Protein Profiling of Primary Human Samples for Pathway Analysis and Patient Stratification

In recent years, scientific progress has led to the advancement of precision medicine, a novel therapeutic strategy. For this purpose, molecular investigation of patient-derived samples is a necessity. Genetic analysis has been the gold standard in this field for years. However, in a cell, proteins and their posttranslational modifications lead to differences in genotype and phenotype. Therefore, the use of genetic information as basis for treatment decisions does not always translate into therapeutic benefits. The integration of proteomic approaches to further elucidate pathophysiological mechanisms is essential. Protein analysis methods need to be flexible to be used in different sample types and provide high sensitivity as well as throughput to complement these novel therapeutic approaches. The recently emerging DigiWest technology allows for detection of numerous proteins and protein variants from a single sample. Here, the DigiWest workflow was adapted and modified for protein analysis from clinically relevant sample types, such as formalin-fixed or fresh frozen tissue extracts and blood samples. A novel serum-screening platform was designed and established. Through the integration of authentic antigens, the parallel detection of immunoglobulins against different pathogenic strains of coronaviruses was achieved. Furthermore, multiplexed protein analysis from minuscule formalin-fixed and paraffin-embedded cervical punch biopsies by DigiWest was established. In vivo treatment effects of non-invasive-physical plasma, a novel therapeutic approach for treatment of cervical intraepithelial neoplasia, were evaluated and upon comparing results to in vitro cell culture experiments, general trends in treatment response could be confirmed. However, differences in protein expression profiles emphasize the need for molecular investigation of treatment effects in vivo. The potential of protein analysis of fresh frozen samples was explored by referencing snap frozen breast cancer biopsies. The multiplexed detection of several infiltrating immune cell markers by DigiWest as prognostic factor was established. A subset of co-expressed immune cell markers, revealing a positive influence on patient outcome was identified and induced changes in several pathways were detected. Overall, sample preparation, assay strategies and analysis tools were adapted to the multiplexed protein analysis of different human sample types via DigiWest and the unique potential of this approach was demonstrated

PKC-mediated phosphorylation and activation of the MEK/ERK pathway as a mechanism of acquired trastuzumab resistance in HER2-positive breast cancer

Protein expression, activation and stability are regulated through inter-connected signal transduction pathways resulting in specific cellular states. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins (phospho-protein profile) in key signal transduction pathways, in breast cancer cellular models of trastuzumab resistance. To this effect, we utilized a multiplex, bead-based protein assay, DigiWest®, to measure around 100 proteins and protein modifications using specific antibodies. The main advantage of this methodology is the quantification of multiple analytes in one sample, utilising input volumes of a normal western blot. The intrinsically trastuzumab-resistant cell line JIMT-1 showed the largest number of concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, b catenin stabilization by inhibitory phosphorylation of GSK3b, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance cellular models. The overexpression of upstream RAS/RAF, however, was confined to JIMT 1; meanwhile, in a cellular model of acquired trastuzumab resistance generated in this study (T15), entry into the ERK pathway seemed to be mostly mediated by PKCa activation. This is a novel observation and merits further investigation that can lead to new therapeutic combinations in HER2-positive breast cancer with acquired therapeutic resistance.peer-reviewe

Computer Controlled Automated Assay for Comprehensive Studies of Enzyme Kinetic Parameters

Stability and biological activity of proteins is highly dependent on their physicochemical environment. The development of realistic models of biological systems necessitates quantitative information on the response to changes of external conditions like pH, salinity and concentrations of substrates and allosteric modulators. Changes in just a few variable parameters rapidly lead to large numbers of experimental conditions, which go beyond the experimental capacity of most research groups. We implemented a computer-aided experimenting framework (“robot lab assistant”) that allows us to parameterize abstract, human-readable descriptions of micro-plate based experiments with variable parameters and execute them on a conventional 8 channel liquid handling robot fitted with a sensitive plate reader. A set of newly developed R-packages translates the instructions into machine commands, executes them, collects the data and processes it without user-interaction. By combining script-driven experimental planning, execution and data-analysis, our system can react to experimental outcomes autonomously, allowing outcome-based iterative experimental strategies. The framework was applied in a response-surface model based iterative optimization of buffer conditions and investigation of substrate, allosteric effector, pH and salt dependent activity profiles of pyruvate kinase (PYK). A diprotic model of enzyme kinetics was used to model the combined effects of changing pH and substrate concentrations. The 8 parameters of the model could be estimated from a single two-hour experiment using nonlinear least-squares regression. The model with the estimated parameters successfully predicted pH and PEP dependence of initial reaction rates, while the PEP concentration dependent shift of optimal pH could only be reproduced with a set of manually tweaked parameters. Differences between model-predictions and experimental observations at low pH suggest additional protonation-sites at the enzyme or substrates critical for enzymatic activity. The developed framework is a powerful tool to investigate enzyme reaction specifics and explore biological system behaviour in a wide range of experimental conditions

Targeted Protein Profiling of In Vivo NIPP-Treated Tissues Using DigiWest Technology

Non-invasive physical plasma (NIPP) is a novel therapeutic tool, currently being evaluated for the treatment of cancer and precancerous lesions in gynecology and other disciplines. Additionally, patients with cervical intraepithelial neoplasia (CIN) may benefit from NIPP treatment due to its non-invasive, side-effect-free, and tissue-sparing character. However, the molecular impact of in vivo NIPP treatment needs to be further investigated. For this purpose, usually only very small tissue biopsies are available after NIPP treatment. Here, we adapted DigiWest technology, a high-throughput bead-based Western blot, for the analysis of formalin-fixed paraffin-embedded (FFPE) cervical punch biopsies with a minimal sample amount. We investigated the molecular effects of NIPP treatment directly after (0 h) and 24 h after in vivo application. Results were compared to in vitro NIPP-treated human malignant cervical cells. NIPP effects were primarily based on an inhibitory impact on the cell cycle and cell growth factors. DigiWest technology was suitable for detailed protein profiling of small, primary FFPE biopsies

Cell Type-Specific Anti-Adhesion Properties of Peritoneal Cell Treatment with Plasma-Activated Media (PAM)

Postoperative abdominal adhesions are responsible for serious clinical disorders. Administration of plasma-activated media (PAM) to cell type-specific modulated proliferation and protein biosynthesis is a promising therapeutic strategy to prevent pathological cell responses in the context of wound healing disorders. We analyzed PAM as a therapeutic option based on cell type-specific anti-adhesive responses. Primary human peritoneal fibroblasts and mesothelial cells were isolated, characterized and exposed to different PAM dosages. Cell type-specific PAM effects on different cell components were identified by contact- and marker-independent Raman imaging, followed by thorough validation by specific molecular biological methods. The investigation revealed cell type-specific molecular responses after PAM treatment, including significant cell growth retardation in peritoneal fibroblasts due to transient DNA damage, cell cycle arrest and apoptosis. We identified a therapeutic dose window wherein specifically pro-adhesive peritoneal fibroblasts were targeted, whereas peritoneal mesothelial cells retained their anti-adhesive potential of epithelial wound closure. Finally, we demonstrate that PAM treatment of peritoneal fibroblasts reduced the expression and secretion of pro-adhesive cytokines and extracellular matrix proteins. Altogether, we provide insights into biochemical PAM mechanisms which lead to cell type-specific pro-therapeutic cell responses. This may open the door for the prevention of pro-adhesive clinical disorders