Brain Tumor Stem Cell (BTSC) Neurosphere Invasion Assay: An In Vitro Assay to Analyze the Effect of Drug Treatment on BTSC Invasion from Neurosphere

Brain tumor stem cells or BTSCs – a subset of cells within brain tumors – possess self-renewal capabilities and contribute to tumor progression and therapy resistance. To determine the effect of drugs on BTSC invasion in vitro, begin with BTSC suspensions in a suitable media in culture flasks. Treat one of the suspensions with the drug of interest.

Incubate the flasks. The media components promote cell proliferation. The cells remain in suspension, thereby forming spherical clusters called neurospheres. On reaching the desired size, collect the neurospheres. Allow them to sediment by gravity and remove media. Then, resuspend the untreated and drug-treated neurospheres in chilled media containing a suitable extracellular matrix protein and the drug.

Plate the suspensions in a chilled multi-well plate. Allow the neurospheres to sediment to the well bottom for better imaging. Next, incubate for matrix protein polymerization, embedding the neurospheres within. Image the neurospheres for an extended duration using live-cell microscopy. In culture, embedded BTSCs secrete proteolytic enzymes, which degrade the matrix proteins, enabling cell migration.

Over time, increased matrix degradation creates an appropriate microenvironment for BTSC invasion from neurospheres into the surrounding matrix. Using suitable software, measure the total surface area of the neurospheres at different time points. The untreated BTSCs display increased surface area, indicating an increased invasion rate than the drug-treated BTSCs.