Analysis and therapeutic blocking of Exosome-miRNA tumor-derived signaling in colorectal carcinoma (Short title: Exo-antimiR)
Hematology, Oncology
Final Report Abstract
Extracellular vesicles (ECVs) are secreted cell-derived membrane particles involved in intercellular signaling and cell-cell communication. By transporting various biomacromolecules, ECVs and in particular exosomes are relevant in various (patho-) physiological processes. ECVs are also released by cancer cells and can confer pro-tumorigenic effects. Their target cell tropism, effects on proliferation rates, natural stability in blood and immunotolerance makes ECVs particularly interesting as delivery vehicles. Polyethylenimines (PEIs) are linear or branched polymers which are capable of forming noncovalent complexes with small RNA molecules including siRNAs or antimiRs, for their delivery in vitro and in vivo. The German part of this German-Russian collaboration project with the Petrov Institute of Oncology, Russia (Dr. Anastasia Malek) aimed at the development of novel exosome-based antimiR delivery and miRNA inhibition strategies for selected miRNA candidates. The Russian collaboration partner analyzed miRNA profiles in exosomes from colon carcinoma patients’ blood, and provided patients’ exosomes. On the German side, the project goals covered (1) the establishment of innovative exosomebased antimiR delivery systems (Exo-antimiRs), (2) the assessment of the newly developed exosome-based antimiR strategy (Exo-antimiRs) for the inhibition of the miRNA candidates, using exosomes from cell culture and, to a lesser extent, from CRC patients, and (3) the extensive exploration of the anti-tumor potential of the newly developed exosome-based antimiR strategy (Exo-antimiRs) in vitro and in vivo. This study thus explored for the first time the combination of PEI-based nanoparticles with naturally occurring ECVs from different cell lines, for the delivery of small RNAs. ECV- modified PEI/siRNA complexes were analyzed by electron microscopy vs. ECV or complex alone. On the functional side, we were able to demonstrate increased knockdown efficacy and storage stability of PEI/siRNA complexes upon their modification with ECVs. This was paralleled by enhanced tumor cell-inhibition by ECV-modified PEI/siRNA complexes targeting Survivin. Pre-treatment with various inhibitors of cellular internalization revealed alterations in cellular uptake mechanisms and biological activities of PEI/siRNA complexes upon their ECV modification. Extending our studies towards PEI-complexed antimiRs against miR-155 or miR-1246, dose-dependent cellular and molecular effects were found enhanced in ECV-modified complexes, based on the de-repression of direct miRNA target genes. Differences between ECVs from different cell lines were observed regarding their capacity of enhancing PEI/siRNA efficacies, independent of the target cell line for transfection. Finally, an in vivo therapy study in mice bearing s.c. PC3 prostate carcinoma xenografts revealed marked inhibition of tumor growth upon treatment with ECVPC3-modified PEI/siSurvivin complexes, based on profound target gene knockdown. In conclusion, the ECV-modification enhanced the activity of PEI-based complexes, by altering pivotal physicochemical and biological nanoparticle properties. Since the improvement of PEI nanoparticles by ECV modification was shown to be mainly based on the introduction of ECVs as an “outer shell” of the complexes, rather than by the expected role of specific ECV proteins serving as ligands for targeted delivery, it became thus obvious that ECVs show properties beyond their protein (ligand) decoration which provide substantial improvement of nanoparticles. This provides the basis for subsequent studies aiming at the identification of the minimally required properties of ECVs for introducing the above-described physicochemical and biological improvement of nanoparticles. Beyond protein decoration, this may also cover lipid composition or other components critical for ECV uptake, and should also allow for the definition and generation of artificial ECVs with the critically required properties for nanoparticle modification.
Publications
- Comparative analysis of RT-qPCR based methodologies for microRNA detection. Klinicheskaya Laboratornaya Diagnostika (Russian Clinical Laboratory Diagnostics). 2018; 63 (11): 722-728
Korobkina E.A., Knyazeva M.S., Kil Yu.V., Titov S.E., Malek A.V.
(See online at https://doi.org/10.18821/0869-2084-2018-63-11-722-728) - Diagnostic value of exosomal mirnas for colorectal cancer. Koloproktologia. 2018; 2: 25-31
Samsonov R., Tarasov M., Burdakov V., Shtam T., Guljaev A., Tkachenko O., Rybakov E. Filatov M., Aigner A., Malek A.
(See online at https://doi.org/10.33878/2073-7556-2018-0-2-25-31) - Functional Properties of Circulating Exosomes Mediated by Surface-Attached Plasma Proteins. J Hematol. 2018;7(4):149–53
Shtam Т, Naryzhny S, Kopylovd A, Petrenko E, Samsonov R, Kamyshinsky R, et al.
(See online at https://doi.org/10.14740/jh412w) - Extracellular vesicle (ECV)-modified polyethylenimine (PEI) complexes for enhanced siRNA delivery in vitro and in vivo. J Control Release. 2020 Mar 10;319:63-76
Zhupanyn P, Ewe A, Büch T, Malek A, Rademacher P, Müller C, Reinert A, Jaimes Y, Aigner A
(See online at https://doi.org/10.1016/j.jconrel.2019.12.032)
