This study investigates the behavior of brain tumor stem cells (BTSCs) in response to drug treatment, focusing on their invasion capabilities in vitro. The methodology involves culturing BTSCs and assessing their invasion through a matrix using live-cell microscopy.
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.
To assess BTSC neurosphere invasion, first, add the compound of interest to the flask at the desired concentration for the predetermined time course before neurosphere invasion plating. When the average neurosphere size reaches approximately 150 to 200 micrometers, tip and gently mix the flask with a pipette, and transfer 500 microliters of the resuspended BTSC neurospheres to one 1.5-milliliter tube per experimental condition on ice.
Aspirate as much of the media from the bottom of each 1.5-milliliter tube as possible without losing the neurosphere pellet and gently resuspend the neurospheres in 500 microliters of freshly prepared 0.4-milligram per milliliter type I collagen solution. Transfer 100 microliters of the collagen suspended neurospheres to three wells per condition to a new 96-well plate on ice and allow the neurospheres to settle for 5 minutes.
It is critical to allow the neurospheres to settle for 5 minutes on ice. Otherwise, the spheres will not be on the same plane of focus for imaging. After 5 minutes of collagen polymerization in the cell culture incubator, transfer the plate into the tray of the live cell imaging system to acquire a reference image of the neurosphere size at the time of plating before the invasion begins. Then, set the software to acquire images every 1 to 2 hours until the rate of invasion has plateaued, typically by 24 hours.
To image the increasing surface area of the BTSCs as they invade the matrix over time, use the 10x objective to acquire four images per well for each set of three replicate wells. To determine the relative cell surface area represented as the percent confluence of the well, set up a processing definition that specifically highlights the cells over the background of the well. Then, collect the data as the total cell surface area for every well at each time point. Determine the mean of the three replicate wells and graph the invasion of the BTSCs by plotting the increasing cell area over time.
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