We examined the peritoneal metastasis of ovarian cancer using both in vitro and in vivo models in this experimental study. We demonstrated that the accumulation of ascites and a reduction in survival were induced by the inoculation of the ES-2 ovarian clear cell carcinoma cell line, a known chemotherapy-resistant cancer cell type, into the peritoneal cavity and that the EGFR pathways were activated in the cancer cells in both the in vitro and in vivo models. Since ES-2 cells harbor a genetic mutation in B-raf, a downstream member of the EGFR pathway and a known resistance factor against anti-EGFR therapy , EGFR interference was thought to be an unfavorable strategy in ES-2 models. Thus, we focused on the management of ascites using a modified liposomal formulation of a chemotherapeutic agent, since doxorubicin liposomes have been applied for ovarian cancer therapy. We demonstrated that IHL-305 was retained within the ascites of the mice and prolonged the survival period of the mice.
Ovarian cancers advance to peritoneal metastasis through the dissemination of the cancer cells from the ovary to the peritoneal cavity. In this study, we used a clear cell type ovarian cancer cell line, ES-2 to establish peritoneal metastasis models (Figure 1). We also examined an adenocarcinoma ovarian cell line, SK-OV-3, and the amount of vascular endothelial growth factor (VEGF) in the spheroid culture supernatant was much higher for the ES-2 cells than for the SK-OV-3 cells (data not shown). VEGF reportedly plays an important role in the progression of peritoneal metastasis . In clinical studies, clear cell type ovarian cancer has exhibited a resistance to chemotherapy . Therefore, we selected ES-2 cells as an aggressive peritoneal metastasis model in this study.
In general, peritoneal metastasis induced the accumulation of ascites, including floating cancer cell aggregates. We focused on the floating cells, which showed anchorage-independent growth and were regarded as comparable to the spheroids of cancer cells in the in vitro peritoneal metastasis model. We detected the activation of Akt in both the in vivo and the in vitro models of peritoneal metastasis (Figure 2). Akt reportedly plays an important role in resistance to anoikis (detachment-induced apoptosis) . In addition, the activation of EGFR was also detected in both models. However, EGF, an activating ligand of EGFR, was not detected in the spheroid culture medium or ascites (data not shown). A three-dimensional culture resulting in the up-regulation of HER2 has been reported . Therefore, alterations in the formation of three-dimensional structures might activate the EGFR pathways.
Platinum and taxane agents are used as standard chemotherapy for the treatment of ovarian cancers. We examined the cytotoxic activity of CDDP, SN-38, PTX and PD153035 against monolayer and spheroids of ES-2 cells. Spheroids of ES-2 cells demonstrated resistance to the agents tested, especially to CDDP and PTX, though these agents exerted potent activity in monolayer cultures (Figure 3A). Cancer cells bearing the B-raf gene mutation have been reported to exhibit resistance to EGFR inhibitors, and ES-2 cells carry this mutation ; therefore, we assumed that the ES-2 cells were resistant to the EGFR inhibitors. Targeting Akt is still remaining but among the tested agents, SN-38 exerted the highest cytotoxic activity (Figure 3B); thus, we applied CPT-11, a prodrug of SN-38, for the treatment of the peritoneal metastasis.
Liposomal formulations improve the pharmacokinetic profile, but the profile of IHL-305 in a peritoneal metastasis model with ascites has not been previously examined. In the present study, a larger amount of IHL-305 was retained in the ascites, compared with CPT-11 (Figure 4B). Generally, therapeutic agents in the peritoneal cavity are absorbed through vessels and lymph nodes. High molecular weight agents are mainly absorbed through lymph nodes, whereas low molecular weight agents are mainly absorbed through vessels . In peritoneal metastasis, cancer cells invading the lymph nodes reduce the absorption by lymph nodes. The absorption rate of IHL-305, a high molecular weight agent, has been shown to be lower than that of CPT-11 (Figure 4B). It is suggested, therefore, that those improved pharmacokinetic profile of IHL-305 leads to an enhancement of the anti-cancer efficacy (Figure 5A). As shown in Table 1, ascites fluids have observed in CPT-11 group on day 31, whereas it has not observed on days 18 and 26. It was thought that this dosage of CPT-11 was not enough to cure the tumor cells completely, although this dosage was recommended dose of CPT-11 in mouse. Thus, the remaining tumor cells in CPT-11-treated mouse proliferated and then release of the ascites fluids was induced. Moreover, IHL-305 prolonged the survival time when administered according to any of the schedules that were examined (Figure 5B). IHL-305 was administered on day 12 after the inoculation of ES-2 cells; at this stage, advanced tumor invasion (omentum, pancreas, etc.) was apparent. Nevertheless, IHL-305 exhibited a therapeutic efficacy against the invaded tumors in addition to the cancer cell aggregates in the ascites.