Round bottom ultra low attachment plate

Organoid-derived CD44(-)PI(-) differentiated ductal-like cancer cells were sorted using flow cytometry (FACS Aria III). Then, we established them with CFSE-labeled endothelial cells in round-bottom ultra-low attachment plates (Corning) and cultured them with 10 μg/ml of JAG1-neutralizing antibody (R&D Systems, MAB12771) and control mouse IgG2b (R&D Systems, MAB004). After 1 day, the assembloids were washed and then embedded in GFR Matrigel and cultured in the basal medium containing JAG1-neutralizing antibody or mouse IgG2b. After 6 days, the assembloids were subjected to a FACS analysis to determine the CIC population.

For the generation of the cell spheroids, cells were seeded into 96-well round-bottom ultra-low attachment plates (Corning) at 2000 viable cells per well. HepG2/LX-2 24:1 spheroids were grown in minimum essential medium (MEM) supplemented as previously described [17 (link)]. The volume of spheroids was determined using the following formula: 4/3 π r³, where “r” is the mean of the long diameter and short diameter of the spheroid divided by two.

3D cultures were established by seeding cancer cells alone (9 000 cells per well) or cancer cells and fibroblasts (SV80 or MRC-5) in a 1:2 ratio (3 000 cancer cells and 6 000 fibroblasts) in a final volume of 100 µL cell culture medium per well of 96-well round-bottom ultra-low attachment plates (#7007, Corning Inc., Corning, NY, US). To encourage efficient generation of multicellular spheroids, the plates were centrifuged at 1019 g for 20 min, and thereafter placed in a cell culture incubator at 37°C, 5% CO₂, 5% O₂. The next day, an additional 100 µL fresh cell medium was added per well. Monocultures were maintained in 200 µL RPMI1640 medium, while co-culture spheroids were cultured in equal volumes of DMEM and RPMI1640 supplemented with FBS, penicillin, streptomycin, and L-glutamine, as described. Culture media were changed every second to every third day by carefully removing 100 µL and adding 100 µL fresh medium. Phase object confluence was used as a surrogate parameter representing the quantification of spheroid formation. Generation of compact spheroid structures was quantified using the IncuCyte ZOOM microscope and the built-in software. Masking was performed using the following settings: segmentation adjustment: 1, Hole fill: 30 000 µm², Filtered minimum area: 5 000 µm².

Differentiated PDAC organoids were dissociated using TrypLE™ Express (Thermo Fisher Scientific, 12605010), and organoid-derived CD44(-) cancer cells were stained for CD44-APCcy7 and sorted using flow cytometry (FACS Aria III). Sorting purity is presented in Figure S1A. For cell tracing in the flow cytometry analysis, HUVECs and PBMCs were stained using carboxyfluorescein succinimidyl ester (CFSE) according to the manufacturer's instructions. Organoid-derived differentiated CD44(-) cancer cells, HUVECs, and PBMCs paired with organoids were mixed at a 1:2:2 ratio and cultured on round-bottom ultra-low attachment plates (Corning) in growth medium supplemented with 10% Matrigel for 24 h. Then, the mixtures were cultured in the basal medium (AdDMEM/F12 with GlutaMax and 5% FBS) for 7 days, as previously described 13 (link).

For generating cell spheroids, cells were seeded into 96-well round-bottom ultra-low attachment plates (Corning) at a density of 2,000 viable cells per well. IHH/LX-2 24:1 spheroids were grown in William’s E medium supplemented as described above. The plates were incubated for 48 h at 37 °C in a humidified atmosphere with 5% CO₂. After 48 h, cells were treated with TGF-β1 or cocktail (oleic acid + TGF-β1 + TNF-α) with or without BMP4 or GREM1 for another 48 h.