A microfluidics workflow for spatial analysis of microenvironmental gradient impact on cancer cell phenotypes

Despite the physiological and pathophysiological significance of microenvironmental gradients, tools for generating such gradients and analysing their impact on cellular phenotypes are lacking. Here we present an integrated microfluidics-based workflow for mimicking extracellular pH gradients characteristic of solid tumors, and studying their multifactorial impact on cancer cells. Our microfluidics device generates a pH gradient across cancer cell 3D cultures in an extracellular matrix. The gradient, validated using pH-sensitive fluorophores can be rapidly controlled to represent spatiotemporal microenvironmental changes, and the device allows high resolution live imaging of, e.g., cell motility and chemotaxis. The device can be reopened, allowing immunofluorescence analysis of phenotypes and spatially resolved analysis of gene expression changes across the pH gradient. The workflow is easily adaptable for other gradients and multiple cell types, making it broadly applicable for integrated analysis of roles of microenvironmental gradients in biology. Overall design: MDA-MB-231 cells grown in a microfluidics device generating a ph gradient from 6.0 to 7.4, in triplicates (one per library)