A new advanced cellular model of functional cholinergic-like neurons developed by reprogramming the human SH-SY5Y neuroblastoma cell line.

Modeling human neuronal properties in physiological and pathological conditions is essential to identify novel potential drugs and to explore pathological mechanisms of neurological diseases. For this purpose, we generated a three-dimensional (3D) neuronal culture, by employing the readily available human neuroblastoma SH-SY5Y cell line, and a new differentiation protocol. The entire differentiation process occurred in a matrix and lasted 47 days, with 7 days of pre-differentiation phase and 40 days of differentiation, and allowed the development of a 3D culture in conditions consistent with the physiological environment. Neurons in the culture were electrically active, were able to establish functional networks, and showed features of cholinergic neurons. Hence here we provide an easily accessible, reproducible, and suitable culture method that might empower studies on synaptic function, vesicle trafficking, and metabolism, which sustain neuronal activity and cerebral circuits. Moreover, this novel differentiation protocol could represent a promising cellular tool to study physiological cellular processes, such as migration, differentiation, maturation, and to develop novel therapeutic approaches.

An Optimized Workflow for the Analysis of Metabolic Fluxes in Cancer Spheroids Using Seahorse Technology.

Three-dimensional cancer models, such as spheroids, are increasingly being used to study cancer metabolism because they can better recapitulate the molecular and physiological aspects of the tumor architecture than conventional monolayer cultures. Although Agilent Seahorse XFe96 (Agilent Technologies, Santa Clara, CA, United States) is a valuable technology for studying metabolic alterations occurring in cancer cells, its application to three-dimensional cultures is still poorly optimized. We present a reliable and reproducible workflow for the Seahorse metabolic analysis of three-dimensional cultures. An optimized protocol enables the formation of spheroids highly regular in shape and homogenous in size, reducing variability in metabolic parameters among the experimental replicates, both under basal and drug treatment conditions. High-resolution imaging allows the calculation of the number of viable cells in each spheroid, the normalization of metabolic parameters on a per-cell basis, and grouping of the spheroids as a function of their size. Multivariate statistical tests on metabolic parameters determined by the Mito Stress test on two breast cancer cell lines show that metabolic differences among the studied spheroids are mostly related to the cell line rather than to the size of the spheroid. The optimized workflow allows high-resolution metabolic characterization of three-dimensional cultures, their comparison with monolayer cultures, and may aid in the design and interpretation of (multi)drug protocols.

Profiling and Targeting of Energy and Redox Metabolism in Grade 2 Bladder Cancer Cells with Different Invasiveness Properties.

Bladder cancer is one of the most prevalent deadly diseases worldwide. Grade 2 tumors represent a good window of therapeutic intervention, whose optimization requires high resolution biomarker identification. Here we characterize energy metabolism and cellular properties associated with spreading and tumor progression of RT112 and 5637, two Grade 2 cancer cell lines derived from human bladder, representative of luminal-like and basal-like tumors, respectively. The two cell lines have similar proliferation rates, but only 5637 cells show efficient lateral migration. In contrast, RT112 cells are more prone to form spheroids. RT112 cells produce more ATP by glycolysis and OXPHOS, present overall higher metabolic plasticity and are less sensitive than 5637 to nutritional perturbation of cell proliferation and migration induced by treatment with 2-deoxyglucose and metformin. On the contrary, spheroid formation is less sensitive to metabolic perturbations in 5637 than RT112 cells. The ability of metformin to reduce, although with different efficiency, cell proliferation, sphere formation and migration in both cell lines, suggests that OXPHOS targeting could be an effective strategy to reduce the invasiveness of Grade 2 bladder cancer cells.