Cancer Research
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Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells
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Summary May 5th, 2022
This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.
Transcript
We developed a rapid and efficient protocol to generate bladder cancer organoids via ex vivo gene editing of normal mouse urothelial cells carrying floxed alleles in genes of interest. This ex vivo method has a one-to-two-week workflow instead of years of mouse breeding, and it is highly efficient in adenovirus transduction on the Cre-mediated gene deletions. To begin, prepare all sterile instruments and reagents, including scissors, forceps, DPBS in 35-milliliter culture dishes, 70%ethanol, gauze, and clean paper towels.
Next, clean all surfaces of the dissection area and cover the dissection area with a clean paper towel. Place the euthanized mouse on the gauze in the dissection area and spray 70%ethanol on the mouse's abdomen, then using sterile dissection scissors, make a lower midline abdominal incision to expose the bladder. Now, use the forceps to grasp and gently pull up the bladder to identify the bilateral ureterovesical junction.
Using scissors, remove the bladder fundus, following the boundary line between the two ureter openings and transfer the bladder into a sterile dish containing 2 milliliters of DPBS, then remove the fatty and connective tissue and put the bladder into a new dish containing 2 milliliters of DPBS. Also remove the adenovirus from the minus 80-degree Celsius storage and keep it on ice. For cell dissociation, prepare all sterile instruments and reagents as described in the manuscript, then in a biosafety cabinet, transfer the bladder to a 35-millimeter dish and wash with 2 milliliters of sterile DPBS.
Collect bladder tissues from four mice into a dish with one milliliter of complete culture medium without charcoal-stripped FBS. Next, using a surgical blade, mince each bladder into four equal pieces, then using forceps, unfold the bladder to expose the urothelium surface to the medium. Transfer the bladder pieces and medium into a 15-milliliter centrifuge tube.
Wash the dish twice with 2 milliliters of complete culture medium without charcoal-stripped FBS and add the wash to the centrifuge tube, bringing the total volume to 5 milliliters. Next, add a collagenase and hyaluronidase mixture and place the tube horizontally in a 37-degree Celsius orbital shaker incubator for 30 minutes at 200 RPM. After tissue digestion, add an equal volume of 10%charcoal-stripped FBS and DPBS into the tube and centrifuge at 200 times g for 5 minutes.
After discarding the supernatant, add 2 milliliters of trypsin substitute into the cell pellet and mix well, then incubate the tube at 37 degrees Celsius and 5%carbon dioxide for 4 minutes. After incubation, pipette the cells up and down 10 times with a standard P1000 tip and add 5 milliliters of 10%charcoal-stripped FBS and DPBS. Strain the dissociated cells through a 100-micrometer sterile cell strainer and collect the cell suspension in a 50-milliliter tube.
Transfer the cell suspension to a 15-milliliter tube and centrifuge at 200 times g for 5 minutes. After removing the supernatant, resuspend the pellet in 1 milliliter of complete culture medium. Count the number of cells using a hemocytometer and plate 0.5 times 10 to the 6th cells into each well of a 24-well plate with 0.5 milliliters of fresh complete culture medium.
Next, take out the matrix extracts of Engelbreth-Holm-Swarm mouse sarcoma cells from minus 20-degree Celsius storage and thaw them on ice. Also, prewarm a 6-well plate in a 37-degree Celsius cell incubator. For adenoviral transduction, add 2 microliters of adenovirus into the 24-well plate and mix well with the dissociated urothelial cells.
To enhance transduction efficacy, perform spinoculation by centrifuging the plate at 300 times g for 30 minutes, then incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 1 hour. Transfer cells and medium from the well into a 15-milliliter tube and centrifuge at 200 times g for 5 minutes. After discarding the supernatant, add 50 microliters of complete culture medium and mix well with the cells at the bottom.
Next, add 140 microliters of matrix extract and gently mix well to avoid air bubbles, then create domes for organoids by quickly adding 50 microliters of the solution per dome into the prewarmed 6-well plate. Incubate the plate and allow the domes to solidify for 5 minutes. After 5 minutes, flip the 6-well plate upside down and continue solidification for an additional 25 minutes.
After incubation, add 2.5 milliliters of complete culture medium to each well and place the plate in an incubator for organoid culture. The green fluorescent protein was detected in nearly 100%of cells, suggesting high efficiency of adenovirus transduction. Hematoxylin and eosin staining and immunohistochemistry of triple knockout organoids tumors showed the morphology of high-grade urothelial carcinoma with positive protein expression of basal subtype markers of CK5 and p63.
The un-recombined floxed alleles were detected in urothelial cells, untreated with adenovirus, whereas the recombined alleles were observed in the triple knockout organoids. In general, 2 to 3 weeks were needed to form a 2-centimeter subcutaneous tumor. The immunohistochemistry of triple knockout organoids, in vivo xenografts, displayed positive expression of CK7, CK5, and p63 proteins.
Negative expression was detected for CK8 and uroplakin proteins. The mesenchyme marker, vimentin, was detected only in the tumor capsule or stroma. The mouse bladder dissociation must not exceed 30 minutes.
A longer dissociation time may increase in the percentage of the stroma cells, such as endothelial cells and the fibroblasts This approach can be combined with cell sorting or cell-type a specific adenovirus to generate the cell type of specific organoids, such as lumen versus basal organoids.
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