Evaluation of miRNA Expression in 3D In Vitro Scaffold-Based Cancer Models

Accumulating experimental evidence suggests that 3D in vitro cancer models strengthen our understanding of vital processes in the tumor microenvironment (TME) and accelerate the drug discovery pipeline. Previous studies examining the effects of specific miRNAs on cancer cells in vitro have involved ectopic expression of miRNA mimics in 2D in vitro culture. Assessment of cell viability and gene expression ensures that upregulation of the chosen miRNA and repression of its target genes have been achieved. However, this 2D culture is overly simplified and lacks the complex cell to extracellular matrix (ECM) interactions observed in the native TME, yielding results often not reproduced when progressed to in vivo studies. Hence, this chapter describes a novel method of overexpressing the miRNA mimic in cells cultured on 3D collagen-based scaffolds adapted from tissue engineering techniques. Cell growth on scaffolds is sequentially monitored via a DNA quantification assay, and overexpression of the miRNA mimic and repression of its target gene is assessed via reverse transcription quantitative PCR (RT-qPCR).