Introduction: TKIs have showed exciting efficacy to treat renal cell carcinoma (RCC), with 70% of patients responding well to initial sunitinib (Suni) dosing. However, 30% of patients are believed to be primarily resistant to treatment with all patients eventually developing resistance. Resistance to treatment has been previously linked to exosomes that contain oncogenic markers. This study sought develop a combination therapy of Tipifarnib (Tipi) + Suni to target exosome conferred drug resistance.
Methods: 786-O, 786-0 Suni Resistant (SR), A498, A498 SR, Caki-2, Caki-2 SR and 293-T cells were cultured, exosomes collected from conditioned media treated with Tipi, and later isolated using differential ultracentrifugation. Cell proliferation and Immunoblot analysis were used for downstream analysis after Tipi, Suni, and Tipi + Suni treatments.
Results: First a Jurkat assay assessed the role of exosomes within the tumor microenvironment. SR exosomes displayed a cytotoxic effect on immune cells which allowed for increased tumor burden. This cytotoxic effect was paralleled with increased expression of PD-L1 on SR exosomes. When comparing PD-L1 expression, PD-L1 was localized on exosomes at higher concentrations than the cell membrane. Next, we employed Tipi which decreased 293-T exosomes by 16% (p < 0.05) while all cancerous cell lines displayed an even greater reduction (p < 0.01). With increasing dose of Tipi, 293-T showed a dose dependent increase in Alix (exosome biogenesis marker) and no change in either nSMase (biogenesis marker) or Rab27a (exosome trafficking marker). Conversely, our cancerous lines displayed a dose dependent decrease in all 3 exosome markers. A proliferation assay was then used to assess the direct cytotoxic ability of Tipi + Suni. After treatment on Suni-sensitive and SR cell lines, the drug combination displayed synergistic ability to decrease tumor growth.
Conclusions: Repurposing drugs has a unique ability to reduce drug discovery costs and improve treatments options for patients. Our data shows that exosomes play a pivotal role within the survival of RCC through PD-L1 expression. Using Tipi to block exosome biogenesis, this study demonstrates that Tipi's exosome targeted effects can be implemented with standard therapy to decrease tumor burden.
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