Abstract B40: Tuning exosomes to modulate macrophage inflammation: A therapeutic strategy for melanoma
Authors: Noel Thomas A. , Hood Joshua L. 
Published: May 2016

Melanoma survival can be dependent in part on tumor associated macrophage (TAM) activity. TAMs can adopt anti- or pro-tumor supportive phenotypes. Melanoma exosomes also can interact with and suppress immune cells. We previously reported that melanoma exosomes naturally home to lymph nodes and prepare them for tumor metastasis. Based on their innate ability to interact with the immune system, we hypothesize that melanoma exosomes might be modified as therapeutic nanocarriers to antagonize macrophage pro-tumor inflammatory processes.

Melittin is a 26 amino acid, alpha helical, cytolytic peptide derived from Apis mellifera (honeybee) venom. Melittin enters cell membranes as a monomer where it oligomerizes, creating pores which cause cell death. Recent work has shown melittin to have immunologic adjuvant properties. Loading melittin into the membrane of exosomes should allow for the release of potentially pathogenic RNA content while keeping the exosome structure largely intact. Further, melittin loaded into exosomes will be non-cytotoxic compared to free melittin given that the peptide’s pore forming activity has been neutralized. By precisely dosing B16-F10 melanoma exosomes with melittin, we plan to preserve exosome biophysical properties (size and zeta potential) that allow exosomes to naturally home to and interact with macrophage cells in vitro.

B16-F10 melanoma exosomes were cultured and isolated by differential centrifugation. Exosomes were loaded with melittin at different concentrations using simple mixing approaches. Modified exosomes were washed and re-isolated by ultracentrifugation. Exosome membrane loading of melittin peptide was determined using RNA UV absorption. We previously determined that pore formation in exosome membranes can be monitored through exosomal RNA release. The extent to which incorporation of melittin influenced exosome biophysical characteristics was determined using an Izon qViro tunable resistive pulse sensing instrument. RAW 264.7 macrophage cell viability in the presence of melittin modified melanoma exosomes was determined using Life Technologies PrestoBlue cell viability reagent. Induction of inflammatory RAW 264.7 macrophage cytokines was determined using a Qiagen Multi-Analyte ELISArray.

Our results demonstrate that melittin can be loaded into melanoma exosome membranes and forms pores as evidenced by RNA release indicating membrane permeability. Loading exosomes with proper amounts of melittin allowed for this permeability without substantially altering key biophysical properties (size and zeta potential) that permit normal trafficking. Melanoma exosomes modified with low concentrations of melittin (0.05 mM) also increased macrophage activity, though to a lesser degree than unmodified melanoma exosomes. Melanoma exosomes modified with high concentrations of melittin (1.0 mM) caused complete macrophage cell death. This suggests an upper limit to melittin loading capacity above which un-neutralized free melittin is likely being carried by exosomes intraluminally.

Macrophage cytokine expression profiles differed between unmodified and melittin modified melanoma exosomes treatments. Unmodified melanoma exosomes induced greater macrophage expression of pro-angiogenic IL-1α, G-CSF and the immunosuppressive cytokine IL-10. IL-1α induction by melittin modified melanoma exosomes was less than that of cells cultured in the absence of either exosome type. Melittin modified exosomes induced no expression of the immunosuppressive cytokine IL-10. Collectively, these findings demonstrate the potential use of melittin modified exosomes to antagonize macrophage pro-tumor inflammatory processes.

Citation Format: Thomas A. Noel, Joshua L. Hood. Tuning exosomes to modulate macrophage inflammation: A therapeutic strategy for melanoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B40.