This protocol can be used and further developed to analyze how ionizing radiation affects tumor cell recruitment, leading to a greater understanding of cancer recurrence. Mammary organoids are powerful models. They mimic basic in vivo characteristics, but they allow for easy isolation of biological variables.
Use of low-adhesion plates negates work-flow challenges associated with protein matrices. Triple-negative breast cancer patients experience local regional recurrence at higher rates following therapy. Studying how radiation influences tumor and immune cell behavior will lead to important insights into recurrence mechanisms.
Demonstrating confocal microscopy of the organoids is Javier Gomez, a graduate student in the Silvera Batista lab. Begin this procedure with removal of abdominal and inguinal mammary glands from sacrificed mice as detailed in the text protocol. 45 minutes after irradiation of the samples as described in the text protocol, place mammary glands in a 35-millimeter sterile cell plate and mince with scalpels.
Mince with approximately 40 strokes until the tissue relaxes and pieces are obtained that are no larger than approximately one square millimeter in area. Now transfer the tissue to collagenase solution in a 50-milliliter centrifuge tube. Use 10 milliliters of collagenase solution per mouse.
Place the sample tubes in a water bath for 30 to 60 minutes at 37 degrees Celsius, vortexing for five seconds every 10 minutes. Digestion is complete when the collagenase solution is cloudy. Spin down the digested solution at 450-times-G for 10 minutes at room temperature.
Now three layers are observed. The supernatant is composed of fat, the middle layer is an aqueous solution, and the bottom is a pellet. The pellet appears red, as it is a mixture of epithelial cells, individual stromal cells, and red blood cells.
Pre-coat all pipettes, pipette tips, and centrifuge tubes with BSA solution prior to contact by addition and removal of BSA solution. BSA solution can be reused, although it should sterile-filtered before each experiment. For additional recovery, transfer the supernatant to a fresh BSA-coated 15-milliliter tube.
Pipette up and down vigorously to disperse the fat layer. Centrifuge the sample at 450-times-G for 10 minutes at room temperature. Aspirate the supernatant, leaving a small amount of media in the tube to avoid aspirating the cell pellet.
Now aspirate the aqueous layer from the tube with the original pellet. Add 10 milliliters of DMEM/F12 to the tube with the original pellet, and transfer to the second tube. Pipette vigorously to combine and resuspend the two pellets.
After centrifuging at 450-times-G for 10 minutes at room temperature, aspirate the supernatant and add four milliliters of DMEM/F12 to the tube. Add 40 microliters of deoxyribonuclease to the suspension and gently shake by hand for two to five minutes at room temperature. Add six milliliters of DMEM/F12 and pipette thoroughly.
After centrifuging the tube at 450-times-G for 10 minutes at room temperature, aspirate the supernatant to the 0.5-milliliter mark. Resuspend the pellet in 10 milliliters of DMEM/F12 and pipette thoroughly. Now centrifuge the tube by pulsing to 450-times-G and stopping four seconds after reaching that speed.
Repeat the washing steps three more times to purify organoids via centrifugal differentiation. The pellet should now be an off-white color consisting of only epithelial organoids. Resuspend the pellet in 10 milliliters of DMEM/F12.
Pipette thoroughly to create a homogenous solution. Transfer 50 microliters to a 30-millimeter Petri dish and view under a phase-contrast microscope at 20X. Count the number of organoids with a tally counter.
Pipette 50 microliters of organoids into each well of a low-adhesion plate. Add 150 microliters of organoid medium to bring the total working volume to 200 microliters. Carefully replace the medium every two days.
Maintain GFP or D-tomato-labeled raw 264.7 macrophages in DMEM media supplemented with 10%FPS and 1%penicillin-streptomycin. Seed the cells into the organoid medium at the desired density. Use live cell phase contrast and fluorescent imaging to monitor macrophage infiltration over time.
Remove organoid medium from the wells by carefully aspirating. Fix the samples with 10%neutral-buffered formalin for 15 minutes at room temperature. Wash the fixed organoids three times for five minutes in 1X PBS.
If desired, fixed samples can be stored at four degrees Celsius for one week for further staining. Following wash, permeabilize the fixed organoids for five minutes. Block the fixed organoids with 5%normal goat serum in 0.1%PBST for one hour at room temperature before washing three times for five minutes with PBS.
Incubate the fixed organoids with anti-cytokeratin-14, E-cadherin, or tight-junction protein one, in 1%NGS in PBST for one hour at room temperature. Then wash three times for five minutes in PBST. Now incubate the organoids with goat anti-rabbit secondary antibody with 1%NGS PBST for one hour at room temperature.
Cover with foil to avoid light exposure. After washing as before, use the nuclear dye to stain nuclei for approximately a half-hour. Wash the stained organoids for five minutes in PBS three times.
If using a chamber slide, mount with a cover slip. Store the organoids wrapped in foil at four degrees Celsius for up to two weeks. Irradiated organoids could be cultured in low-adhesion plates, or within basement membrane, but the most rapid growth occurred in low-adhesion plates.
Organoids recapitulated mammary gland characteristics. Constructs that are morphologically similar to ducts and lobes were observed. Growth trends indicated that when organoids were immediately seeded after digestion and sorting, non-irradiated organoids grew faster than irradiated organoids, most likely due to cell growth arrest resulting from mechanisms of DNA damage repair.
Organoids expressed epithelial characteristics, which were evaluated through immunofluorescent staining. Irradiated organoids expressed epithelial markers. Cytokeratin-14, a marker of myoepithelium, was expressed strongly on the surface of irradiated organoids.
Additionally, E-cadherin and tight-junction protein one were expressed within cellular junctions of organoids. These proteins are essential for proper cell adhesion. After irradiation, organoids continued to retain their epithelial characteristics.
Fluorescent staining of organoids could be visualized within low-adhesion plates using fluorescence microscopy. However, the clearest visualization was obtained via confocal microscopy. Corrected total fluorescence intensity was calculated by subtracting the background, and normalizing by organoid area.
Growing organoids in the 96-well low-adhesion plates also simplified co-culture experiments. When seeded at concentrations typical in the mammary gland, macrophage colocalization increased with irradiated organoids. When performing this procedure, the most important things to remember are to not over-digest the organoids, and to appropriately purify the centrifugal differentiation.
Following this procedure, additional co-culture experiments can be performed. Organoids can be co-cultured with tumor cells, other immune cells, and stromal cells to recreate the irradiated microenvironment associated with recurrence. This method will be important for evaluating how normal tissue damage specifically influences immune cell dynamics, and will eventually be used to understand tumor cell recruitment mechanisms.
Mammary organoids have elucidated mechanisms of mammary gland development, and have robustly recapitulated breast cancer in vitro. We hope that our model provides insights about radiation-induced tumor cell recruitment.
