Lectin-induced aggregation is a low-cost and easily performed method for isolation of exosomes from urine. Isolated exosomes can be further analysed in terms of miRNA content. The miRNA profile of urinary exosomes reflects development of prostate cancer and may present a promising diagnostic tool.

Exosomes, the subclass of small membrane extracellular vesicles, have great diagnostic and therapeutic potential, but the lack of standardized methods for their efficient isolation and analysis limits the introduction of exosomal technologies into clinical practice. This review discusses the problems associated with the isolation of exosomes from biological fluids, as well as the principles of traditional and alternative methods of isolation. The aim of the presented review is to illustrate the variety of approaches based on the physical and biochemical properties of exosomes that can be used for exosome isolation. The advantages and disadvantages of different methods are discussed.

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Exosomes are a type of extracellular vesicles (EVs) secreted by multiple mammalian cell types and involved in intercellular communication. Numerous studies have explored the diagnostic and therapeutic potential of exosomes. The key challenge is the lack of efficient and standard techniques for isolation and downstream analysis of nanovesicles. Conventional isolation methods, such as ultracentrifugation, precipitation, filtration, chromatography, and immune-affinity-based approaches, rely on specific physical properties or on surface biomarkers. However, any of the existing methods has its limitations. Various parameters, such as efficacy, specificity, labor input, cost and scalability, and standardization options, must be considered for the correct choice of appropriate approach. The isolation of exosomes from biological fluids is especially challenged by the complex nature and variability of these liquids. Here, we present a comparison of five protocols for exosome isolation from human plasma: two chemical affinity precipitation methods (lectin-based purification and SubX™ technology), immunoaffinity precipitation, and reference ultracentrifugation-based exosome isolation method in two modifications. An approach for the isolation of exosomes based on the phenomenon of binding and aggregation of these particles via clusters of outer membrane phosphate groups in the presence of SubX™ molecules has been put forward in the present study. The isolated EVs were characterized based upon size, quantity, and protein content.

Резюме. Охарактеризованы видовой состав, чувствительность к антимикробным препаратам, частота встре-чаемости и типы карбапенемаз карбапенемаза-продуцирующих грамотрицательных бактерий, выделеных в ФГБУ «РНЦРХТ» МЗ РФ с февраля 2014 г. по апрель 2016 г. Скрининг карбапенем-резистентных бактерий проводился путем посева биоматериала на хромогенные среды (CHROMagar KPC, DRG, Франция), а их ви-довая идентификация -методом матричной лазерной десорбционной времяпролетной масс-спектрометрии (MALDI-TOF MS) на анализаторе VITEK MS (bioMérieux, Франция). Чувствительность грамотрицательных бактерий к антимикробным препаратам определялась на анализаторе VITEK-2 (bioMérieux, Франция). Полу-ченные результаты интерпретировались в соответствии с критериями EUCAST v. 6.0, 2016. Гены, кодирующие карбапенемазы групп КРС, ОХА-48/162, VIM, IMP, NDM, OXA-51, OXA40/24, OXA-23 и OXA-58 выявлялись методом мультиплексной ПЦР в реальном времени (АмплиСенс, Россия). Выделено 813 клинически значи-мых штаммов бактерий (602 пациента), в том числе 405 грамотрицательных, среди которых 5,1% (21 штамм от 16 пациентов) нечувствительных к меропенему и/или имипенему: Klebsiella pneumoniae (n = 5), Enterobacter cloacae (n = 2), Serratia marcescens (n = 1), Pseudomonas aeruginosa (n = 3), Acinetobacter baumannii (n = 10). Из кли-нического материала 4-х пациентов одновременно выделено до 3-х штаммов разных видов нечувствительных к карбапенемам бактерий. У 84% нечувствительных к карбапенемам изолятов обнаружены гены, кодирую-щие приобретенные карбапенемазы: у A. baumannii -OXA40/24 (n = 8); у K. pneumoniae -OXA-48 (n = 1), КРС (n = 1) и NDM (n = 2); у P. aeruginosa -VIM (n = 1) и KPC (n = 1); у E. cloacae -КРС (n = 1). OXA-48 карба-пенемаза обнаружена также в одном карбапенем-чувствительном штамме K. pneumoniae. Все карбапенема-за-продуцирующие штаммы имели фенотип множественной резистентности к антимикробным препаратам. Результаты исследования показали, что хотя доля карбапенем-нечувствительных штаммов среди грамотри-цательных бактерий сравнительно невелика, все они обладают множественной устойчивостью к антими-кробным препаратам и в большинстве из них обнаружены гены приобретенных карбапенемаз. Видовой со-став и типы обнаруженных приобретенных карбапанемаз характерны для территории России. У K. pneumoniae и P. aeruginosa не выявлено превалирования какого-либо одного типа карбапенемаз, в то время как у всех штаммов A. baumannii обнаружен ген OXA40/24 карбапенемазы.

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In 2016 we have developed novel approach for isolation of circulating cell-free DNA employing the proprietary bi-functional substance SubX® that tightly binds DNA and forms sandwich [cfDNA- SubX®-magnetic particles]. These sandwich aggregates are easily separated from bio-liquid with magnet followed by cfDNA isolation by speedy protocol. Our approach does not require Proteinase K digestion and 2-3-fold dilution of bio-liquid with lysis/binding buffer in contrast to all commercially available kits. Our kit has been evaluated in various research and clinical labs for cfDNA isolation from serum, plasma and urine of patients with different forms of cancer. SubX®-approach also allows for isolation of cfDNA from large volumes of urine samples without its concentration. We demonstrated linear increase of the DNA yield with liquid biopsy volume. Another unique property of the SubX® is that both ends of the molecule display lipid binding groups. This feature allows each molecule of SubX® to anchor two exosomes (i.e. dimerize). Excess of SubX® molecules in the solution results in aggregation of up to 10-15 exosomes and formation of micron-size particles that are easily precipitated in a brief 14K x g (or 30 min 2K x g) centrifugation step. A specially designed buffer allows for reconstitution of the pelleted exosomes back to monomer format for downstream applications. Flow cytometry with specific surface marker CD9 confirmed that SubX®-aggregated vesicles are exosomes. Analysis of membrane-bound protein markers and microRNA profile of exosomes isolated from plasma of prostate cancer patients will be presented. Difference in SubX® binding strength for cfDNA and exosomes allows for consequent isolation from the [cfDNA/exosomes- SubX®-magnetic particles] pellet first exosomes, then cfDNA. Eventually, we combined both approaches in one kit for isolation of cfDNA and exosomes from the same sample. This simultaneous isolation protocol is simple and fast, it allows to employ minimal volume of valuable bio samples as well as to reduce labor and cost. The kit was validated in serum and urine samples from patients with ovarian and pancreatic cancer. Exosome nature of isolated vesicles was confirmed by three independents methods - light scattering, immunological and electron microscopy. Thus, one could analyze both genetic (DNA and RNA) as well as protein markers present in the same liquid biopsy sample. Citation Format: Andrei G. Malykh, Anastasia Malek, Anna Lokshin, Vladimir Evtushenko. Simultaneous isolation of exosomes and cfDNA from liquid biopsies using universal kit based on SubX-MatrixTM technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1618.

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