Schematic representation of multicellular 3D spheroid development protocol and their use in different experimental analysis. (A–F) Normal lung epithelial cells (BEAS-2B), lung adenocarcinoma cells (A549 and NCI-H460), MRC-5 fibroblasts, and THP-1 monocytes are used to prepare multicellular tumor spheroids (BMT, AMT, and HMT) in DMEM+10% FBS growth medium. After optimization, as detailed in the result section, 10,000 cells per 25 μl drop in a ratio of 5:4:1 (BEAS-2B/A549/NCI-H460: MRC-5: THP-1) are taken to prepare spheroids using hanging drop method. (G) Different techniques are used for spheroid analysis; g(a). Spheroids frozen at low temperature (-80°C) were sectioned and stained with fluorescent-labeled antibodies for immunofluorescence analysis of cell proliferation, hypoxia, and cell plasticity in the spheroid microenvironment. g(b) Whole-mount analysis was performed for spheroid characterization (cell diameter, uniformity, cell viability), and functional analysis (gene expression and sprouting assays). g(c) Dissociation of multicellular spheroids by enzymatic digestion resulted in cell suspension, which was used for colony formation assay (cell proliferation), and flow cytometric analysis of cell surface markers (phenotypic characterization). Cell suspension was also used for magnetic bead-based separation of a single cell population for gene expression analysis (phenotypic characterization).
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