Fig. 3

Ovarian cancer Leader cells are enriched in response chemotherapeutics commonly used to treat disease. LC-T2A-GFP cells were seeded as a: A-I monolayer; 10,000 cells/single well of a 96-well plate adhered and serum starved overnight or J-S spheroids; 2500 cells/single well in 96-well ultra-low adhesion plates; once formed, spheres were transferred into 96-well flat-bottom imaging plates using wide bore tips. Cells were subsequently treated with olaparib (B, K), rucaparib (C, L), cyclophosphamide (D, M), cisplatin (E, N), paclitaxel (F, O), doxorubicin (G, P), carboplatin (H, Q), and topotecan (I, R) in spheroid and monolayer format for 72 h, with untreated controls (A, J) and a representative spheroid at assay commencement shown (S). 72 h post treatment, duplicate wells were imaged on RFP and GFP channels at 4X magnification to determine LC^GFP+ status and spheroid outgrowth using the Cytation™ 3 Multimode Imager. Spheroids were imaged on 5 focal planes (Z-stacked) and analysis was performed on focus-stacked images. Scale bar 1000 μm