Background: The extracellular signal-regulated kinase (ERK) pathway regulates cell growth, and is hyper-activated and associated with drug resistance in hepatocellular carcinoma (HCC). Metabolic pathways are profoundly dysregulated in HCC. Whether an altered metabolic state is linked to activated ERK pathway and drug response in HCC is unaddressed. Methods: We deprived HCC cells of glutamine to induce metabolic alterations and performed various assays, including metabolomics (with 13 C-glucose isotope tracing), microarray analysis, and cell proliferation assays. Glutamine-deprived cells were also treated with kinase inhibitors (e.g. Sorafenib, Erlotinib, U0126 amongst other MEK inhibitors). We performed bioinformatics analysis and stratification of HCC tumour microarrays to determine upregulated ERK gene signatures in patients. Findings: In a subset of HCC cells, the withdrawal of glutamine triggers a severe metabolic alteration and ERK phosphorylation (pERK). This is accompanied by resistance to the anti-proliferative effect of kinase inhibitors, despite pERK inhibition. High intracellular serine is a consistent feature of an altered metabolic state and contributes to pERK induction and the kinase inhibitor resistance. Blocking the ERK pathway facilitates cell proliferation by reprogramming metabolism, notably enhancing aerobic glycolysis. We have identified 24 highly expressed ERK gene signatures that their combined expression strongly indicates a dysregulated metabolic gene network in human HCC tissues. Interpretation: A severely compromised metabolism lead to ERK pathway induction, and primes some HCC cells to pro-survival phenotypes upon ERK pathway blockade. Our findings offer novel insights for understanding, predicting and overcoming drug resistance in liver cancer patients.

Time-resolved cell culture assays circumvent the need to set arbitrary end-points and reveal the dynamics of quality controlled experiments. However, they lead to the generation of large data sets, which can represent a complexity barrier to their use. We therefore developed the Time-Resolved Cell Culture Assay (TReCCA) Analyser program to perform standard cell assay analyses efficiently and make sophisticated in-depth analyses easily available. The functions of the program include data normalising and averaging, as well as smoothing and slope calculation, pin-pointing exact change time points. A time-resolved IC50/EC50 calculation provides a better understanding of drug toxicity over time and a more accurate drug to drug comparison. Finally the logarithmic sensor recalibration function, for sensors with an exponential calibration curve, homogenises the sensor output and enables the detection of low-scale changes. To illustrate the capabilities of the TReCCA Analyser, we performed on-line monitoring of dissolved oxygen in the culture media of the breast cancer cell line MCF-7 treated with different concentrations of the anti-cancer drug Cisplatin. The TReCCA Analyser is freely available at www.uni-heidelberg.de/fakultaeten/biowissenschaften/ipmb/biologie/woelfl/Research.html. By introducing the program, we hope to encourage more systematic use of time-resolved assays and lead researchers to fully exploit their data.

A fiber optic biosensor was constructed from Pleurotus ostreatus laccase for the detection of polyphenolic compounds. Laccase was immobilized on the surface of the commercially available fiber optic oxygen sensor spots by using 3-aminopropylsilanetriol, glutaraldehyde, and amino-modified carboxycellulose. A diffusion layer containing tetramethyl orthosilicate (TMOS), trimethoxymethylsilane (Tri-MOS), and polyvinyl alcohol (PVA) was added to the immobilized laccase layer. The consumption of oxygen as a result of laccase activity was monitored using a fiber optical measuring setup with catechol as a model substrate. The optimal enzyme amount was determined as 1.5 mg per 50 µL of enzyme layer mixture, and with one diffusion layer and at pH 6.9, optimum detection conditions were attained. The biosensors have high reproducibility, stability (at least 85 days if stored in PBS at 4 °C), and convenient measurement duration (ca. 25 min between two successive measurements). The biosensor was found to have a broad linear working range for catechol (40-600 µM) and to be applicable to a flow-through system. In summary, an easy-to-produce, reproducible, and stable laccase sensor with a broad linear working range was produced. The sensor has potential in the food industry as well as in environmental monitoring for the detection of phenolic compounds.

At Abcam we are constantly improving our manufacturing processes and adopting more robust antibody validation technologies as they are developed. Recently we have implemented the use of human knockout (KO) cell lines to interrogate the specificity of our antibodies on a large scale. This has been possible through a partnership with Horizon Discovery and the use of their KO cell lines generated using CRISPR/Cas9 technology. PTEN, is a well-known tumour suppressor that plays a critical role in the host immune response. Recent research has highlighted the importance of this protein with regards to a) lineage stability and homeostasis in Treg cells (1) and b) negative regulation of the NK cell immune response (2, 3). In both cases PTEN acts by regulation of the PI3-kinase signalling pathway. We have identified several monoclonal antibodies to both PTEN and PI3K and confirmed their specificity by KO validation. We performed this analysis in either denatured (western blot) or native (immunocytochemistry) conditions. Our KO models represent true negative controls and provide confidence that these antibodies are binding to their intended target. This technology has allowed us to develop a growing range of over 650 KO-validated antibodies, including a number that are applicable to immunology/oncology research.