AFM-TEM correlation microscopy and its application to lipid nanoparticles.

So far, only a few articles have demonstrated the possibility of correlated AFM-TEM imaging - sequential imaging of the same individual objects using atomic-force microscopy (AFM) and transmission electron microscopy (TEM). The current work contributes to the development of this approach by giving a step-by-step procedure, which yields pairs of correlated AFM-TEM images. We describe the application of correlation AFM-TEM microscopy to lipid nanoparticles (small extracellular vesicles and liposomes). The sizes of individual particles measured by the two methods were in good agreement, taking the tip broadening into account. The correlated AFM-TEM imaging can be valuable for single-particle analysis and nanometrology.

Comparison of Methods for MicroRNA Isolation from Extracellular Vesicles Obtained from Ascitic Fluids.

Secreted extracellular vesicles (EVs) contain active biomolecules, including miRNAs, composition of which reflects epigenetic changes occurring in cells during pathological processes, in particular, malignant transformation. The accumulated pool of data on the role of EVs in carcinogenesis has stimulated investigations of the EV-derived cancer markers. The most important factor limiting development of this scientific direction is lack of "gold standards" both for methods of EV isolation from biological fluids and for analyzing their molecular content, including composition of miRNAs. Here we first examined efficacy of various methods for small RNA isolation from EVs contained in ascitic fluid for subsequent miRNA analysis. Comparison of different commercial kits showed advantages of the methods based on phenol-chloroform extraction: Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit. Analysis of the small RNA transcriptome showed presence of various classes of molecules in the EVs, among which proportion of miRNAs averaged 6% and reaching 10% with the Total Exosome RNA & Protein Isolation Kit. The PureLink miRNA Isolation Kit demonstrated the lowest efficiency. The miRNeasy Advanced Serum/Plasma Kit showed the highest concentration of the small RNA fraction, miRNA proportion of which, however, did not exceed that obtained with the miRNeasy Serum/Plasma Kit and Total Exosome RNA & Protein Isolation Kit. Moreover, RT-PCR analysis of the individual molecules showed lower levels of each of investigated miRNAs (miR-1246, miR-200b-5p, miR-200c-3p, and miR-23a-3p) when using the miRNeasy Advanced Serum/Plasma Kit. In conclusion, Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit can be considered as optimal kits in terms of performance based on combination of the studied characteristics, including small RNA concentration, percentage of microRNA according to bioanalyzer and sequencing results, and levels of individual miRNAs detected by RT-PCR.

Isolation and Characterization of Extracellular Vesicles from Gastric Juice.

EVs are involved in local and distant intercellular communication and play a vital role in cancer development. Since EVs have been found in almost all body fluids, there are currently active attempts for their application in liquid diagnostics. Blood is the most commonly used source of EVs for the screening of cancer markers, although the percentage of tumor-derived EVs in the blood is extremely low. In contrast, GJ, as a local biofluid, is expected to be enriched with GC-associated EVs. However, EVs from GJ have never been applied for the screening and are underinvestigated overall. Here we show that EVs can be isolated from GJ by ultracentrifugation. TEM analysis showed high heterogeneity of GJ-derived EVs, including those with exosome-like size and morphology. In addition to morphological diversity, EVs from individual GJ samples differed in the composition of exosomal markers. We also show the presence of stomatin within GJ-derived EVs for the first time. The first conducted comparison of miRNA content in EVs from GC patients and healthy donors performed using a pilot sampling revealed the significant differences in several miRNAs (-135b-3p, -199a-3p, -451a). These results demonstrate the feasibility of the application of GJ-derived EVs for screening for miRNA GC markers.

Extracellular Vesicles from Uterine Aspirates Represent a Promising Source for Screening Markers of Gynecologic Cancers.

Extracellular vesicles (EVs), including exosomes, are key factors of intercellular communication, performing both local and distant transfers of bioactive molecules. The increasingly obvious role of EVs in carcinogenesis, similarity of molecular signatures with parental cells, precise selection and high stability of cargo molecules make exosomes a promising source of liquid biopsy markers for cancer diagnosis. The uterine cavity fluid, unlike blood, urine and other body fluids commonly used to study EVs, is of local origin and therefore enriched in EVs secreted by cells of the female reproductive tract. Here, we show that EVs, including those corresponding to exosomes, could be isolated from individual samples of uterine aspirates (UA) obtained from epithelial ovarian cancer (EOC) patients and healthy donors using the ultracentrifugation technique. First, the conducted profiling of small RNAs (small RNA-seq) from UA-derived EVs demonstrated the presence of non-coding RNA molecules belonging to various classes. The analysis of the miRNA content in EVs from UA performed on a pilot sample revealed significant differences in the expression levels of a number of miRNAs in EVs obtained from EOC patients compared to healthy individuals. The results open up prospects for using UA-derived EVs as a source of markers for the diagnostics of gynecological cancers, including EOC.

CRABP1 and CRABP2 Protein Levels Correlate with Each Other but Do Not Correlate with Sensitivity of Breast Cancer Cells to Retinoic Acid.

Retinoic acid (RA) binding proteins, CRABP1 and CRABP2, are molecular chaperones that mediate intracellular activity of RA, the key promoter of cell differentiation with tumor suppressor activity. One of the main functions of CRABP2 is delivery and transfer of RA to the nuclear receptors RAR/RXR, which leads to activation of the transcription of a wide range of retinoid-responsive genes. The functions of CRABP1 are less studied but are apparently associated with sequestration of RA in cytoplasm and limitation of its transcriptional activity, suggesting involvement of this protein in the development of RA resistance. The mechanisms regulating activity of CRABP1 are also poorly understood. Comparison of the CRABP1 level in tumor cell lines of various origins, performed for the first time here, showed absence of the CRABP1 protein in the cell lines of tumors considered to be RA-resistant, and pronounced production of this protein in the RA-sensitive cells. However, analysis carried out with a panel of breast cancer cell lines with different levels of RA-sensitivity showed that there was no correlation between the production of CRABP1 protein and the sensitivity of the cells to RA. At the same time, we found strong correlation between the expression of CRABP1 and CRABP2 proteins in all studied cell types, regardless of their origin and RA-sensitivity/resistance. Moreover, suppression of the CRABP1 level in both RA-sensitive and RA-resistant cells was shown in the cells with cells with knockdown of CRABP2 gene. The revealed CRABP2-dependent regulation of CRABP1 production is a new mechanism of the intracellular retinoic signaling system.

Stomatin is highly expressed in exosomes of different origin and is a promising candidate as an exosomal marker.

Proteins involved in the organizing of lipid rafts can be found in exosomes, as shown for caveolin-1, and they could contribute to exosomal cargo sorting, as shown for flotillins. Stomatin belongs to the same stomatin/prohibitin/flotillin/HflK/C family of lipid rafts proteins, but it has never been studied in exosomes except for extracellular vesicles (EVs) originating from blood cells. Here we first show the presence of stomatin in exosomes produced by epithelial cancer cells (non-small cell lung cancer, breast, and ovarian cancer cells) as well as in EVs from biological fluids, including blood plasma, ascitic fluids, and uterine flushings. A high abundance of stomatin in EVs of various origins and its enrichment in exosomes make stomatin a promising exosomal marker. Comparison with other lipid raft proteins and exosomal markers showed that the level of stomatin protein in exosomes from different sources corresponds well to that of CD9, while it differs essentially from flotillin-1 and flotillin-2 homologs, which in turn are present in exosomes in nearly equal proportions. In contrast, the level of vesicular caveolin-1 as well as its EV-to-cellular ratio vary drastically depending on cell type.