Resources - Webinars

Automated, Efficient Imaging of 3D Glioma Tumoroid Invasion to Screen for MT1-MMP Inhibitors in 96-Well Format

Related Products: Cytation 5, Gen5 for Imaging

April 20, 2022

Presenters: Gregg Fields, Ph.D. Executive Director, I-HEALTH Professor, Florida Atlantic University, Ania Knapinska, Ph.D. Director of Research and Development, Alphazyme and Brad Larson, Product Manager - MarCom, Agilent

Malignant gliomas, including glioblastoma multiforme (GBM) and astrocytomas, are the most common primary brain tumors in the United States. Membrane type 1 Matrix Metalloproteinase (MT1-MMP) has been shown to be crucial for the progression, invasion, migration and angiogenesis of tumors.

For many years, the simplest model to study tumor invasion consisted of culturing cells in a two-dimensional (2D) monolayer. Despite the simplicity of the model, there were numerous limitations, such as the fact that cells in monolayers behave differently than cells cultured in a three-dimensional (3D) manner. Newer, more advanced cell models have been developed to better mimic the in vivo cell environment and invasion, particularly embedding 3D tumor models into a hydrogel to allow invasion into the ECM. This type of model allows for more complex development that more closely mimics tumor development, and leads to increased production of matrix degrading enzymes, such as matrix metalloproteinases. While incorporation of 3D cell models has proven to deliver results more indicative of an in vivo response, this can lead to increased cellular imaging times and storage capacity needs. This is because multiple images need to be captured across x-, y-, and z-planes to accurately visualize invasion.

In this webinar we describe a “hit pick” method where PMT-based fluorescent signal from test wells is compared to that from untreated positive control wells in order to trigger cellular imaging of “hit” test wells. A pro-fluorescent FAM-fTHP-9 substrate, preferentially targeted and degraded by MT1-MMP, was developed to serve this purpose. MT1-MMP activity leads to cleavage of the substrate, and an increase in fluorescent signal detected by the plate reading capabilities of the Agilent BioTek Cytation 5 cell imaging multimode reader. Only test wells containing MT1-MMP inhibitors that yield a statistically lower fluorescent signal compared to uninhibited control wells are then triggered to be imaged. The result was an easy-to-perform, robust method to reduce image capture time and storage requirements.