Normal goat serum (ngs)

Spheroids were embedded in an optimum cutting temperature compound (OCT), frozen on dry ice, and stored at −80 °C. Frozen sections (10 μm) were cut using Cryostat CM1950 (−20/−19 °C). All staining incubations were performed at RT and all reagents were rinsed once with PBS. Sections were fixed in PFA 4% for 20 minutes and washed 3 times for 5 minutes. Sections were then incubated for 30 minutes in 3% normal goat serum (Vector Laboratories, California) for blocking. Blocking was removed and anti-CD31 (Abcam, MA, USA, 1:50) in 3% normal goat serum was added for an overnight incubation at 4 °C. Slides were washed 3 times for 5 minutes and incubated with a secondary antibody labeled with Alexa fluor 488 (goat anti rabbit, Abcam, MA, USA) (1:100) in 3% normal goat serum for 1 hour. Sections were then washed 3 times before incubation with DAPI. Mounting media was applied on the slides and samples were visualized using a fluorescent microscope (Olympus IX-73). Immunohistochemically staining was performed using DAB kit as detailed in the Supplementary Information.

To test substrate accumulation in GM05653 patient cells, we used a commercially available GM1 antibody for immunostaining. Briefly, GM05653 cells were grown for 7–10 days to permit substrate accumulation and then fixed (4% paraformaldehyde in PBS) and permeabilized using normal goat serum (Thermo, catalog no. 50062Z) with 0.01% Triton X-100. Cells were then blocked in normal goat serum and then stained with an anti-GM1 antibody (Abcam, catalog no. ab23943, diluted 1:250 in normal goat serum) overnight at 4 °C. Cells were imaged on an ImageXpress high-content microscope as described previously. Granular immunostaining with anti-GM1 antibody was quantified using a granularity algorithm within MetaMorph software. GM1 granularity was calculated as total granule area multiplied by average granule intensity divided by the number of cells and expressed as arbitrary units.

At predetermined time points from their seeding, spheroids were fixed in 4% PFA and left at room temperature (RT) on an orbital shaker for 1 h. Spheroids were then washed and left in phosphate-buffered saline (PBS) for 5 min, repeated 3 times. Next, spheroids were removed from their molds by gentle pipetting using trimmed tips and transferred into 1.5 mL mini test tubes to ensure complete exposure to solution in the next steps which were all performed at room temperature (RT) on an orbital shaker. Fixed samples were incubated for 15 min in 0.1% Triton X-100 for permeabilization and were then washed and left in PBS for 5 min, repeated 3 times. Next, co-cultured spheroids were incubated with a blocking medium containing 3% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h. Blocking was removed and primary antibodies: Mouse anti-human fibroblasts monoclonal antibody (Chemicon, Temecula, CA, USA, CBL271,1:100) and conjugated anti-CD31 antibody (Abcam, Cambridge, UK, ab215912, [JC/70A] Alexa Fluor 647, 1:100) were added successively in the 3% normal goat serum for an overnight incubation at 4 °C on an orbital shaker. Spheroids were washed 3 times in PBS, as previously mentioned, and incubated with a secondary labeled antibody: H&L [Cy3^®] (Abcam, ab97035, goat anti-mouse igG H&L pre-adsorbed, 1:100) in 3% normal goat serum for 1 h followed by PBS washing.