Automated biomarker identification in 3D cell models using microfluidics-based Pu·MA System and CellVoyager CQ1 confocal imaging system

Physiologically relevant 3D cell models are essential for pre-clinical research and drug discovery because they better represent physiological processes and disease phenotypes. Biomarker studies have become a cornerstone in preventive and personalized medicine. There is an increased interest in using cellular biomarkers to complement genomic ones. Development of functional assays and identification of cellular biomarkers in 3D models is critical as they are expected to have a higher clinical importance than in 2D culture systems. Immunofluorescence staining (IF) is a widely used method to identify and quantify biomarker expressions and their cellular localizations. One of the challenges faced by researchers is that IF staining of delicate spheroids/organoids tend to be manual and tedious. Culture systems like hanging drop or ultra-low attachment are difficult to handle for IF staining due to a risk of losing or damaging spheres. There is a further technical challenge of plate compatibility and locating spheroids for confocal microscopy, which is typically used for imaging of stained spheres. The commonly used approach of sphere embedding, and sectioning is cumbersome, time consuming and prone to errors. We present a breakthrough advancement in biomarker staining and detection – the automated immunofluorescence staining in 3D cell models using our proprietary microfluidic-based Pu·MA System combined with high resolution confocal imaging technology from Yokogawa. In this study we demonstrate the application of this automated workflow in two different models: MCF7 breast cancer spheres and TU-BcX-4IC (4IC) patient-derived triple negative breast cancer tumoroids. We investigated the expression of E-cadherin (an epithelial cell-cell adhesion protein), Vimentin (intermediate filament protein in mesenchymal cells) and F-actin (cytoskeletal component). The staining was performed in two different extracellular matrices: Matrigel and Xeno-free engineered hydrogel Vitrogel demonstrating the compatibility of the Pu·MA Flowchip with different ECMs. The workflow consists of 1) loading of spheres to the flowchip, 2) filling up the flowchip with reagents, 3) automated execution of IF staining steps and 4) confocal high-resolution imaging using CellVoyager CQ1 System within the flowchip. Confocal images were acquired with a 20X long working distance dry objective using 405nm (Nuclei Hoechst), 488nm (E-Cadherin or Vimentin) and 561nm (Phalloidin) channels. A 120 µm z-stack of images separated by 10 µm was acquired. The epithelial-like and low malignant MCF-7 cells shows intense peripheral E-cadherin staining prevalent at cell-cell contact areas, and no detectable expression of Vimentin. The highly aggressive 4IC tumoroids showed loss of E-cadherin expression while being positive for Vimentin. F-actin distribution and organization was detected by Phalloidin staining. The workflow presented here has high utility and value for studying the interplay between cadherin cell adhesion machinery, Vimentin and associated partners like F-actin, its role in epithelial-to-mesenchymal transition and metastasis, as well as modulation dynamics by pharmacological agents. Overall, in combination with other automated 3D assays, the Pu·MA System enables examining a large set of parameters including biomarkers, signaling molecules, cell morphological changes, proliferation indices, and toxicity.