Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

The overall goal of the following experiment is to establish an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis. This is achieved by generating transgenic human multiple myeloma cells for analysis in 3D in vitro and in vivo models. The cells are transfected with lentivirus providing GFP for visualization and blastin resistance for selection of transfected cells.

As a second step 3D viroids. With the transfected cells, mesenchymal cells and extracellular matrix are generated, which allows for large scale screening of anti-cancer drugs in a complex 3D in vitro environment. Next 3D tumor.

PHE can be grown in vivo in chicken embryos in order to test effects of novel drugs on tumor growth, invasion and angiogenesis. Ultimately, tumor size and angiogenic responses can be documented daily and after euthanasia. The tumors can be examined by GFP, Eliza and immunohistochemistry.

The main advantage of this technique over existing methods like mirroring xenograft models is the ECX over handling and imaging of human tumors. Human tumor cells with supportive human thro cells and extracellular matrix compounds can grow in vivo and chicken embryos that still lack adaptive immune responses. This method can help develop novel therapies and enables fast preclinical screening for the treatment of patients suffering from multiple myeloma.

The visual demonstration of this method is critical because human multiple myeloma cells easily undergo apoptosis due to stress and cheek embryos can die when mishandled what due to appropriate exor culture conditions Demonstrating the procedure and all its critical steps will be Cornelia. Hes and experience technician from our laboratory. For this protocol, prepare cultures of multiple myeloma cell lines, OPM two RPMI 8 2 2 6, and human mesenchymal stem cells from bone marrow incubate the cells normally in supplemented medium following the text protocol transfect a million multiple myeloma cells with 100, 000 lentiviral particles.

In a 24 well plate with complete growth medium after three days, start selecting for transfected cells by adding two micrograms of blastin for every milliliter of culture medium or use 500 microliters of neomycin per milliliter for commercially available EGFP lentivirus after two weeks of selection, look for clusters of cells expressing EGFP. Collect, floating or adherent cell clusters by carefully Resus suspending them in a centrifuge tube pellet, D-E-G-F-P cells at 1000 GS for five minutes and expand them out as sublines. After selection and propagation of GFP expressing multiple myeloma cells, always check their ability before using them for further experiments.

To begin chill some collagen type one solution and 10 XDMM on ice. Once cooled, mix a one-ten volume of the 10 X-D-M-E-M medium into the collagen solution. Then add 0.2 normal sodium hydroxide dropwise to neutralize the solution to a pH of 7.4.

Keep the solution on ice and confirm the pH with a test strip. Next, mix the transgenic multiple myeloma cell lines with the human mesenchymal cells. Now centrifuge the cell mixture in 15 milliliter tubes at 1000 Gs.For five minutes, discard the supernatant and using a P 1000 pipette re suspend the pellet in a milliliter of the cold collagen mixture.

Using a P 100 tip immediately transfer 30 microliter aliquots of the mixture onto sterile paraffin. In a 24 well culture plate, allow the mixture to polymerize for 30 minutes at 37 degrees Celsius, thus forming steroids. Next, overlay the steroids with a milliliter of prepared media with different treatments.

For example, a drug can be applied at different concentrations. After 72 hours of incubation at 37 degrees Celsius, document the PHE by fluorescent stereo microscopy. Then using wide flat jaw forceps, transfer each steroid to a reaction tube to measure the GFP expression.

In preparation for this procedure, incubate chicken eggs in an egg incubator at 37 degrees Celsius and 70%humidity for three days. Then open the eggs and transfer the embryos to sterilized square 10 centimeter plastic, weighing boats with cell culture platelets To achieve higher survival rates of chicken embryos in X OVO culture. Use plastic waiting boats instead of 10 centimeter culture tissue.

Moreover, sterilize and prevet with long PBS waiting boats before use. Continue incubating the eggs X ovo for six more days during which the CAM will develop. Now prepare multiple myeloma and mesenchymal cell PHE as in the previous procedure, but in a six well plate, then using forceps, transfer the steroid as on plants to the untreated surface of the cam about two centimeters away from the embryo of nine day old chicken embryos.

Position four on plants onto each chicken embryo. Let the ex ovo embryos with the on plants sit in the egg incubator for five days. Then document the xenografts by fluorescent stereo microscopy.

After documentation, use ophthalmic scissors and forceps to remove the xenografts with some adjacent camp tissue. To measure GFP expression or to perform immunohistochemical analysis, begin by transferring each multiple myeloma steroid or excise xenograft into 0.5 milliliters of RIPA with protease inhibitors. Then use a homogenizer on ice to blend the tissues once homogenized, freeze and thaw the homogenates using liquid nitrogen and then a 37 degrees Celsius water bath.

Do this three times, then centrifuge the homogenates at four degrees Celsius for 20 minutes at 12, 000 G and store the supernatants. Next, dilute some supernatant at one to 20 in the assay buffer of the ELIZA kit. Then measure the GFP levels with biotinylated anti GFP antibodies using a kit.

Begin with an overnight fixation of the excise xenografts in para formaldehyde. The next day dehydrate and embed the fixed xenografts. First, load them into cassettes.

Then move the cassettes through a graded alcohol series. Let each bath persist for a full hour and end the process with paraffin embedding. Next, make five micron section xenografts using a benchtop rotary microtome bake the collected paraffin sections on glass slides overnight at 56 degrees Celsius.

The next day, de parize the sections using a decreasing graded alcohol series to double distilled water. The slide should be in each bath for just 10 minutes. Now follow the text protocol to incubate with the respective primary and secondary antibodies and perform calor metric detection with enzyme and substrate after the antibody staining counterstain with hematin and mount dissects with a synthetic mounting medium.

Two EGFP expressing multiple myeloma. Cell lines were established in cultivated for three days in the presence of the drug. Bortezomib tumor cells and steroids were then visualized by the expression of GFP on a stereo fluorescence microscope.

Tumor cell mass after drug treatment was quantified after homogenization of PHE and measuring EGFP contents by A GFP Eliza. In another experiment, three day old chicken embryos were cultivated X ovo for six days. At day nine, PHE of multiple myeloma cells were grafted on.

Four on plant grafts were attached to each embryo. The target substance bortezomib was applied topically at one mol after five days, the on plants formed tumors compared to controls. Bortezomib inhibited the growth of multiple myeloma cells in graft based on visualization of fluorescent tumor mass.

Single graft from three different animals were then excised, homogenized, and thereafter measured by GFP Eliza. Compared to controls, bortezomib treated tissue had significantly less myeloma cell mass. After watching this video, you should have a good understanding of how to perform experiments if your cancer cells of interest in ex over cultivated chicken embryos to analyze tumor growth and genesis and invasion into host tissue After its development.

This technique paved the way for researchers in the field of multiple myeloma to explore noble compounds or drugs in a easy to handle model organism, which is requisite for preclinical drug testing.