Flat bottom ultra low attachment plates

For the initial screening of the twenty-one Nectin-4 peptides, 50 ul of the peptides at 300 µg/mL (for a final working concentration of 150 µg/mL peptide per well) in complete media were added to 96-well flat-bottom ultra-low attachment plates (Corning, Corning, NY, USA). DMSO was used as a negative control. Fifty microliters of NIH:OVCAR5 cells at a concentration of 1.0 × 10⁵ cells/mL in complete media were added to each well. The plates were then incubated at 37 °C in 5% CO₂ for 48 h, with photographs taken manually at 24 and 48 h on a Nikon^® TE200 microscope. A single screening experiment was conducted with 8 technical replicates at each condition to determine which peptides had the most significant effect on spheroid formation.
Experiments were then repeated using round-bottom ultra-low attachment plates in which the cells were added to the wells containing the peptides at a final concentration of 150 µg/mL. The plates were placed in the IncuCyte^® Zoom instrument for 24-48 h of live cell imaging, as described above. Experiments with the peptides selected for further study were performed in triplicate, with 8–16 technical replicates at each treatment condition.

Cell proliferation was determined in 2D and spheroid culture systems. A total of 1 × 10³ cells were seeded in standard (2D) or round-bottom ultra-low attachment (Corning, spheroid) 96-well plates. Spheroid formation was induced by centrifugation at 300 × g for 3 min. Cell confluency and spheroid growth were monitored for 5 days using an IncuCyte S3 system (Sartorius) with 4× (2D, whole well scan) or 10× magnification (spheroid). Confluence masks were generated by using the IncuCyte analysis software. CellTiter Glo (Promega) was used to determine cell viability according to manufacturer’s protocols. For anoikis resistance assays, 1 × 10³ cells were seeded in flat-bottom ultra-low-attachment plates (Corning) and cultured in media supplemented with 1% fetal bovine serum for 5 days. Colony formation was determined by bright-field microscopy (IncuCyte S3, 4× magnification) and cell viability by CellTiter Glo. Invasion assays were performed by monitoring tumor cell infiltration in Matrigel. Pre-formed spheroids of 1 × 10³ cells were embedded in invasion matrix (Trevigen; 6 mg/ml) by centrifugation at 300 × g for 3 min. Infiltration was monitored for 24 h using an IncuCyte S3 system. The relative invasive area and the invasive front were determined by using the IncuCyte analysis software.

Gastruloids were generated as described in ref. ^{23 (link)}. In brief, 300 mESCs were plated in 40 μL N2B27 medium in 96-well Ultra-Low Cluster Round Bottom Ultra-Low Attachment plates (7007, Corning). After 48 h, 150 μL of N2B27 supplemented with 3 μM CHIR-99021 (S1263, Selleckchem) were added to each well. Then after 72 h, the medium was changed with 150 μL of fresh N2B27. At 96 h, gastruloids were transferred 1:1 in 100 μL of medium in 24-well Flat Bottom Ultra-Low Attachment plates (3473, Corning), containing 700 μL of fresh N2B27 supplemented with 30 ng/mL bFGF (Recombinant Human FGF-basic, 100-18 C, Peprotech), 5 ng/ml VEGF (Recombinant Human VEGF165, 100-20, Peprotech), and 0.5 mM ascorbic acid (Sigma A4544) (N2B27 +++). Then, 50% of the medium was changed daily, at 120 h with 400 μL of fresh N2B27 +++ and at 144 h with N2B27, until 168 h.