We describe strategies and step-by-step protocols to establish serial 3D patient-derived organoids of bone metastatic prostate cancer. Our optimized protocols are practical to set up 3D cultures for experiments using limited starting material directly from patients or patient-derived xenograft tumor tissues. The 3D organoids from this protocol can be used as ex vivo models to understand basic mechanisms of bone metastatic prostate cancer pathology and to test treatment.
The 3D organoid culture media in this protocol is specific for prostate-derived cells. Other parts of our protocols are applicable to different types of tumor tissues and metastatic sites. The doming technique may prove to be the most difficult part of this process.
Practicing the doming technique will ensure a consistent size of the domed mixture of organoids, media, and Matrigel, and minimize bubble formation during sample suspension. Visually demonstrating this method provides a better understanding of how to handle the viscous Matrigel for successfully culturing lower density organoids from a low number of cells. Start by resuspending the cell pellet in the appropriate volume of basement membrane for a 24-well plate setup.
Pipette up and down gently to ensure that the cells are resuspended, then pipette the appropriate volume of the cell suspension into the center of a pre-warmed tissue culture plate. Invert the plate and immediately place it upside down in the cell culture incubator set at 37 degrees Celsius and 5%carbon dioxide for 15 minutes. Pipette the appropriate volume of pre-warmed medium containing 10 micromolar Y-27632 dihydrochloride into each well.
To form a floating dome from the attached round dome, detach the dome from the plate using a cell scraper. Cut a two by four inch piece of paraffin film and place it on top of the divots of an empty tip holding rack from a one milliliter plastic pipette tip box. Use a gloved index finger to gently press down on the paraffin film to trace the divots taking care not to break through the film.
Spray the paraffin film with 70%ethanol and turn on the UV lamp in the cell culture hood to sterilize the prepared film for at least 30 minutes. Meanwhile, resuspend the pre-processed cells in 20 microliters of basement membrane. Then pipette the suspension into the divots formed in the paraffin film.
Place the solidified beads and paraffin film into a six-well plate and pipette three to five milliliters of pre-warmed medium containing 10 micromolar Y-27632 dihydrochloride into each well while gently brushing beads off of the paraffin film. To process the organoids for histology, remove existing media from the well taking care not to aspirate the basement membrane domes. Add an equal volume of cell recovery solution and incubate the plate for 60 minutes at four degrees Celsius.
After the incubation, dislodge the basement membrane dome using a pipette and crush it with a pipette tip. Collect the dissociated dome and the cell recovery solution into a 1.5 milliliter tube. Centrifuge the tube at 300 times g in four degrees Celsius for five minutes, then remove the supernatant and set it aside.
Save all the supernatants until the final step when the presence of organoids is confirmed at the desired volume of cold PBS and gently pipette up and down to mechanically dislodge the pellet without disrupting the organoids. Repeat the centrifugation and remove the supernatant. Fix the pellet in a matched volume of 4%PFA for 60 minutes at room temperature.
After fixation, repeat the centrifugation step and PBS wash. Then slowly add 200 microliters of warm 2%agarose and immediately but gently detach the cell pellet from the tube wall without disrupting it using a 25 gauge needle attached to a one milliliter syringe. For the agarose spin down method, it is critical to detach the cell pellet from the wall of the tube right after adding agarose using a 25 gauge needle.
Once the agarose has completely solidified, detach it from the tube with a 25 gauge needle attached to a one milliliter syringe and transfer it to a new 1.5 milliliter tube. Fill the tube with 70%ethanol and proceed with the conventional protocol for tissue dehydration and paraffin embedding. This protocol was successfully used to establish 3D organoids from patient-derived xenograft models of bone metastatic prostate cancer as well as directly from the cancer tissue.
The organoids displayed different phenotypes that manifested as cysts, spheroids, and higher complexity organoids. An entire dome of basement membrane and organoids can be seen in a stitched image from 25 high resolution, 10X magnification images. To further investigate the tumor tissue, immunofluorescent cytochemistry was performed on a five micrometer thick paraffin section of organoids targeting the basal epithelial cell marker cytokeratin-5, luminal epithelial cell marker cytokeratin-8 and DAPI.
This protocol can be optimized for other applications such as Western blotting, co-culture and flow cytometry to explore characteristics of 3D organoids and the effects of drug treatments. These experiments could address mechanisms of drug resistance and the efficacy of novel therapeutics alone or in combination with current therapies. The 3D organoids from this technique retain inter-subject and intra-subject heterogeneity and therefore are a more accurate model of the disease in patients.
This technique paves the way for researchers to explore mechanisms of drug resistance, tumorigenesis, metastasis and therapeutics that may be more predictable of the disease progression in patients and their response to therapy.
