住セルイメージ投射アッセイ研究神経膠腫脳腫瘍幹細胞の遊走と浸潤に

This method can help answer key questions in the field of glioblastoma brain tumor stem cell or BTSC biology by facilitating the identification of novel approaches to inhibit migration and invasion. The main advantage of these techniques is that they allow the quantification of BTSC migration and invasion over time under multiple conditions. The implications of this technique extend toward glioblastoma therapy as the migration and invasion of cancer cells into normal brain tissue contributes to disease recurrence.

Though this method can provide insight into how BTSCs migrate and invade, it can also be applied to cancer stem cells derived from other tumor types. Begin by thawing a vial of cryogenically-preserved BTSCs in a beaker of 70%ethanol placed inside a 37-degrees Celsius water bath just until the last of the ice has thawed. Dilute the thawed cells and 10 milliliters of media in a 15-milliliter conical tube for their centrifugation and resuspend the BTSC pellet in eight milliliters of complete media.

Transfer the cells into a T25 culture flask for 10 to 14 days of culture under non-adherent conditions, monitoring the flask daily, and supplement the cells with one to two milliliters of fresh complete media as necessary. When the neurospheres reach an approximately 250-micrometer diameter, use a pipette to flush media across the bottom of the flask to detach any adherent cells and transfer the supernatant to a 15-milliliter conical tube for centrifugation. To disassociate the BTSC neurospheres into single cells, resuspend the pellet in one milliliter of prewarmed cell detachment solution and incubate for seven minutes at 37 degrees Celsius, followed by 40 triturations with a P1000 micropipette set to 800 microliters.

When the cells are fully dissociated, add five milliliters of PBS supplemented with antibiotics to the tube for another centrifugation and resuspend the pellet in one to four milliliters of complete media depending on the size of the pellet. Then seed two to three times 10 to the fifth cells in eight milliliters of complete media per T25 flask and transfer the flasks to the cell culture incubator for one to two weeks of culture. To assess the effects of a drug treatment of interest on migration, first pretreat the cells with the appropriate concentration of the drug for a suitable experimental period.

To prepare the chemotaxis plate, coat three membrane insert wells and three bottom reservoir wells per condition of a 96-well chemotaxis plate with 75 and 225 microliters of 0.2 milligrams per milliliter type I collagen respectively for one hour at 37 degrees Celsius. At the end of the incubation, aspirate the collagen solution from each well without scratching the collagen coating and wash the wells two times with sterile PBS. After the last wash, remove the insert from the plate and add 225 microliters of growth factor-free media supplemented with 10%FBS to each bottom reservoir well of the chemotaxis plate.

Then gently return the top insert to the bottom reservoir at an angle to avoid creating bubbles and add 2.5 times 10 to the third dissociated BTSCs in 50 microliters of growth factor-free media to each insert well. It is important to fully dissociate the BTSCs into single cells and to count them carefully before plating to avoid having cell clumps or too many cells in the top insert. To image BTSC migration along the FBS gradient, place the plate into a live-cell imaging system and set the automated imaging software to acquire microscopic images of the plate every one to two hours for up to 72 hours.

When the image acquisition is complete, select a new processing definition to distinguish the BTSCs from their insert pores on the bottom side of the membrane, modifying the analysis if it does not accurately distinguish the cells from the background membrane as necessary. To quantify the cells that have migrated through the pores, collect the data as the cell surface area on the bottom side of the membrane for each of the three replicate wells per condition, then graph the cell migration as the area of the cells migrated in micrometers squared over time. To assess BTSC neurosphere invasion, first add the compound of interest to the flask at the desired concentration for the pre-determined 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 five minutes.

It is critical to allow the neurospheres to settle for five minutes on ice. Otherwise the spheres will not be on the same plane of focus for imaging. After five 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 one to two 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.

BTSCs plated as single cells can be grown as free-floating neurospheres. The neurospheres can then be treated with a drug of interest and plated for chemotaxis analysis of the effects of therapeutic interventions on BTSC migration. If the BTSCs are not fully dissociated, the cells do not migrate through the pores and remain as small neurospheres.

If more than 2.5 times 10 to the third BTSCs are plated per well, the cells clump rather than migrate to the bottom sides of the membranes. Further, it is essential to avoid making bubbles in the membrane insert wells while plating cells or when placing the membrane insert into the bottom reservoir, as bubbles prevent accurate cell imaging and quantification. BTSC invasion from neurospheres into the surrounding matrix can be imaged and measured over time in a 96-well plate format.

The effect of therapeutic interventions on BTSC invasion can be assessed by quantifying the surface area of the cells as they invade into the matrix over time. Embedding BTSC neurospheres with a greater than 200-micrometer diameter leads to a plane of focus that is too large to accurately quantify all of the spheres in the field of view. Similarly, a failure to maintain the matrix at four degrees Celsius while embedding the neurospheres in collagen for the invasion assay can also lead to spheres that are not on the same plane of focus with an extracellular matrix that does not solidify evenly, preventing an accurate data acquisition.

While attempting this procedure, it's important to remember to optimize the drug treatment conditions and image acquisition times for each BTSC culture being investigated. This procedure can also be modified to answer additional questions about how cancer stem cells from other tumor types migrate and invade in vitro. This technique can help pave the way for researchers in the field of cancer biology to explore in vitro migration and invasion in glioblastoma BTSCs.