Fig. 19

FL118 in vitro (ex vivo) and in vivo efficacy for RCC: A FL118 inhibition of the FHpRCC cell growth. FHpRCC UOK262 cells in 96-well plates were treated with and without FL118, SN-38 and topotecan for 72 h with a series of drug concentrations as shown. Cell viability was then determined using MTT assay. The data is the mean ± SD from three independent assays. B IC₅₀ values derived from two FHpRCC Type 2 cell lines (UOK262, NCCFH1), one Type 1/2 pRCC cell line (ACHN) and two ccRCC cell lines (UOK161, UOK111) are shown. IC50 were calculated from cell growth inhibition determined using MTT assay. C, D 50–100 RCC cells were seeded in a total of 2 ml complete media in each well of 12 Well-plates and allowed to attach for 24 h prior to treatment. Cells were then treated with vehicle or FL118 (1, 5, 10 nM) for 24 before changing the complete media without FL118. Cells were then allowed to grow for 2 weeks in the incubator with complete media changed every 72 h. when the colonies were of sufficient size (> 50 cells), media was aspirated, and wells containing colonies were washed with 1X PBS and then fixed using 1 ml of 100% methanol for 5–10 min. Methanol was then aspirated, and colonies were stained using 1 ml of 0.5% Crystal violet for 20 min. Plates were then washed with tap water until cleared and dried before images were taken. E Inhibitory effects of FL118 on UOK161 cell-established xenograft tumor growth. Xenograft tumor establishment: UOK161cells grown in complete medium were harvested and 5-million cells mixed with 50% matrigel per site were injected in the flank area of SCID (severe combined immunodeficiency) mice. After the tumors reached 100–200 mm³ (designated as day 0), vehicle or FL118 were orally administrated (arrowed) with two doses (2 mg/kg, 8 mg/kg) on days 0, 7 and 14. Of note, FL118 MTD is 10 mg//kg with the weekly schedule. F Mouse body weight changes