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

1. Brain Tumor Stem Cell Neurosphere Invasion Assay

1. Seed 200,000 cells in 8 mL of complete media in a T25 flask 1–2 weeks before planning to perform an invasion assay.
   NOTE: The timing from plating to performing the assay will depend on the proliferation rate of the BTSC culture used.
   1. For pretreated cells, add the compound of interest to the flask at the desired concentration for the predetermined time course before the neurospheres are ready to be plated for invasion.
2. Wait until the average neurosphere size is approximately 150–200 μm; typically, this takes 7–14 days, depending on the BTSC culture being used.
   NOTE: It is typical to observe a distribution of sphere sizes in the flask.
3. Tip and gently mix the flask with a pipette and move 500 μL of the BTSC neurospheres to a 1.5 mL conical tube for each treatment condition; this will be used to plate three replicate wells.
   NOTE: Steps 1.3.1–1.3.2 are optional but recommended for the first invasion experiment on a new BTSC culture.
   1. To determine the density of the neurospheres that will end up in the well, prepare a 1.5 mL tube as in step 1.3. Gently mix the neurospheres and move 100 μL into a separate 96-well plate and allow the neurospheres to settle by gravity for 5 min.
   2. Check the number of neurospheres under the microscope to ensure multiple neurospheres are in the region of the well that will be imaged without overcrowding it.
      NOTE: Invading neurospheres require enough room to triple in diameter without interacting with neighboring neurospheres. The number of neurospheres per well can be adjusted by counting the number of neurospheres per well and adding more or fewer neurospheres from the T25 flask to the 1.5 mL conical tube in step 1.3.
4. Place the 1.5 mL conical tube with the cells from step 1.3 on ice and perform steps 1.5–1.7 on ice.
5. Let the neurospheres settle by gravity to the bottom of the 1.5 mL conical tube for 5 min and prechill a labeled 96-well plate on ice.
6. Resuspend the neurospheres in the extracellular matrix protein.
   1. Prepare 500 μL of a 0.4 mg/mL type I collagen solution on ice for each treatment condition: add 459 μL of media (without growth factors), 40 μL of the 5 mg/mL collagen stock solution, and 0.92 μL of sterile 1N NaOH (Table 1).
      NOTE: 500 μL of the collagen solution is required per treatment condition: 100 μL for each of the three replicate wells and 200 μL of excess. Drug treatments need to be added here at the desired final concentration, subtracted from the media component of the solution. This protocol describes the use of type I collagen as the extracellular matrix protein; however, any extracellular matrix protein can be tested. In addition, for BTSC cultures that have slower rates of invasion, growth factors or FBS can be supplemented in the media to increase the rate of invasion.
   2. After the neurospheres from step 1.5 have settled, aspirate as much of the media as possible without losing the neurosphere pellet that has settled at the bottom. Gently resuspend the neurospheres in 500 μL of the collagen solution prepared in step 1.6.1.
7. Add 100 μL of the collagen and neurosphere mixture to three replicate wells of the 96-well plate on ice. Allow the neurospheres to settle on ice for 5 min.
8. Transfer the 96-well plate to a 37 °C incubator for 5 min to allow for collagen polymerization.
9. Immediately prepare to image the 96-well plate.
   1. Place the 96-well plate into the tray of a live-cell imaging system or on the stage of a microscope to acquire an image as a reference for the neurosphere size at the time of plating, before the invasion begins.
   2. Acquire images every hour until the rate of invasion has plateaued; typically, this occurs within 24 h.
      NOTE: The imaging interval can be modified depending on the equipment being used. The imaging interval and total elapsed time can also be modified for BTSC cultures that have different rates of invasion.
10. Determine the increasing surface area of BTSCs as they invade the matrix over time.
    NOTE: The surface area of cells at the time of plating, calculated as the mean of three replicate wells, needs to be similar (less than 20% variation across all wells) between the different treatment conditions to ensure the differences in the BTSC invasion are not strongly affected by the number or size of the neurospheres plated.
    1. For the live-cell analysis system, use a 10X objective for the image acquisition and acquire four images per well for the 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. Using commercial software (see Table of Materials), select a New Processing Definition and set the segmentation adjustment to 0.8, the area minimum to 300 μm², and the eccentricity maximum to 0.99. Modify this analysis if it does not accurately distinguish cells from the background.
       NOTE: If using other methods to image neurosphere invasion over time, it is recommended to obtain multiple fields of view for the three replicate wells over time. However, not imaging the exact same field of view at each interval will increase the standard error of the data collected. Computer software can be used to help measure the surface area of the invading cells in each image.
    2. Collect data as the total cell surface area (either relative or absolute) for every well at each time-point and determine the mean of the three replicate wells. Graph the invasion of BTSCs by plotting the increasing cell area over time.

Table 1: Preparation of the 0.4 mg/mL type I collagen solution. Listed are the known starting and desired final concentrations of the type I collagen solution. The volumes listed are sufficient to plate neurospheres into three replicate wells, plus two for excess, and can include a single treatment condition, as described in detail in protocol step 1.