Phenotypical and functional evaluation of dendritic cells after exosomal delivery of miRNA-155
Introduction
Dendritic cells (DCs) are specialized antigen-presenting cells, which present antigens to natural killer (NK) cells, B-cells and T-cells. Therefore, DCs is considered as an outstanding candidate cells for immunotherapy of cancers [1]. However, the clinical efficiency of DC vaccination therapy needs to be improved, especially, with regards to the types of antigens which are used to prime DCs and the utilization of macromolecules such as microRNAs (miRNAs) for better activation and maturation of DCs [[2], [3], [4], [5]].
Exosomes are nano-sized homogenous membrane vesicles (30–120 nm) derived from the exocytosis of intraluminal vesicles (within multi-vesicular bodies), then attached to the plasma membrane to be released into the extracellular space. Many cell types produce exosomes through this mechanism [[6], [7], [8]]. Since the exosome innately transport RNA molecules among cells and body fluids, it has been hypothesized that this feature might be suitable for gene delivery. Thus, exosomes have been previously used to transfer miRNA and other oligonucleotides to the target cells [9,10]. In comparison with other gene transport methods including lipid nanoparticles, viruses and polymeric nanoparticles, several advantages have been demonstrated for exosomes gene delivery system. Exosomes are not subject to attack by complement, opsonins, coagulation factors and antibodies in the body fluids [8,[11], [12], [13]]. There are different strategies which are used for loading antigen to dendritic cells including exosomes, viral vector-delivered whole antigen, tumor lysate, whole antigen–protein and using DNA or RNA [15,22,26,27]. Tumor cell-derived exosomes have a broad antigen repertoire for priming DCs in which they can expand both CD8+ and CD4+ T cells against cancer cells. Apart from the mentioned advantages of gene delivery, tumor cell-derived exosome contains several DC's maturation factors including HSP and GM-CSF, that can mature DC effectively [3,22,[26], [27], [28]].
miRNAs are a group of small noncoding RNAs, 20–25 nucleotide-long, and has key regulatory roles in different cell processes including cell survival, proliferation, apoptosis, tumor growth and metastasis [17]. In recent years, research to improve RNA-based therapy has been meaningfully increased [9,14,21,26,27]. It is noteworthy that, the main problem is an immune stimulation against the synthetic delivery vehicle and synthetic miRNAs, particularly if needed to repeat the dosing several times to treat disease [18]. Natural carriers can overcome these obstacles during miRNA delivery to cells. Exosomes as a natural RNA carrier might provide an available source of effective delivery plans [9].
So far, many miRNA studies on DCs have focused on the miRNA-155. miRNA-155 could modulate the IL-1 signaling pathway in stimulated human DCs [19]. miR-155 could enhance IL-12p70 and NF-κB production in DCs [20]. But the role of miR-155 in antigen presentation by DCs and DC maturation remains controversial [19,21,22].
To the best of the authors' knowledge, there is no experiment on delivering the miRNA-155 by tumor cells derived exosome into dendritic cells.
Here, delivery of miRNA-155 to DCs with exosomes, for simultaneous miRNA-155 delivery and antigen priming of DCs was optimized. Following treatment with miRNA-155 mimic loaded cancer derived exosomes, DCs were evaluated functionally and phenotypically.
Section snippets
Cell culture and exosome isolation
CT26 cell line was obtained from Pasteur Institute (Tehran, Iran). Then, cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) (Gibco, NY, USA) complemented with 100 U/ml penicillin (Gibco®, NY, USA), 10% fetal bovine serum (Gibco®, NY, USA) and 100 mg/ml streptomycin (Gibco, NY, USA) then incubated at 37 °C in 5% CO2. CT26 cells were adapted to the serum-free medium during a two-week period. Adapted cells line were grown to 70% confluences, and then incubated for 72 h in
CT26 cell-derived exosomes characterization
The average size of exosomes was 47 nm, as measured by dynamic light scattering (DLS), which was in the range of exosome size (Fig. 1A).
The spherical shape and size of isolated and electroporated exosomes (<100 nm) were identified by scanning electron microscopy (Fig. 1B–C). The CT26 cell lines exosome was examined by atomic force microscopy and the size range (<100 nm) and circular shape in 2-dimensional structure were confirmed (Fig. 1D).
Optimization of miRNA-155 mimic loading on CT26 cell-derived exosomes
To optimize the miRNA loading procedure, tumor
Discussion
The purpose of this study was to optimize delivering of the miRNA-155 mimic by tumor cells derived exosome into DCs in vitro and evaluate the effect of overexpression of miRNA-155 mimic on DCs.
miRNA-155 could enhance IL-12p70 and NF-κB production in DCs. Considering the noticeable miRNA-155 role's in the signaling pathways of DCs, miRNA-155 mimic was chosen in this study as the exogenous bio-macromolecules [24,29,38].
In previous studies, it was indicated that exosomes can naturally transfer a
Conclusion
In conclusion, the present study optimized the loading status for tumor exosomes, and employed them as transporter to transport miRNA-155 into DCs. It is shown that the DCs with overexpression of miRNA-155 mimic in term of phenotype and function is similar to mDCs. In addition, the miRNA-155 mimic can induce immunogenic DCs, which can activate and proliferate lymphocyte as well as LPS. Finally, it can be stated that miRNA-155 could be a candidate for dendritic cell maturation.
Acknowledgments
The present research is financially supported by “Research Department of the School of Medicine, Shahid Beheshti University of Medical Sciences” (Grant No. 8993) and “National Institute for Medical Research Development” (Grant No. 962574).
References (41)
- et al.
Dendritic cell therapy in advanced gastric cancer: a promising new hope?
Surg. Oncol.
(2012) - et al.
Exosomes: small vesicles participating in intercellular communication
Int. J. Biochem. Cell Biol.
(2012) - et al.
Exosomes as nanocarriers for immunotherapy of cancer and inflammatory diseases
Clin. Immunol.
(2015) - et al.
Exosomes and their roles in immune regulation and cancer
Semin. Cell Dev. Biol.
(2015) - et al.
Biological gene delivery vehicles: beyond viral vectors
Mol. Ther.
(2009) - et al.
A miR-155-dependent microRNA hierarchy in dendritic cell maturation and macrophage activation
FEBS Lett.
(2014) - et al.
Antibacterial activity, inflammatory response, coagulation and cytotoxicity effects of silver nanoparticles
Nanomedicine
(2012) - et al.
An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow
J. Immunol. Methods
(1999) - et al.
MicroRNA-155 modulates the pathogen binding ability of dendritic cells (DCs) by down-regulation of DC-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN)
J. Biol. Chem.
(2009) - et al.
Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles
J. Control. Release
(2013)
Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes
Blood
Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages
Nanomedicine
Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages
Nanomedicine
Silencing of c-Fos expression by microRNA-155 is critical for dendritic cell maturation and function
Blood
Development of a dendritic cell (DC)-based vaccine for patients with advanced colorectal cancer
Hepato-Gastroenterology
Autologous tumor lysate-pulsed dendritic cell immunotherapy with cytokine-induced killer cells improves survival in gastric and colorectal cancer patients
PLoS One
Improved Vaccine Efficacy of Tumor Exosome Compared to Tumor Lysate Loaded Dendritic Cells in Mice
Cancer immunotherapy via dendritic cells
Nat. Rev. Cancer
Immunomodulatory effects of mesenchymal stem cell–derived exosomes on experimental type-1 autoimmune diabetes
J. Cell. Biochem.
Isolation and characterization of exosomes from cell culture supernatants and biological fluids
Curr. Protoc. Cell Biol.
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