Automated image segmentation for detecting cell spreading for metastasizing assessments of cancer development

The automated segmentation of cells in microscopic images is an open research problem that has important implications for studies of the developmental and cancer processes based on in vitro models. In this paper, we present the approach for segmentation of the DIC images of cultured cells using G-neighbor smoothing followed by Kauwahara filtering and local standard deviation approach for boundary detection. NIH FIJI/ImageJ tools are used to create the ground truth dataset. The results of this work indicate that detection of cell boundaries using segmentation approach even in the case of realistic measurement conditions is a challenging problem

DEVELOPMENT OF ANALYTIC TOOLS FOR HIGH-THROUGHPUT LABEL-FREE MICROSCOPY OF CELL MOTILITY AND CELL SPREADING

High-throughput microscopy is an approach that emerged a decade ago. It is an efficient tool to solve numerous tasks in cell biology and drug discovery in combination with automated microscopy. Automated microscopy is suited to perform direct observation of living cells and allows to obtain consistent and straightforward results from dynamic processes. The aim of various images processing tools is to study cell migration. However, there is a deficiency in the tools to perform accurate and efficient image processing of unlabeled cells, compared to fluorescently labeled ones. Labelfree microscopic techniques like Phase contrast and DIC result in low contrast images, therefore the tools designed for fluorescent image segmentation are inefficient. To overcome this limitation, many methods to automatically analyze images of wound healing were developed within the last decade, but these tools require manual tuning of parameters and lack of automation to process stacks of thousands of images. This work focuses on building an efficient image processing pipeline for brightfield image segmentation. The solution proposed herein is a filtering sequence to modify the bright-field image intensity histogram so that it resembles that of fluorescent images. In addition, a conditional operator was introduced, which is a logic loop to test the quality of wound gap segmentation in unsupervised mode. This allowed achieving >95% accuracy of segmentation in the un-supervised mode. The processing pipeline for cell spreading is further enhanced by the transformation of image coordinates to reduce the dimensionality of the image, and to simplify the image edge to a single direction gradient. The tools developed in this Ph.D. project were applied to evaluate the effect of inhibiting microtubule dynamics on cell motility and spreading. It was confirmed that wound healing closure occurs in a non-linear manner for the majority of cell lines studied. A piecewise regression analysis was performed to specify the period when the wound closure occurs at constant velocity. Further, it was found that the observations made in the first 12 hours after scratching the cell layer were optimal for obtaining precise measurements of the wound edge for both normal and cancer cells. A reduction in cell motility in response to microtubule inhibitors' action occurred in a dose-dependent manner at concentrations below the apparent cytotoxic doses. It was demonstrated that the speed of wound closure observed in 24 hours did not depend on a change in cell proliferation induced by the microtubule drugs. Therefore, the image processing pipeline developed in this Ph.D. research project can significantly reduce time consumption for RAW data processing and at the same time greatly increase the precision in analyzing motility-based assays

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

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

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

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

Abstract 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

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

Abstract 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 β-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/β-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