Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Today we're going to introduce a larvae Zebrafish PDX Model. This model is significant because they can better mimic a similar cellular composition in the xenograft for assessing drug responses and also further improves the consistency of the results with our comparative analysis. There are two main advantages of this technique.

Firstly, we use various standard chemical dyes to label cells into different fluorescence, allowing the real-time tracing of the cellular compositions and the ability of the anagraph phase. Secondly, it also improves the general survival time of human cells in zebrafish, their genetic modification, which provides a longer observation window for drug testing. This technique provides a potential model for personalized medicine for patients with pancreatic cancer and also for the pre-assessment of pre-clinical tumor drug.

This method can also be used to trace other tumor cells or no tumor cell xenografted in zebrafish in vivo and to improve the cell survival for longer observation time. To prepare the injection plate, prepare a 50 milliliter solution of 1%agarose in E3 solution. Boil the solution until the agarose dissolves.

Pour the entire solution into a 10 centimeter Petri dish and then place the Zebrafish embryo fixation mold onto the surface. When the agarose solution solidifies completely, remove the mold. To prepare injection needles, use a needle puller to pull a 10 centimeter glass capillary with an inner dimension of 0.9 millimeters into two needles.

Using a microscope and forceps, cut the end of the needle to create an opening. First, place one to two pairs of adult Zebrafish into a mating tank around seven to nine PM.At eight AM the next morning, collect the fertilized eggs. Transfer the fertilized eggs to a Petri dish containing 40 milliliters of fresh E3 solution.

Incubate at 28.5 degrees Celsius. First, transfer the collected pancreatic cancer sample into a Petri dish. Rinse the tissue five to six times with PBS and use a scalpel to cut the tissue into one millimeter cubed pieces.

Next, transfer the shredded tissue into a 50 milliliter tube containing five milliliters of HBSS. Add collagenase type four, hyalurodinase, and DNAse one at the final concentrations shown here. Pipette the mixture up and down to mix well.

Incubate the mixture at 37 degrees Celsius in a 5%carbon dioxide incubator for 15 to 20 minutes. Pipette the mixture up and down a few times every five minutes. Add seven milliliters of DMEM to the tube.

Centrifuge at 110 times G and at four degrees Celsius for five minutes. Then, decant the supernatant and re-suspend the tumor mixture in DMEM. Plate the mixture into two six centimeter Petri dishes containing three milliliters of full growth media named group one and group two.

Incubate both groups at 37 degrees Celsius in a 5%carbon dioxide incubator. After 48 hours, add 100x inhibitor of pancreatic cancer fibroblasts into the medium of group one to remove the overgrown fibroblasts, leaving the cancer cells as the major cell types. Continue incubating using the previous conditions.

After producing the lentivirus, seed the cells to be infected into a 12-well plate with 30 to 40%density. Culture the cells overnight at 37 degrees Celsius in a 5%carbon dioxide incubator. The next day, replace the medium with 500 microliters of serum-free medium containing polybrene at a concentration of eight micrograms per milliliter.

Incubate at 37 degrees Celsius with 5%carbon dioxide for four hours. Then, add an additional 100 microliters of the lentivirus to the medium and incubate again for 12 hours. After 12 hours, replace the medium with two milliliters of complete medium and incubate for an additional 36 hours.

After 36 hours, check the fluorescence markers. Harvest the infected cells and mix them at a one-to-one ratio with a final concentration of one million cells per milliliter. Centrifuge the cells at 110 times G for five minutes and re-suspend the cell mixture in 50 microliters of injection solution.

After anesthetizing the Zebrafish larvae, transfer them into the injection plate, which is filled with modified E3.Take up 25 microliters of the mixed cell suspension into the microcapillary needle and insert the needle into the microinjection manipulator. Set the injection pressure and time and inject 50 to 80 cells into the yolk sac of 48 hours post-fertilization Zebrafish. First, transfer the post-zenografted Zebrafish larvae into 40 milliliters of mix solution at 32 degrees Celsius.

Place 10 zenografted larvae into each well of a 12-well plate. Next, divide the larvae into four groups. Treat the control group with E3 containing 0.1%DMSO and treat the other groups with Gemcitabine and/or Navitoclax.

Incubate all larvae at 32 degrees Celsius for two days. After anesthetizing the zenografted larvae post-treatment, place them in 3%methyl cellulose. Use a fluorescence or confocal microscope to image the larvae from the lateral view.

Then, use image J and graph pad software to quantify the intensity of red and green fluorescence signals. In this study, primary cancer tissue cells are seeded into complete medium after digestion with or without the addition of pancreatic cancer fibroblasts inhibitors. Cancer cells and fibroblasts are enriched as two distinct populations.

The population without inhibitors is dominated by fibroblasts while cancer cell growth prevails after the addition of inhibitors. Two lentiviral packaging vectors are constructed which express green or red fluorescent proteins in BCL2L1. The virus-based fluorescence labeling represents the survival status of the cells.

The mixed cancer cells and fibroblasts are injected into the Zebrafish yolk sac at 48 hours post-fertilization and are then treated with Gemcitabine and/or Navitoclax for two days. The cell viabilities in different populations and cellular composition of the zenograft are changed as the responses to the drug treatment. When processing different types of tumors, there are several steps that may need to be objected, including cell digestion time, drug treatment dosage, and so on.

This technique helps researchers to employ Zebrafish from a standard CGX or PDS assay. It allows for the mixing and injection of different cells at fixed ratios to quantify the progress bands by comparing them to cell growth. Personal protection is of great significance, especially when packaging lentivirus.

Wear masks and gloves when appropriate and try not to mix on skin.