Using picoliter droplet deposition to track clonal competition in adherent and organoid cancer cell cultures.

Clonal growth and competition underlie processes of key relevance in etiology, progression and therapy response across all cancers. Here, we demonstrate a novel experimental approach, based on multi-color, fluorescent tagging of cell nuclei, in combination with picoliter droplet deposition, to study the clonal dynamics in two- and three-dimensional cell cultures. The method allows for the simultaneous visualization and analysis of multiple clones in individual multi-clonal colonies, providing a powerful tool for studying clonal dynamics and identifying clonal populations with distinct characteristics. Results of our experiments validate the utility of the method in studying clonal dynamics in vitro, and reveal differences in key aspects of clonal behavior of different cancer cell lines in monoculture conditions, as well as in co-cultures with stromal fibroblasts.

High-Content and High-Throughput Clonogenic Survival Assay Using Fluorescence Barcoding.

The Clonogenic Survival Assay (CSA) is a fundamental tool employed to assess cell survival and proliferative potential in cancer research. Despite its importance, CSA faces limitations, primarily its time- and labor-intensive nature and its binary output. To overcome these limitations and enhance CSA's utility, several approaches have been developed, focusing on increasing the throughput. However, achieving both high-content and high-throughput analyses simultaneously has remained a challenge. In this paper, we introduce LeGO-CSA, an extension of the classical CSA that employs the imaging of cell nuclei barcoded with fluorescent lentiviral gene ontology markers, enabling both high-content and high-throughput analysis. To validate our approach, we contrasted it with results from a classical assay and conducted a proof-of-concept screen of small-molecule inhibitors targeting various pathways relevant to cancer treatment. Notably, our results indicate that the classical CSA may underestimate clonogenicity and unveil intriguing aspects of clonal cell growth. We demonstrate the potential of LeGO-CSA to offer a robust approach for assessing cell survival and proliferation with enhanced precision and throughput, with promising implications for accelerating drug discovery and contributing to a more comprehensive understanding of cellular behavior in cancer.

p(HEMA)-RR241 hydrogel membranes with micron network for IgG depletion in proteomic studies.

Serum proteins can generally be considered a good source for the illness' indication and are precious resources to detect diseases such as inflammation, cancer, diabetes, malnutrition, cardiovascular diseases, Alzheimer's, other autoimmune diseases, and infections. However, one of the biggest difficulties for proteomic studies is that the majority of serum protein mass consists of only a few proteins. Albumin and Immunoglobulin (IgG) constitute 80% of total serum protein. In this study, dye ligand affinity-based hydrogel membranes were proposed as new materials with micron mesh structures. Micron mesh p(HEMA) hydrogel membranes were synthesized by using the UV-photopolymerization method, then modified with Reactive Red 241 (RR241) dye ligand to increase the affinity towards IgG. Characterizations of synthesized micron mesh p(HEMA)-RR241 hydrogel membranes were also performed. It was demonstrated by the characterization studies that; the dye was successfully incorporated into the membrane structure with the amount of 119.38 mg/g. The hydrophilic property of the hydrogel membrane was demonstrated by swelling tests and the swelling value of dye modified membrane was found to be 8 times higher than that of the plain membrane. Micron network structure, as well as the porosity, were demonstrated with SEM/ESEM studies. Optimization of IgG adsorption conditions was also studied at different parameters (pH, temperature, ion strength, initial IgG concentration). Optimum pH, temperature, and ionic strength were found to be 6.5, 25 °C, 0.05 M, respectively, and the maximum IgG absorption value was 10.27 mg/g. Finally, it was shown that the proposed materials can be used repeatedly by 5 adsorption-desorption cycles.