Extracellular vesicles in gastrointestinal cancer in conjunction with microbiota: On the border of Kingdoms.

Extracellular vesicle (EV) production is a universal feature of metazoan cells as well as prokaryotes (bMVs - bacterial microvesicles). They are small vesicles with phospholipid membrane carrying proteins, DNA and different classes of RNAs and are heavily involved in intercellular communication acting as vectors of information to target cells. For the last decade, the interest in EV research has exponentially increased though thorough studies of their roles in various pathologies that was not previously possible due to technical limitations. This review focuses on research evaluating the role of EV production in gastrointestinal (GI) cancer development in conjunction with GI microbiota and inflammatory diseases. We also discuss recent studies on the promising role of EVs and their content as biomarkers for early diagnosis of GI cancers. The bMVs have also been implicated in the pathogenesis of GI chronic inflammatory diseases, however, possible role of bMVs in tumorigenesis remains underestimated. We propose that EVs from eukaryotic cells as well as from different microbial, fungi, parasitic species and edible plants in GI tract act as mediators of intracellular and inter-species communication, particularly facilitating tumor cell survival and multi-drug resistance. In conclusion, we suggest that matching sequences from EV proteomes (available from public databases) with known protein sequences of microbiome gut bacteria will be useful in identification of antigen mimicry between evolutionary conservative protein sequences. Using this approach we identified Bacteroides spp. pseudokinase with activation loop and homology to PDGFRα, providing a proof-of-concept strategy. We speculate that existence of microbial pseudokinase that 'mimics' PDGFRα may be related to PDGFRα and Bacteroides spp. roles in colorectal carcinogenesis that require further investigation.

The Frequent Sampling of Wound Scratch Assay Reveals the "Opportunity" Window for Quantitative Evaluation of Cell Motility-Impeding Drugs.

Wound healing assay performed with automated microscopy is widely used in drug testing, cancer cell analysis, and similar approaches. It is easy to perform, and the results are reproducible. However, it is usually used as a semi-quantitative approach because of inefficient image segmentation in transmitted light microscopy. Recently, several algorithms for wound healing quantification were suggested, but none of them was tested on a large dataset. In the current study, we develop a pipeline allowing to achieve correct segmentation of the wound edges in >95% of pictures and extended statistical data processing to eliminate errors of cell culture artifacts. Using this tool, we collected data on wound healing dynamics of 10 cell lines with 10 min time resolution. We determine that the overall kinetics of wound healing is non-linear; however, all cell lines demonstrate linear wound closure dynamics in a 6-h window between the fifth and 12th hours after scratching. We next analyzed microtubule-inhibiting drugs', nocodazole, vinorelbine, and Taxol, action on the kinetics of wound healing in the drug concentration-dependent way. Within this time window, the measurements of velocity of the cell edge allow the detection of statistically significant data when changes did not exceed 10-15%. All cell lines show decrease in the wound healing velocity at millimolar concentrations of microtubule inhibitors. However, dose-dependent response was cell line specific and drug specific. Cell motility was completely inhibited (edge velocity decreased 100%), while in others, it decreased only slightly (not more than 50%). Nanomolar doses (10-100 nM) of microtubule inhibitors in some cases even elevated cell motility. We speculate that anti-microtubule drugs might have specific effects on cell motility not related to the inhibition of the dynamic instability of microtubules.

On the Relationship Between EB-3 Profiles and Microtubules Growth in Cultured Cells.

Microtubules are dynamic structures undergoing rapid growth and shrinkage in living cells and in vitro. The growth of microtubules in vitro was analyzed with subpixel precision (Maurer et al., Current Biology, 2014, 24 (4), 372-384); however, to what extent these results could be applied for microtubules growing in vivo remains largely unknown. Particularly, the question is whether microtubule growth velocity in cells could be sufficiently approximated by a Gaussian distribution or its variability requires a more sophisticated description? Addressing this question, we used time-lapse microscopy and mathematical modeling, and we analyzed EB-3 comets forming on microtubules of cultured cells with subpixel precision. Parameters of comets (shape, form, and velocity) were used as topological characteristics of 3D voxel objects. Using regression analysis, we determined the real positions of the microtubule tips in time-lapse sequences. By exponential decay fitting of the restored comet intensity profile, we found that in vivo EB-3 rapidly exchanges on growing microtubule ends with a decoration time ∼ 2 s. We next developed the model showing that the best correlation between comet length and microtubule end growth velocity is at time intervals close to the decoration time. In the cells, EB comet length positively correlates with microtubule growth velocity in preceding time intervals, while demonstrating no correlation in subsequent time intervals. Correlation between comet length and instantaneous growth velocity of microtubules remains under nocodazole treatment when mean values of both parameters decrease. Our data show that the growth of microtubules in living cells is well-approximated by a constant velocity with large stochastic fluctuations.

Composite Cryogel with Polyelectrolyte Complexes for Growth Factor Delivery.

Macroporous scaffolds composed of chitosan (CHI), hydroxyapatite (HA), heparin (Hep), and polyvinyl alcohol (PVA) were prepared with a glutaraldehyde (GA) cross-linker by cryogelation. Addition of PVA to the reaction mixture slowed down the formation of a polyelectrolyte complex (PEC) between CHI and Hep, which allowed more thorough mixing, and resulted in the development of the homogeneous matrix structure. Freezing of the CHI-HA-GA and PVA-Hep-GA mixture led to the formation of a non-stoichiometric PEC between oppositely charged groups of CHI and Hep, which caused further efficient immobilization of bone morphogenic protein 2 (BMP-2) possible due to electrostatic interactions. It was shown that the obtained cryogel matrix released BMP-2 and supported the differentiation of rat bone marrow mesenchymal stem cells (rat BMSCs) into the osteogenic lineage. Rat BMSCs attached to cryogel loaded with BMP-2 and expressed osteocalcin in vitro. Obtained composite cryogel with PEC may have high potential for bone regeneration and tissue engineering applications.

Analysis of Microtubule Dynamics Heterogeneity in Cell Culture.

Microtubules (MTs) are dynamic components of the cytoskeleton playing an important role in a large number of cell functions. Individual MTs in living cells undergo stochastic switching between alternate states of growth, shortening and attenuated phase, a phenomenon known as tempered dynamic instability. Dynamic instability of MTs is usually analyzed by labeling MTs with +TIPs, namely, EB proteins. Tracking of +TIP trajectories allows analyzing MT growth in cells with a different density of MTs. Numerous labs now use +TIP to track growing MTs in a variety of cell cultures. However, heterogeneity of MT dynamics is usually underestimated, and rather small sampling for the description of dynamic instability parameters is often used. The strategy described in this chapter is the method for repetitive quantitative analysis of MT growth rate within the same cell that allows minimization of the variation in MT dynamics measurement. We show that variability in MT dynamics within a cell when using repeated measurements is significantly less than between different cells in the same chamber. This approach allows better estimation of the heterogeneity of cells' responses to different treatments. To compare the effects of different MT inhibitors, the protocol using normalized values for MT dynamics and repetitive measurements for each cell is employed. This chapter provides detailed methods for analysis of MT dynamics in tissue cultures. We describe protocols for imaging MT dynamics by fluorescent microscopy, contrast enhancement technique, and MT dynamics analysis using triple color-coded display based on sequential subtraction analysis.

Dysregulation of YAP by ARF Stimulated with Tea-derived Carbon Nanodots.

YAP is a downstream nuclear transcription factor of Hippo pathway which plays an essential role in development, cell growth, organ size and homeostasis. It was previously identified that elevation of YAP in genomics of genetic engineered mouse (GEM) model of prostate cancer is associated with Pten/Trp53 inactivation and ARF elevation hypothesizing the essential crosstalk of AKT/mTOR/YAP with ARF in prostate cancer. However, the detailed function and trafficking of YAP in cancer cells remains unclear. Using GEM microarray model, we found ARF dysregulates Hippo and Wnt pathways. In particular, ARF knockdown reduced non-nuclear localization of YAP which led to an increase in F-actin. Mechanistically, ARF knockdown suppressed protein turnover of ß-catenin/YAP, and therefore enhanced the activity of AKT and phosphorylation of YAP. Moreover, we found tea-derived carbon dots can interact with ARF in nucleus that may further lead to the non-nuclear localization of YAP. Thus, we reported a novel crosstalk of ARF/ß-catenin dysregulated YAP in Hippo pathway and a new approach to stimulate ARF-mediated signaling to inhibit nuclear YAP using nanomaterials implicating an innovative avenue for treatment of cancer.