Heterotypic Spheroid Formation and Characterization

3 × 10⁵ OVCAR3 cells were seeded with 3 × 10⁵ CAFs in 6-well ultra-low attachment plates (Corning Cat. No. 3471). At the time of seeding, the plate was kept inclined for 30 min to help the OC cells and CAFs aggregate and interact. Thereafter, plates were reverted to the usual horizontal position and cultured for 7 days to allow the heterotypic spheroids to grow. Spheroid fixation, blocking, and antibody staining were done as described by Condello et al.^{49 (link)}. Briefly, spheroids were fixed and permeabilized in suspension for 3 h at 4 °C in PBS containing 4% PFA (Boston BioProducts BM-155) and 1% Triton X-100 (Thermo Scientific Cat. No. 85112). Spheroids were dehydrated with increasing concentrations of methanol (25%, 50%, 75%, 95%, 100%) and rehydrated in the opposite sequence, then stained with ALDH1 (1:100, BD Bioscience Cat. No. 611194), Wnt5a (1:200, CST Cat. No. 2530) and Vimentin (1:500, Thermo Fisher Scientific Cat. No. PA1-10003) antibodies. Nuclei were visualized by Hoechst 33342 (Life Technologies Cat. No. H3570). The primary antibodies were probed with 1:1000 Alexa Fluor 488 conjugated anti-mouse IgG (Cell Signaling Technology, cat. No. 4408S), Alexa Fluor 594 conjugated anti-rabbit IgG (Cell Signaling Technology, cat. No. 8889) or Alexa Fluor 647 conjugated anti-chicken IgG (Invitrogen, cat. No. A-21449).