This protocol provides standard and reproducible methods to assess pharmacological responses in prostate organoid cultures. Prostate organoid cultures provide an in vitro system that preserves many aspects of in vivo biology and pharmacological response by allowing cells to adopt a three-dimensional structure in a basement membrane matrix. We are particularly excited about using these methods to assess how genotype dictates pharmacological response in the prostate and to model drug resistance.
These methods can be applied to organoid culture systems that use the dome plating method. However, the components in the media such as growth factors may differ depending on tissue type. Visual demonstration will allow for a more specific and detailed discussion of the protocol steps and how they differ from the many other published organoid culture systems available to researchers.
Organoid culture is typically more time consuming than two-dimensional cell culture. Be sure to allocate extra time for these methods. Start by isolating prostate organoids from mouse or human tissue.
Mince and enzymatically digest the tissue to produce a single cell suspension, then collect the cells by centrifugation at 300 times g for five minutes. Count the cells and resuspend them in the basement membrane matrix. Then plate them at the appropriate density in the matrix domes on pre-warmed organoid culture plates.
Domes should be two millimeters apart from one another and from the side of the well. Once solidified, gently add media from the side of the well to avoid disrupting the matrix. After the domes have solidified, add media on top of the dome so that they are completely covered.
Grow organoids to the desired quantity for downstream applications determining cell number with standard counting methods. When the organoids are ready to use, draw up the medium with a Pten00 pipette and pipette it up and down to disrupt the basement membrane matrix. Transfer the suspension to a 15 milliliter conical tube and centrifuge at 300 times g for five minutes.
Aspirate the supernatant and wash the cell pellet with five milliliters of PBS. Repeat the centrifugation, then resuspend the pellet in four milliliters of trypsin replacement and allow it to digest for five to 10 minutes while shaking at 37 degrees Celsius. Add an equal volume of organoid medium with 10%FBS to inhibit the trypsin replacement and centrifuge the tube at 300 times g for five minutes.
Aspirate the supernatant and resuspend the cells in one milliliter of PBS. Then strain the cells with a 40 micrometer filter to ensure single cell suspension and count them using a hemocytometer. Dilute the cell suspension to 100 cells per 10 microliters using organoid medium with 10 micromolar rho kinase inhibitor Y27632.
Transfer 1, 100 cells to a new conical tube and add 285 microliters of basement membrane matrix which will result in a 70%matrix concentration. Next, seed the cells in 35 microliters of matrix domes in a pre-warmed 24 well plate making sure to plate three to five replicates per sample. Flip the plate and place it in a cell incubator to solidify the basement matrix.
After 10 minutes, remove the plate from the incubator and add medium containing the rho kinase inhibitor. Refresh the medium every two days and after seven days, count the number of organoids established per dome and calculate the percent of organoids formed out of the total number of cells. After isolating the organoids as previously described, seed 1, 000 to 10, 000 cells in the matrix dome using organoid formation efficiency and growth speed as a proxy for determining the final cell number.
Then seed 35 microliters of matrix domes in a 24 well plate and let the dome solidify. Add medium containing the rho kinase inhibitor and the drug of choice. Perform a log 10 incremental to determine half maximal inhibitory concentration using the vehicle in which the drug was dissolved as a control.
Refresh the medium every two to three days and analyze the organoids on day seven to determine pharmacological response to the drug by performing a cell viability assay as described in the manuscript. To plate organoid fragments without trypsinization, use a Pten00 pipette to aspirate the medium and pipette up and down to disrupt the basement membrane matrix. When the matrix is fully disrupted, transfer the suspension to a 15 milliliter conical tube and centrifuge at 300 times g for five minutes.
Aspirate the supernatant and add five milliliters of PBS. After the wash, resuspend the organoids in one milliliter of PBS and disrupt the organoids by trituration with a glass Pasteur pipette. Quantify the number of organoid fragments and seed five replicates with 100 fragments as previously described.
Seven days later, perform a cell viability assay according to manuscript directions. Wild type prostate basal cells demonstrated superior organoid formation compared to luminal cells. A minor increase in organoid formation was achieved with CRISPR-Cas9-mediated loss of Pten or P53 and loss of both further increased formation capacity.
The effects of anti-androgenic molecules on growth were tested in murine organoids with different genotypes. Loss of P53 did not cause resistance to the anti-androgenic molecules, but loss of Pten increased resistance. Dual loss of P53 and Pten, however, resulted in complete resistance.
Androgen receptor inhibition also altered phenotypes. Wild type Pten deleted and P53 deleted organoids demonstrated the decrease in organoid lumen size while organoids with loss of both Pten and P53 were phenotypically unaffected. As expected, when these cells were grafted subcutaneously in the flank, only the double deleted Pten and P53 organoids grew.
Two distinct human prostate cancer organoids MSKPCA2 and MSKPCA3 were tested for their response to anti-androgenic molecules. Proliferation of MSKPCA2 organoids was strongly inhibited whereas MSKPCA3 organoids remained unaffected. MSKPCA2 expressed high levels of androgen receptors and FKBP5 as well as hallmark luminal proteins.
MSKPCA3 also expressed basal and mesenchymal markers and showed no expression of FKBP5 suggesting that these organoids model a non-luminal androgen-independent phenotype. Organoids needs to be disrupted periodically either by trypsinization or trituration with a glass pipette in order to remain viable. Frequency depends on species origin and genotype.
Any type of next generation sequencing or proteomics experiment can be performed following the procedures described. These experiments would investigate the molecular basis of the phenotypes and pharmacological responses. This three-dimensional organoid culture technique allows researchers to study pharmacological responses in a highly controlled genetic context in vitro.
This can be a reliable alternative to lengthy in vivo studies.
