Fig. 5.

Sunitinib induces cell cycle arrest and suppresses proliferative tumor cones in RB organoids. A, B Percentage organoid cells in G0/G1, S, and G2/M cell cycle phases after sunitinib treatment at the IC₁₀ (2 µM and 3 µM, respectively) of RB170 (A) and RB654 (B) for 24 or 72 h compared with vehicle. C IC₅₀ of sunitinib, topotecan, and melphalan in RB170 and RB654 organoids. D–G Representative staining of RXRG (green), Ki67 (red), and nuclei (DAPI, blue) in RB170 organoids treated for 24 h with vehicle or the indicated drugs at their IC₅₀. H Percentage non-cycling and cycling tumor cones and the proportion of proliferative tumor cones in RB170 calculated from the data shown in (D–G). I–M Same as (D–H) but for RB654 organoids. The number of RXRG + tumor cells (non-cycling) and RXRG + Ki67 + tumor cells (cycling) was determined from a total number of DAPI-positive tumor cells. The ratio of RXRG + Ki67 + cells to RXRG + cells indicated a proportion of proliferative tumor cones. Data (A, B, H, M) obtained from three independent experiments; means ± SEM are shown. For immunofluorescence data (D–M), 6 tumor organoid gel drops for each treatment in different experiments were used; about 6000 cells counted from 15 microscope fields (5 fields/experiment) for each treatment. Scale bar: 50 μm (D–G, I–L)