Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

This method can help answer key questions in the field of drug discovery to identify anti-cancer effect and gain better mechanistic insight into novel molecules using in vitro and in vivo 3D culture systems. The main advantage of this technique is the varieties of pharmacological effect of compounds in a more physiologically relevant in vivo model. To begin, plate 20, 000 A549 cells per square centimeter in 15 milliliters of RPMI 1640 cell culture medium, supplemented with 10%FBS and 1%antibiotics, in a 75 square centimeter flask.

Incubate the culture at 37 degrees Celsius and 5%CO2. On the day of the experiment, use a hemocytometer to determine the cell concentration. Collect 50, 000 cells in a 1.5 milliliter microcentrifuge tube by centrifugation at 400g for seven minutes and resuspend the cells in 100 microliters of sterile 1x PBS.

Using a multi-pipet, dispense 1.1 milliliters of semi-solid methylcellulose-based medium, or MCBM, into tubes, preparing one tube for each condition. Then, add 110 microliters of FBS to each tube. Next, save 1000 cells per milliliter.

Then, pipet 2.4 microliters of the stock cell solution into the tubes of MCBM and FBS. After adding the cells, hold the tubes vertically and vortex them at the highest speed for one minute to thoroughly mix the MCBM and cells. Now, add test compound OT48 alone or in combination with A1210477 to the samples.

Prepare a separate tube as a control for the solvent used, such as DMSO. Then, vortex the samples for one minute. For each assay, pipet one milliliter of the mixture into the center well of a 12 well cell culture plate.

Then, use one milliliter of sterile water or PBS to fill the empty wells. Incubate the plates at 37 degrees Celsius and 5%CO2 for 10 days. Then, add 200 microliters of a five milligram per milliliter MTT stock solution to each well.

After incubating the plates for ten minutes, check for viable colonies which will turn violet. Using a white background plate, capture images using the following settings:For method, choose digitize. For exposure type, choose precision.

For exposure time, choose manual and one second. For sensitivity and resolution, choose high resolution. Then, save the captured images in TIFF format.

With ImageJ software, quantify viable colonies by selecting menu image, type, 8-bit. Select menu adjust and threshold. Set the threshold to control and keep it constant for all conditions.

Then, select menu analyze, analyze particles. Save the data and analyze it with a statistic software for graphical representation. After preparing stock reagents according to the text protocol, separate one female and one male zebrafish in a partition tank filled with UV sterilized and filtered tap water at 28.5 degrees Celsius and place them in the dark.

After 24 hours of separation, remove the partition to initiate mating. Transfer fertilized eggs from the mating tank to a Petri dish containing five milliliters of fresh Danios solution. Use five milliliters of Danios solution to wash the eggs three times by using a pipet to carefully aspirate the eggs and transfer them to five milliliters of fresh solution.

Then, incubate the eggs at 28.5 degrees Celsius for eight hours. Change to Danios solution containing 0.03%PTU to inhibit pigmentation and sort out opaque unfertilized eggs to prevent contamination. 24 hours post-fertilization, use sharp forceps to dechorionate the embryos.

Then, incubate the embryos in Danios solution with PTU up to 48 hours post-fertilization. After seeding A549 cells and incubating them with compounds according to the text protocol, 24 hours before the end of treatment, add four micromolar of the fluorescent cell tracker dye, CM-Dil, to stain the cells and incubate them for two hours. Following the incubation, use 0.05%Trypsin-EDTA to trypsinize the cells.

Then, dilute ten to the sixth cells in 50 microliters of phenol red PBS solution prior to injection. To carry out microinjection of cancer cells, pull a 1.0 millimeter glass capillary using the following program settings:Use a syringe to cut the capillary to produce a sharp edge. Then, load the microcapillary with 20 microliters of the cell phenol red solution.

After anesthetizing zebrafish according to the text protocol, inject 100 to 200 cells into the yolk sack by injecting six to ten nanoliters via three to five injections. Microinjections should be done very carefully and precisely to ensure that the correct volume of the cells is injected and injection injury is minimal and the carbon dioxide pressure should be optimal to avoid a fissure laterally. After injection, allow the fish to recover for ten to 30 minutes in five milliliters of fresh Danios solution containing PTU.

Transfer the fish into 24 well plates with one milliliter of Danios PTU solution, and incubate them at 28.5 degrees Celsius for 72 hours. Following the incubation, after anesthetizing the fish according to the text protocol, immobilize them on a glass slide with a drop of 3%methyl cellulose. Take 5x fluorescent images at a wavelength of 620 to 750 nanometers.

Use ImageJ software to quantify the size of fluorescence tumors by selecting analyze and measure. Then, save the fluorescence values and analyze them with a statistics software for graphical representation. In this experiment, lung cancer cell line A549 was seeded in MCBM to form colonies after treatment with OT48 alone or in combination with BH3 mimetic, A1210477, at the concentration seen here.

The results showed that the combination significantly reduced the number, size and total surface area of the colonies after ten days of incubation. Here, after treatment with OT48 alone or in combination with BH3 mimetic, A1210477, the cells were allowed to form spheroids using the U-bottom plate technique. Following a three day incubation, the spheroids were imaged and quantified, and 3D images were generated.

This figure illustrates the inhibitory potential of OT48 and/or A1210477 on tumor formation from the quantification of fluorescent tumors generated when treated cells were injected into the zebrafish yolk sack. While attempting this procedure, it is important to remember to keep the number of fish for each condition around ten or more to avoid the important variations affecting the statistical evaluation of the data. Following this procedure, other methods like histological analysis can be performed in order to asses target protein expression in zebrafish xenograft.

This technique paves the way for researchers in the field of drug discovery to explore the efficacy of compounds in an in vivo micro-environment in zebrafish xenograft. After watching this video, you should have a good understanding of how to validate drug efficacy in a 3D culture environment.