br Next we assessed the effect of A on
Next, we assessed the effect of 673A on tumor-initiation capacity. Chemotherapy-resistant SKOv3 MitoPY1 were treated in vitro with cisplatin (1 mg/ml), DSF, 673A (12.5 mM), or cisplatin and 673A. Forty-eight hours after treatment, live (propidium iodide [PI] and annexin-V ) cells were FACS-isolated, and 5,000 cells were injected into mice. Although treatment with DSF had no effect on cancer growth, 673A demonstrated a 4–5-fold reductions in tumor growth versus no treatment or cisplatin therapy (Figure 6Di). Furthermore, with a limiting dilution assay, 673A alone was able to reduce tumor initiation 50% and, when combined with cisplatin therapy, eliminated tumor initiation capacity (Figures 6Di and 6Dii).
673A Synergizes with Chemotherapy In Vivo to Eradicate Cancer in Mice
We next evaluated the anti-tumor effect of 673A in vivo with Ovsaho, HEY-1, and A2780 cell-line xenografts. Three days after cancer cell inoculation, the mice (n = 10 per treatment group)
were treated daily with vehicle or 673A (20 mg/kg) for 21 days. The 673A treatment reduced tumor burden 40%–60% (Fig-ure 7Ai) and significantly decreased tumor cell Ki67 expression (Figure 7Bi–ii). H&E analysis of 673A-treated tumors revealed necrotic morphology (Figure 7B). We next assessed combination therapy on OVCAR8 cells, which are 50% CD133+. We chose a lower dose of 673A because OVCAR8 CD133+ cells are very sen-sitive in vitro. Combined treatment of 673A at 4 mg/kg (n = 10) daily with three weekly doses of cisplatin reduced tumor burden 3.5-fold versus cisplatin only (Figure 7C). qRT-PCR analysis of tumors revealed a significant upregulation of UCP1 and UCP3 in 673A-treated tumors (Figure 7D). Hematologic, renal, and liver function tests revealed no additional toxicity with the addition of 673A to cisplatin (Table S4). Next, we determined the ability of 673A to eliminate ‘‘minimal residual disease.’’ CaoV3 cells (1 3 105) were injected subcutaneously in mice. After 7 days, the mice were treated with cisplatin, 673A, or combination ther-apy, as above, for 3 weeks, and tumor development over the next 6 months was evaluated. Ninety percent of mice treated with cisplatin developed tumors. In contrast, only 50% of mice treated with 673A alone and 40% of mice treated with cisplatin and 673A developed tumors (Figure 7E). In a parallel study that used intraperitoneal (i.p.) injection of 5 3 104 FACS-sorted ALDH+ SKOv3 cells, 100% of mice treated only with cisplatin died, whereas 62% of mice treated with cisplatin and 673A
were disease free at 6 months (Figure 7F). We also examined the effect of 673A with the LSL-K-rasG12D/+PtenloxP/loxP genetic
mouse model of endometrioid ovarian cancer (Dinulescu et al., 2005). Two weeks after intra-bursal adeno-CRE virus-mediated tumor initiation, mice were treated with vehicle, carboplatin (25 mg/kg) weekly, or a combination of carboplatin weekly and 673A (20 mg/kg) daily for 7 weeks. Although only 16% of mice treated with carboplatin alone were tumor free after therapy, 27% of animals treated with combined carboplatin and 673A therapy were tumor free at the conclusion of the experiment (Figure 7G). Next, we examined the effect of combined 673A and carboplatin or 673A and cisplatin treatment with two chemo-therapy-resistant patient-derived xenograft (PDX) models. The addition of 673A to either carboplatin or cisplatin not only pre-vented tumor growth but also induced tumor regression, reducing final tumor weight more than 2-fold (Figure 7H). r> Finally, we confirmed on-target activity in vivo. Immunohisto-chemistry (IHC) demonstrated a reduction of ALDH1A1-express-ing cells in responding 673A-treated tumors (Figure S5Ai). In addition, ALDEFLUOR activity in splenocytes of animals being treated with 673A was decreased 3-fold (Figure S5Aii). To further