Supplementary Materials
Anal Tumor Digestion
In a biosafety cabinet, 1x phosphate buffered saline ((PBS), Corning, Tewksbury, MA, USA) was aspirated from a sterile tube containing the mouse anal tumor tissue and chelation buffer was then added to cover the tissue and placed on ice for one hour.1,2 All stocks and buffers were prepared as described in Pasch et. al., 2019 supplemental materials.3
Next, the tumor was removed and placed in a new sterile tube. Digestion buffer was added to fully cover the tumor, and the tube was placed in a 37°C incubator for approximately 1 hour. The tissue in the tube was checked every 20 minutes and shaken or mechanically disrupted with a pipette tip until properly digested.
Then, the tube with digested cells was placed in the centrifuge at 1800 rpm, 4°C, and 5 minutes with low brake. The supernatant was aspirated in order not to disturb the cells from the bottom of the tube.
Spheroid Plating
The cells were resuspended in ADF stock and subsequently transferred via pipette to a new tube.
A 1:1 solution of 50 μL of cold Matrigel (Corning, Tewksbury, MA, USA) or Extracellular Matrix (ECM) gel (Sigma Aldrich, Saint Louis, MO, USA) and 50 μL cell suspension was gently mixed in a new 1.5mL tube. A total of 50 μL of the gel and cell suspension mixture was carefully pipetted into the center of a pre-warmed 24-well cell culture plate. This process was repeated until all 24 wells were filled.
Spheroid Maturation
The 24-well cell culture plate was placed in an incubator at 37°C for one hour to allow time for the gel and cell suspension mixture to set. After ensuring solidification, 450 μl of warm ADF feed was carefully pipetted in each well taking care to avoid the Matrigel. ADF feed was created from a mixture of 12 mL ADF stock and 12 μL 1000x Epidermal Growth Factor (EGF) stock (Thermo Fisher Scientific, Grand Island, NY, USA). The cell culture plate was returned to the incubator overnight for a minimum of 12 hours. The following day, individual wells were assessed for signs of contamination. Plate wells found to be contaminated were excluded from the remainder of the experiment. Next, cells were passaged by aspirating old media and adding a fresh 450 μL of media to the well to break up the Matrigel/ECM droplet to release cells. The mix of cells in media were combined into a single 15mL conical tube, spun in a centrifuge at 1800 rpm, 4°C, and five minutes with low brake. The supernatant was aspirated so as to not disturb the cells at the bottom of the tube. Spheroid plating was performed as previously described.
After another 24 hours, the wells were again assessed for contamination, and excluded if contaminated. Media was replaced 24 hours post-passage.
1. Foley TM, Payne SN, Pasch CA, et al. Dual PI3K/mTOR Inhibition in Colorectal Cancers with APC and PIK3CA Mutations. [published online ahead of print, 2017 Feb 9]. Mol Cancer Res. 2017;15:317–327. 10.1158/1541-7786.MCR-16-0256 PubMed</jrn>
2. Xue X, Shah YM. In vitro organoid culture of primary mouse colon tumors. J Vis Exp.
2013;e50210:e50210. 10.3791/50210 PubMed</jrn>
3. Pasch CA, Favreau PF, Yueh AE, et al. Patient-Derived Cancer Organoid Cultures to Predict Sensitivity to Chemotherapy and Radiation. Clin Cancer Res. 2019;25:5376–5387.
10.1158/1078-0432.CCR-18-3590 PubMed</jrn>
