Recursos - Pôsteres Científicos
Automated Monitoring and Analysis of Kinetic Live Cell 3D Spheroid-Based Tumor Invasion within a Hydrogel MatrixDownload
April 17, 2018
Authors: Brad Larson, BioTek Instruments, Inc., Winooski, VT; Jan Seldin, Greiner Bio-One, Inc., Monroe, NC
Metastasis is the main cause of death in cancer patients and one of the most complex biological processes in human diseases. The development of therapies designed to forestall the metastatic activity of tumors has been met with multiple challenges. First is the initial focus on single target remedies. As many types of cancers develop multiple mutations during tumor progression, individual cancers are often little affected by this type of drug. Conversely, 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. 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 seeded in microplates. A third impediment is the inclusion of proper incubation time. Traditionally, in vitro assessments of anti-cancer drugs have included short-term incubations, sometimes limited to 48 hours. In vivo treatments, however, can consist of a multi-week regimen. Pre-clinical tests must also, then, extend a similar length of time. A final hindrance which is necessary to overcome is the proper capture and analysis of microscopic images. The ability to monitor the entire tumoroid with a degree of sensitivity across multiple z-planes as cells invade into the matrix, as well as quantify changes over time, is critical. The information presented here will demonstrate a combined procedure for the generation of 3D spheroidal tumoroid structures, creation of a suitable invasion matrix, automated kinetic image-based monitoring of tumor invasion, and cellular analysis of captured z-stacked images.
U-87 and LN-229 glioblastoma multiforme (GBM) cell lines, were used in this study as they have demonstrated phenotypic differences and metastatic ability1. Notably, the growth suppressing PTEN gene is mutated in U-87 cells, and functions normally in LN-229 cells. Additionally the human cytomegalovirus phosphotransferase protein UL-97 inhibits DNA elongation and replication; and is absent from U-87 cells, and present in LN-229 cells2. This supports a more aggressive growth and invasion pattern for U-87 cells. Both cell types were co-cultured with fibroblasts to create 3D tumoroids more closely representing in vivo tumor conditions, and allowed to invade through a protein matrix. 17-allylamino-17-demethoxygeldanamycin (17-AAG), known to inhibit the function of heat shock protein 90 (Hsp90), a chaperone protein that stabilizes proteins required for tumor growth, and was used here to inhibit potential tumor invasion3. Quantification of kinetic captured images was used to characterize the invading potential of inhibited and uninhibited tumoroid cultures.
Figure 1. BioSpa8 and Cytation 5 integrated as the BioSpa Live Cell Imaging System, along with the MultiFlo FX Multi-Mode Dispenser.