Combination therapy targeting the tumor microenvironment is effective in a model of human ocular melanoma

Abstract

Background: Ocular melanoma is the leading intraocular malignancy. There is no effective treatment for metastatic ocular melanoma. We sought a treatment targeting the tumor microenvironment as well as the tumor cells.

Methods: Migration of HUVEC cells, the ability of HUVEC cells to form tubes, and proliferative capacity of a human ocular melanoma cell line were tested in the presence of lenalidomide and sorafenib alone and in combination. The compounds were also tested in a rat aortic ring assay and were tested in a highly aggressive human ocular melanoma xenograft model.

Results: Lenalidomide and Sorafenib inhibit HUVEC ability to migrate and form tubes and when used in combination the inhibition is increased. The agents alone and in combination inhibit outgrowth in the rat aortic ring model. The combination of the agents improved the inhibition over either single agent. In a xenograft model, combination therapy inhibited tumor growth over inhibition by single agent alone in a significant fashion (p < 0.004: lenalidomide and p < 0.0035: sorafenib). Furthermore, spontaneous lung metastasis development was completely inhibited in the combination treated animals. Sixty percent of vehicle treated animals developed lung metastases compared to 50% of lenalidomide treated animals, and 33% of sorafenib treated animals.

Conclusion: Lenalidomide and sorafenib are effective at targeting endothelial cells, inhibiting growth of ocular melanoma cells and can inhibit growth of tumors in a xenograft model as well as inhibit development of metastases. Combining these agents works in an additive to synergistic way to inhibit the growth of tumors and development of metastases.

Figure 1

(A) Migration assay data for single agent lenalidomide, sorafenib and combination treated HUVEC reveals a dose response curve that has significant inhibition at all concentrations. Treatment of HUVEC with a 1:1 combination of lenalidomide and sorafenib showed superior inhibitive efficacy than monotherapy with either compound. There was no detectable difference between lenalidomide or sorafenib monotherapy for the migration assay. Each experimental condition was repeated six times. (B) Tube formation assay showed inhibition of HUVEC ability to form tubes with a maximal inhibition with lenalidomide treatment at 0.001 uM (p = 0.005). The tube formation capabilities of HUVEC were more profoundly inhibited by sorafenib than lenalidomide at all tested concentrations. The combination of compounds showed superior inhibitory efficacy at all concentrations. Each experimental condition was repeated four times. (C) Lenalidomide inhibited the rat aortic ring assay more effectively at lower concentrations than did sorafenib, and its inhibition was present at across all concentrations. Combination treatment with both compounds was more effective at all evaluable concentrations. Images are representative of those used for data derivation. Each experimental condition was repeated eight times.

Figure 2

Evaluation of 92.1's proliferative potential was done with RT-CES (A and B). Each experimental condition was repeated eight times. This technology yielded kinetic growth data which showed combination treatment with lenalidomide and sorafenib (1:1) to have an anti-proliferative effect, which is first apparent at 0.001 μM and most profoundly independent from either single agent's effect at 0.01 μM (A). Lenalidomide alone showed no appreciable activity against proliferation regardless of concentration. Sorafenib alone exhibited anti-proliferative activity which was initially evident at 0.1 μM (B), 10–100× more concentrate than when a similar effect is produced by combination therapy. When therapy was evaluated with the xenograft model, the combination treatment cohort showed the most tumor growth delay Each treatment group represents eight animals. (C). Lenalidomide and sorafenib both displayed tumor growth stasis which was significantly different from carrier treated animals and equivocal from each other. Combinatory therapy showed significant growth retardation relative to either monotherapy. This pattern of anti-tumor efficacy was also seen in the analysis of metastatic frequency. Each treatment group represents eight animals (D).