3D-on-2D: A Physiologically Relevant and Gel-Free In Vitro Coculture Method to Assay Antimetastatic Agents

Metastasis of cancer cells leads to 90% of lethality among cancer patients. A crucial step in the hematogenous spread of metastatic cancer is the detachment of cells from the primary tumor followed by invasion through nearby blood vessels (Wong and Hynes. Cell Cycle 5(8):812-817, 2006). This is common to several solid tumors, including medulloblastoma (Van Ommeren et al. Brain Pathol 30:691-702, 2020). Because invasion is a crucial step in metastasis, the development of assays studying invasion are important for identifying antimetastatic drugs. There is always a need to develop better 3D in vitro models that not only mimic the complexity of in vivo architecture of solid tumors and their microenvironment, but are also simple to execute in medium to high throughput. We developed an in vitro coculture invasion assay that relies on the binary interaction between cancer cells and endothelial cells for research on tumor invasion and antimetastatic drug discovery. The goal of the current protocol is to use the simplicity of a two-dimensional endothelial cell culture to create a gel-free physiological substratum that can facilitate cancer cell invasion from a 3D cancer spheroid. This provides a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. Using this assay, we can compare the effect of invasion inhibitors/activators on cancer spheroids. The results are analyzed by manual scoring of images for the presence or absence of sprouting from cancer spheroids. This enables simple and fast analysis of metastasis, which facilitates multiparameter examination.