Multimodal Phasor Approach to Study Breast Cancer Cell Invasion in a 3D Spheroid Model.

We implemented a multimodal set of functional imaging techniques optimized for deep-tissue imaging to investigate how cancer cells invade surrounding tissues and how their physiological properties change in the process. As a model for cancer invasion of the extracellular matrix, we created 3D spheroids from triple-negative breast cancer cells (MDA-MB-231) and nontumorigenic breast epithelial cells (MCF-10A). We analyzed multiple hallmarks of cancer within the same spheroid by combining a number of imaging techniques, such as metabolic imaging of nicotinamide adenine dinucleotide by fluorescence lifetime imaging microscopy (NADH-FLIM), hyperspectral imaging of a solvatochromic lipophilic dye (Nile Red), and extracellular matrix imaging by second harmonic generation (SHG). We included phasor-based bioimage analysis of spheroids at three different time points, tracking both morphological and biological properties, including cellular metabolism, fatty acid storage, and collagen organization. Employing this multimodal deep-imaging framework, we observed and quantified cancer cell plasticity in response to changes in the environment composition.