Zebrafish patient-derived xenograft is a powerful preclinical model to predict cancer patient chemosensitivity profile in vivo. The protocol describe the generation of the model starting from a patient surgical specimen. By xenografting a small tissue fragment rather than isolate cells into zebrafish embryos, it's possible to maintain the tumor microenvironment which is the crucial step for tumor progression and the response to chemotherapy.
The big advantage of the model is given by its potential to predict the patient's prognosis in a clinical relevant timeframe of just one week acting as a tool for personalized medicine. Begin the sample processing by washing the whole tumor tissue collected from the patient with five milliliters of fresh tumor medium. Pipette it up and down 10 times using a plastic Pasteur pipette.
Carefully aspirate and discard the washing medium before repeating the wash three times. Immerse the sample in one to two milliliters of fresh tumor medium. Cut the tumor sample into one to two millimeter cube pieces using a scalpel blade, and then place them in a sterile five milliliter plastic tube with tumor medium.
After setting the McIlwain Tissue Chopper to 100 micrometer thickness, place the tumor pieces on the circular plastic table of the chopper and chop them. Rotate the table by 90 degrees and repeat the chopping. Collect the chopped tumor fragments in a 15 milliliter plastic tube containing the tumor medium.
Centrifuge the chopped tumor fragments at 300 G for three minutes before aspirating and discarding the supernatant. Incubate the fragments with the cell trace solution or with an alternative fluorescent cell tracker such as CM-DiI or Deep Red for 30 minutes in a 37 degrees Celsius water bath. During incubation, gently resuspend the fragments every 10 minutes.
At the end of the staining, remove any free dye by adding a tumor medium supplemented with at least 1%of protein. The volume will be five times the staining volume. Centrifuge the fragments at 300 G for three minutes and discard the supernatant.
Add one milliliter of Dulbecco's Phosphate Buffered Saline or DPBS and repeat this washing step three times. Once done, suspend the fragments in five milliliters of DPBS in a 60 millimeter Petri dish. To establish the zebrafish-based patient-derived xenografts or zPDX, anesthetize the two days post fertilization embryos.
In a Petri dish having three agarose cylinders, lay a zebrafish embryo on a cylinder exposing one side. Remove the excess solution to keep the embryo in a thin film. Using sterile forceps, transfer the piece of stained tumor tissue fragments from a 60 millimeter Petri dish to the 1%agarose support where the embryo is laid.
Then pick up the tissue and place it on the embryo yolk before pushing it into the perivitelline space using the heat pulled glass microneedle. Add some drops of E3 1%penicillin streptomycin to the embryo to bring it back into the liquid. After repeating the implantation of tumor tissue in all the embryos, remove the agarose supports from the Petri dish and incubate the embryos at 35 degrees Celsius.
Two hours post implantation, check the embryos for the correct xenografts with positive staining using a fluorescent stereo microscope. Discard dead embryos and the embryos without the tumor fragments completely inside the perivitelline space. Once done, randomly distribute the embryos in six multi-well plates, equally divided into groups according to the experimental plan.
Dilute the drugs by mixing them into E3 1%pen-strep to prepare the FOLFOXIRI cocktail. After two hours of implantation, remove the media from each well and add the drug cocktail. Place the embryos into the incubator at 35 degrees Celsius for three days with daily changes of the medium to renew the drugs.
For imaging, capture images under a 40X objective of the confocal microscope. Use the resolution of 1024 by 512 pixels with a Z spacing of five micrometers. The whole mount images of zPDXs acquired to analyze the apoptosis detected in cyano showed that the combined therapy increased cell apoptosis compared to the control group group.
The case study reported that a statistically significant increase in the fraction of apoptotic cells in implanted xenografts was identified for the FOLFOXIRI-treated group compared to the control group. The protocol show an example using pancreatic cancer tissue, but zebrafish patient-derived xenografts can be established from different types of cancer. In additions, since the model has a low ethical impact, it is easily exploitable for in vivo screening of new drugs.
The method is technically challenging and requires an expert operator. Prior expertise in zebrafish embryo manipulation and microinjection will speed up the training of the technique.
