96 well ultra low attachment plate

Cell culture was performed as previously described^{46 (link),47 (link)}. The human PDAC cell lines PK-1, PANC-1, PK-59, and MIA PaCa-2 were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). PK-45P, PK-8, T3M-4, and KP-4 human PDAC cells were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. Cells were grown in growth medium (RPMI 1640 medium containing 10% fetal bovine serum) at 37 °C under a humidified 5% CO₂ atmosphere. For EMT induction, cells were cultured for 48 h in growth medium containing 10 ng/mL TGF-β1 (Peprotech, Rocky Hill, NJ, USA). For 3D culture, cells in growth medium were plated at 1.0 × 10⁴ cells/well or 3.0 × 10³ cells/well in 24-well ultra-low attachment plates (Corning Inc. Kennebunk, ME, USA) or 96-well ultra-low attachment plates (Thermo Fisher Scientific, Waltham, MA, USA), respectively. The spheres were aspirated after 7 days using micropipettes and placed in microcentrifuge tubes for use in further experiments. Photographs of the spheres were taken using a sphere analyzing device, Cell3iMager duos (SCREEN Holdings Co., Ltd., Kyoto, Japan).

Gold (III) chloride trihydrate (HAuCl₄·3H₂O, ≥99.9% trace metals basis), tri-sodium citrate (for molecular biology, ≥99%), tannic acid (ACS reagent), SH-PEG2k-NH₂, Foetal Bovine Serum (FBS), Amphotericin-β, Penicillin-Streptomycin, and Dulbecco’s Modified Eagle’s Medium (D5796) were all acquired from Sigma-Aldrich, Johannesburg, South Africa. TrypLE™ Select Enzyme (1X) (12563-029) ThermoFisher, Johannesburg, South Africa). Hoechst 33258, Caspase 3, and 9 Multiplex Assay kit (Ab219915) as well as 96-well ultra-low attachment plates (174929) were purchased from ThermoFisher, Johannesburg, South Africa. Annexin V/PI apoptosis detection kit (556570) was procured from BD Biosciences, The Scientific Group, Johannesburg, South Africa).

PK-8, PK-45P, T3M-4, and KP4 human PDAC cells were provided by the RIKEN BioResource Research Center through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. PK-59, PK-1, PANC-1, and MIA PaCa-2 human PDAC cell lines were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). The characteristics of the eight PDAC cells are summarized in the Supplementary Table S1^{9 (link),26 (link)–34 (link)}. HPDE6 cells were kindly provided by Prof. Toru Furukawa (Tohoku University, Sendai). Cells were grown in growth medium (RPMI-1640 medium containing 10% fetal bovine serum) at 37 °C in a humidified 5% CO₂ atmosphere. To form spheres, cells (3 × 10³ cells/well) were plated in 96-well ultra-low attachment plates (Thermo Fisher Scientific, Waltham, MA, USA) with growth medium. After 7 days, the spheres were photographed using a phase contrast microscope (Eclipse TS-100, Nikon, Tokyo, Japan). Spheres were then aspirated using micropipettes and used for further experiments. Using a Mycoplasma PCR Detection Kit (iNtRON Biotechnology Inc., Jungwon-Gu, South Korea), it was confirmed that all cells were not infected with mycoplasma.

The human PDAC cell lines PANC-1 and PK-59 were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). PANC-1 and PK-59 cells were grown in growth medium (RPMI 1640 medium containing 10% fetal bovine serum) at 37°C under a humidified 5% CO₂ atmosphere. For EMT induction, cells were cultured for 72 h in growth medium containing 2 ng/mL TGF-β1 (Peprotech, Rocky Hill, NJ, USA). For 3D culture, cells (3.0 × 10³ cells/well) in growth medium were plated in 96-well ultra-low attachment plates (Thermo Fisher Scientific, Waltham, MA, USA). After 7 days, the spheres were aspirated using micropipettes and placed in microcentrifuge tubes for use in further experiments.

The human PDAC cell lines PK-1, PANC-1, and PK-59 were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University (Sendai, Japan). The PK-8 human PDAC cell line was provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. Cells were grown in a growth medium (RPMI 1640 medium containing 10% fetal bovine serum) at 37 °C under a humidified 5% CO₂ atmosphere. For sphere formation by 3D culture, cells in the growth medium were plated at 1.0 × 10⁴ cells/well or 3.0 × 10³ cells/well into 24-well ultra-low attachment plates (Corning Inc., Kennebunk, ME, USA) or 96-well ultra-low attachment plates (Thermo Fisher Scientific, Waltham, MA, USA). The spheres were aspirated after seven days using micropipettes and placed in microcentrifuge tubes for use in further experiments. To inhibit gp130 ligand-triggered signaling, the cells were treated with SC144 (Selleck Chemicals, Houston, TX, USA) or DMSO as a vehicle control. The size (area) of spheres was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).