Real-Time and 3D Quantification of Cancer Cell Dynamics: Exploiting a Bioengineered Human Bone Metastatic Microtissue

: The study of dynamic processes in the bone metastatic compartment has been challenged by the restrictive access and limited live imaging capabilities that in vivo bone models provide. In this protocol, we show the use of a human bone metastatic bioengineered microtissue for the quantitative investigation of cancer cells in an in vitro bone-like microenvironment. Using live cell epifluorescence microscopy, traditional- and spinning disc-confocal laser scanning microscopy, we demonstrate how to obtain multidimensional real-time data of fluorescently labeled cancer cells in the metastatic microenvironment. Using 4D imaging data processing software such as ImageJ and Imaris, we show how to transform qualitative images and videos into quantitative data of cancer cell attachment, morphology, proliferation, and migration in vitro in the human bone metastatic microtissue.