DiscussionMetastasis is the main cause of death in cancer patients and one of the most complex biological processes in human diseases (Hanahan et al., 2011). The development of therapies designed to forestall the metastatic activity of tumors has been met with multiple challenges. The first being the initial focus on single target remedies. As many types of cancers develop multiple mutations during tumor progression (Wood et al., 2007), individual cancers are often little affected by this type of drug. The advancement of novel methods that allow for the discernment of the effect a potential therapy has on the invasive phenotype of a particular type of cancer has proven invaluable to circumventing these early failures. A second hurdle is the choice of an appropriate cell model. Tumors in vivo exist as a three-dimensional (3D) mass of multiple cell types, including cancer and stromal cells (Mao et al., 2013). Therefore, incorporating a 3D spheroid-type cellular structure that includes co-cultured cell types forming a tumoroid, provides a more predictive model than the use of individual cancer cells cultured on the bottom of a well in traditional two-dimensional (2D) format. A final hindrance which is necessary to overcome is the proper analysis of microscopic images captured of the tumor invasion process. Being able to observe changes in tumoroid size, as well as individual cell movement through the matrix in a quantitative way is critical.
Here we demonstrate a method for the generation of 3D spheroidal tumoroid structures, creation of a suitable invasion matrix, image-based monitoring of tumor invasion, and cellular analysis of captured images. Spheroid Microplates, coated with an ultra low attachment surface, were incorporated for tumoroid formation, and performance of the invasion process. Tumor invasion tracking was performed via digital microscopy using a novel cell imaging multi-mode reader. Multiple cellular analysis methods will be presented using either brightfield or fluorescence microscopy to track all forms of tumor invasion, including changes to the original tumoroid, invadopodia development, and cell migration.