomic based UPLC-QToF method for evaluation of lipid composition in cultured cells of normal and cancerous origin using electrospray quadrupole traveling wave ion mobility Q-ToF mass spectrometry. Cell lines were selected based on their lipid metabolism, including three breast cancer cell lines: SK-Br-3 (overexpresses FASN), T47D (TP53-mutated), MCF7 (estrogen receptor-positive); and one pancreatic cancer cell line, ASPC-1(overexpresses 5-and 12-lipoxygenases). Furthermore, we analyzed the immortalized breast epithelial stem cell line D492 along with its subline, D492M, that has undergone epithelial to mesenchymal transition (EMT). Principal component analysis (PCA) revealed distinct differences in lipid composition between all the cell lines tested, each cell line showed a clear cluster of lipid components and patterns that were reproducible between experiments. The Sk-Br-3 was most clearly separated from the other two breast cancer cell lines. Further analysis revealed significant differences in levels of phosphocholins (PC) that are elevated in Sk-Br-3compared to other cell lines. Further investigations of the data have led to identification of certain PC that are different between cell lines. Significant differences were detected in the lipid composition of D492 and D492M. In conclusion, different levels of lipid-synthesizing enzymes are reflected in distinct lipid profiles in breast cancer cells. The observed differences in lipid profiles of the breast epithelial cell line and its mesenchymal-like subline may provide an insight into membrane changes associated with EMT and help evaluate lipidomes of tumor samples from patients in terms of invasive potential. Citation Format: Finnur F. Eiriksson, Manuela Magnusdottir, Skarphedinn Halldorsson, Margret Thorsteinsdottir, Helga M. Ogmundsdottir. Lipidomic analysis: a powerful tool for evaluating lipid metabolism in cultured cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1021.