(1048-A) Development of a MALDI-TOF MS high-throughput capable screening assay for iPSC macrophage phenotyping and drug compound characterization
Monday, February 5, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Over the past decade, there has been a significant demand to develop rapid screening assays that offer label-free, physiologically relevant readouts. Among these techniques, MALDI-TOF MS has played a pivotal role for expanding the label-free biochemical assay capability within the high-throughput (HT) screening environment. Looking forward, we anticipate growing demand for cell-based assays capable of efficiently assessing the effectiveness of drug compounds or stimuli in a HT manner. Despite the existence of such workflows for prokaryotic cells, little progress has been achieved in establishing methodologies to analyze eukaryotic cell models, particularly with respect to primary cells. Here, I will present the development of a novel, fast, and robust workflow that distinguishes primary macrophage cell phenotypes based on cell-specific fingerprints in the metabolite and lipid mass region of the MALDI-TOF MS spectrum. This method will prove invaluable in the field of drug discovery, here used to facilitate the screening of small molecule compounds that act on targets in the macrophage activation pathways. The model utilizes induced pluripotent stem cell derived macrophages (iPSCM) from multiple human donors that are stimulated into a pro-inflammatory phenotype by using bacterial and cytokine stimuli. In brief, the cells were prepared and treated with the compounds, after which they were frozen, lysed and spotted onto the MALDI-TOF MS target plate. The matrix was spotted onto the target plate once the samples were dried. The samples were dried again and acquired by MALDI-TOF MS at a speed of one second per sample. We implemented the use of liquid handling automation equipment at different stages which enabled cell culture in 96 well plates and data acquisition from as little as 4000 cells. Optimization was undertaken in three main areas: cell culture, sample preparation and MALDI-TOF MS analysis. A wide range of cell lysis buffers, encompassing physiological and organic solvents, were tested. The MALDI-TOF MS acquisition was improved by evaluating sample applications, matrices, and acquisition modes. Using the optimized method, we obtained nearly two distinct populations: non-inflammatory and pro-inflammatory cell phenotypes across multiple human donors in PCA. The workflow was miniaturized and adapted for compatibility with automation equipment, enabling us to show robust and reproducible separation between iPSCM phenotypes. To evaluate the screening potential of the assay, two proof-of concept screenings are planned: firstly, a set of 90 compounds with broad, known mechanisms of action (MoA) and secondly, a curated collection of inhibitors targeting both upstream and downstream inflammatory signaling pathways. These screenings will evaluate assay performance across a broad range of MoAs but also reflect more subtle changes. This work will be followed by a platform performance comparison against conventional label-based methods and will be combined with proteomics results to gain a deeper understanding of the cell phenotypes.