ER+ breast tumor organoid media (BTOM-ER)

For BTOM-ER growth media, 2.145 ml of Reagent A, 50 ml of Reagent B, and 1.0% penicillin–streptomycin were added to 100 ml of DMEM/F-12. Please see Table 1 for components of Reagent A and Reagent B and Additional file 1 for further details on components and media recipes.

Establishment of PDX organoid cultures

PDX tumor tissue was placed in a 10 cm dish, and 1–5 ml of resuspension media (DMEM-F12, 1.0% Penicillin–streptomycin, and 1.0% BSA) was added to preserve the viability of the cells during processing. Tissue was then minced using sterile surgical scalpels into fragments ranging from 0.5 to 1.0 mm. Minced tissue was then transferred to a 15 ml conical tube and pelleted by centrifugation at 1500 rpm for 5 min at 4 degrees centigrade. After the supernatant was carefully removed, 2–10 ml of digestion media (DMEM-F12, 0.1 mg/ml Collagenase/Dispase, and 1.0% Penicillin–streptomycin) was added, and the tube was placed in a 37 degrees centigrade shaker for 30–90 min, with checking every 15 min to check progress and ensure proper digestion of the tissue. The tissue is then pelleted again and resuspended in Accutase for 15–30 min to digest tissue into organoid fragments further. The tissue is then put through a 250 mm strainer to remove debris or large tissue fragments while allowing organoids to pass through. Organoids are pelleted and gently resuspended in BTOM-ER containing 5% Matrigel and ROCK-inhibitor (10 mM Y267632, Tocris), and then plated on top of a solidified Matrigel-coated well. Media was changed every 3–4 days.

Passaging of PDXOs

After the removal of BTOM-ER, digestion media is added to each well, and the plate is incubated at 37 degrees centigrade for 1.5 h. When the Matrigel has become a slurry and organoids are dispersed into small clumps or clusters, cold resuspension media is added at a 1:1 volume ratio to each well, and the suspension is transferred to a 15 ml conical tube. After centrifugation at 1500 rpm for 5 min, the supernatant is removed, and the pellet is resuspended in Accutase and incubated for 20–30 min at 37 degrees centigrade. After incubation, resuspension media is added to the suspension. After centrifugation and removal of the supernatant, the pellet is resuspended in BTOM-ER and plated as done during the establishment of cultures.

Immunohistochemistry (IHC) and imaging of PDXOs

Immunohistochemistry was performed as previously described [13]. Briefly, organoid tissue sections were generated by plating 20,000 cells/well of an 8-chamber slide. After the formation of mature organoid structures (typically 7–10 days post-plating), organoids were fixed in 4% PFA for 2 h. After treatment with hematoxylin solution for 15 min, organoids were washed twice with water. The organoids were then scraped onto a solidified layer of histogel, with additional histogel added on top to create a sandwich within a cryomold. Once solidified, the histogel sandwich was transferred to a tissue cassette and fixed in 10% formalin for 16–24 h, followed by a brief wash in 70% EtOH. The cassettes were stored in 70% EtOH until submitted for sectioning. To obtain brightfield images of organoids in culture, cells were plated at a density of 25,000 cells/well, and images were taken at 20 ×, 10 ×, and 4 × magnification using Spot Imaging software.

Proliferation and doubling time of PDXOs

Organoids were digested as above and treated with TrypLE instead of Accutase to generate single cells. Each assay well in a 96-well plate was pre-coated with 30 ml of Matrigel and was allowed to solidify in a 37 degrees centigrade incubator for at least 10 min before plating cell suspensions on top. Cells were resuspended in BTOM-ER at a density of 50,000 cells/ml, and 100 ml was added to each well in triplicate. On the indicated days, corresponding wells were assessed for viability using 3D Cell Titer Glo (Promega). Viability measurements were normalized to Day 0, and doubling time was calculated using GraphPad Prism software.

Western blot analysis

Organoids were grown in six-well plates at a density of 250,000 cells/ml. Once organoids reached confluency, each well was washed with ice-cold PBS. Matrigel was broken up by pipetting, and the slurry was transferred into a 15 ml conical tube. An additional 1.0 ml of PBS was added to the well to collect any remaining organoids. Organoids were pelleted by centrifugation at 3000 rpm for 5 min, and the supernatant was removed. The pellet was resuspended with 1.0 ml of Cell Recovery Solution (Corning) and incubated for one hour on ice. The released organoids were pelleted by centrifugation, and the supernatant was removed. After washing the pellet once with ice-cold PBS, the cells were resuspended with RIPA buffer, and western blotting analysis was performed. Signals were detected using Amersham Imager System via chemiluminescence.

Estrogen responsiveness and dependence of PDXOs

For both estrogen responsiveness and dependence, organoids were digested and plated as above 40–50,000 cells/ml, depending on the organoid line. Three days after plating, media was refreshed, and wells were treated with either 1.0 nM of b-estradiol or vehicle control (EtOH) using the Tecan D300e drug dispenser. For dependence, media was replaced with fresh BTOM-ER or phenol red-free BTOM-ER. Media was refreshed every 2–3 days. After 10 days, organoids were assessed for viability using 3D Cell Titer Glo (Promega). Viability measurements were analyzed using GraphPad Prism software.

Endocrine therapy treatment of PDXOs

Organoids were prepared as above, and on day three, media was refreshed, and wells were treated with either vehicle control or indicated concentrations of fulvestrant (Selleckchem) using the Tecan D300e drug dispenser. Media was refreshed, and plates were retreated every 2–3 days. After 5 days of treatment, organoids were assessed for viability using 3D Cell Titer Glo (Promega).

Statistics and reproducibility

Error bars were generated by Standard Error Mean (SEM) calculations. For experiments with two conditions, an unpaired one-tailed Student’s T-test was performed. For experiments with three or more conditions, one-way ANOVA followed by a Bonferroni comparison was used. Doubling time was calculated by applying a non-linear regression for exponential growth for each condition.