Dictyostelium as model system for studies of the actin cytoskeleton by molecular genetics.

The actin cytoskeleton is an essential structure for most movements at the cellular and intracellular level. Whereas for contraction a muscle cell requires a rather static organisation of cytoskeletal proteins, cell motility of amoeboid cells relies on a tremendously dynamic turnover of filamentous networks in a matter of seconds and at distinct regions inside the cell. The best model system for studying cell motility is Dictyostelium discoideum. The cells live as single amoebae but can also start a developmental program that leads to multicellular stages and differentiation into simple types of tissues. Thus, cell motility can be studied on single cells and on cells in a tissue-like aggregate. The ability to combine protein purification and biochemistry with fairly easy molecular genetics is a unique feature for investigation of the cytoskeleton and cell motility. The actin cytoskeleton in Dictyostelium harbours essentially all classes of actin-binding proteins that have been found throughout eukaryotes. By conventional mutagenesis, gene disruption, antisense approaches, or gene replacements many genes that code for cytoskeletal proteins have been disrupted, and altered phenotypes in transformants that lacked one or more of those cytoskeletal proteins allowed solid conclusions about their in vivo function. In addition, tagging the proteins or selected domains with green fluorescent protein allows the monitoring of protein redistribution during cell movement. Gene tagging by restriction enzyme mediated integration of vectors and the ongoing international genome and cDNA sequencing projects offer the chance to understand the dynamics of the cytoskeleton by identification and functional characterisation of all proteins involved.     

Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell-to-cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)-β1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc-1) cells. TGF-β1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E-cadherin from cell-to-cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT-specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG-β1-treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF-β1 and may contribute to EMT.     

Moving in the right direction-nanoimaging in cancer cell motility and metastasis

Although genetic and protein manipulations have been the cornerstone for the study and understanding of biological processes for many decades, complimentary nanoscale observations have only more recently been achieved in the live-imaging mode. It is at the nano measurement level that events such as protein-protein interactions, enzymatic conversions, and single-molecule stochastic behavior take place. Therefore, nanoscale observations allow us to reinterpret knowledge from large-scale or bulk techniques and gain new insight into molecular events that has cellular, tissue, and organismal phenotypic manifestations. This review identifies pertinent questions relating to the sensing and directional component of cancer cell chemotaxis and discusses the platforms that provide insight into the molecular events related to cell motility. The study of cell motility at the molecular imaging level often necessitates the use of devices such as microinjection, microfluidics, in vivo/intravital and in vitro chemotaxis assays, as well as fluorescence methods like uncaging and FRET. The micro- and nanofabricated devices that facilitate these techniques and their incorporation to specialized microscopes such as the multiphoton, AFM, and TIR-FM, for high-resolution imaging comprise the nanoplatforms used to explore the mechanisms of carcinogenesis. In real-time observations, within a milieu of physiological protein concentrations, true states of dynamic and kinetic fluxes can be monitored.     

Dancing to a somewhat different rhythm: Cell migration along the natural basement membrane

Much of our understanding of the events which underlie cell migration has been derived from studies of cells in tissue culture. One of the components that mediates this process is the dynamic actin-based microfilament system that can reorganize itself into so-called stress fibers that are considered essential components for cell motility. In contrast, relatively few studies have investigated cell movement along an extracellular matrix (ECM) which is known to influence both cellular organization and behavior. This opinion/viewpoint article briefly reviews cell migration during corneal endothelial wound repair along the tissue’s natural basement membrane, Descemet’s membrane. Because the tissue exists as a cell monolayer it affords one an opportunity to readily explore the effect of cell/matrix influences on cell motility. As such, cell movement along this substrate differs somewhat from that found in vitro and migrating endothelial cells also demonstrate an ability to move along the ECM without the benefit of having an organized actin cytoskeleton.     

High-resolution cell outline segmentation and tracking from phase-contrast microscopy images

Accurate extraction of cell outlines from microscopy images is essential for analysing the dynamics of migrating cells. Phase-contrast microscopy is one of the most common and convenient imaging modalities for observing cell motility because it does not require exogenous labelling and uses only moderate light levels with generally negligible phototoxicity effects. Automatic extraction and tracking of high-resolution cell outlines from phase-contrast images, however, is difficult due to complex and non-uniform edge intensity. We present a novel image-processing method based on refined level-set segmentation for accurate extraction of cell outlines from high-resolution phase-contrast images. The algorithm is validated on synthetic images of defined noise levels and applied to real image sequences of polarizing and persistently migrating keratocyte cells. We demonstrate that the algorithm is able to reliably reveal fine features in the cell edge dynamics.     

Applications of quantitative digital image analysis to breast cancer research

Our studies of radiogenic carcinogenesis in mouse and human models of breast cancer are based on the view that cell phenotype, microenvironment composition, communication between cells and within the microenvironment are important factors in the development of breast cancer. This is complicated in the mammary gland by its postnatal development, cyclic evolution via pregnancy and involution, and dynamic remodeling of epithelial-stromal interactions, all of which contribute to breast cancer susceptibility. Microscopy is the tool of choice to examine cells in context. Specific features can be defined using probes, antibodies, immunofluorescence, and image analysis to measure protein distribution, cell composition, and genomic instability in human and mouse models of breast cancer. We discuss the integration of image acquisition, analysis, and annotation to efficiently analyze large amounts of image data. In the future, cell and tissue image-based studies will be facilitated by a bioinformatics strategy that generates multidimensional databases of quantitative information derived from molecular, immunological, and morphological probes at multiple resolutions. This approach will facilitate the construction of an in vivo phenotype database necessary for understanding when, where, and how normal cells become cancer.     

Intravital dual-colored visualization of colorectal liver metastasis in living mice using two photon laser scanning microscopy

A major challenge of cancer biology is to visualize the dynamics of the metastatic process in secondary organs at high optical resolution in vivo real-time. Here, we presented intravital, dual-colored imaging of liver metastasis formation from a single cancer cell to metastatic colonies in the living liver of living mice using two photon laser scanning microscopy (TPLSM). Red fluorescent protein expressing murine (SL4) or human (HT29) colorectal cancer cell lines were inoculated to the spleen of green fluorescent protein expressing mice. Intravital TPLSM was performed by exteriorizing and fixing the liver lobe of living mice. This was repeated several times for the long-term imaging of the same mouse. Viable cancer cells in the living liver of living mice were visualized intravitally at a magnification of over 600×. Single cancer cells were arrested within hepatic sinusoids 2 h after injection. Platelet aggregation surrounding a cancer cell was observed, indicating a phenomenon of tumor-cell induced platelet aggregation. Cancer cells were extravasated from hepatic sinusoids to the space of Disse. Protrusions of Kupffer cells surrounding a cancer cell were observed, indicating that Kupffer cells appear to phagocytose cancer cells. SL4 cells formed liver metastatic colonies with extensive stromal reaction. Liver metastases by HT29 cells were observed as a cluster of micrometastatic nodules. High-resolution, dual-colored, real-time visualization of cancer metastasis using intravital TLPSM can help to understand spatiotemporal tumor-host interactions during metastatic processes in the living organs of living animals.     

Cell-surface ultrastructural changes during the in vitro neuron-like differentiation of rat bone marrow-derived mesenchymal stem cells

The neuron-like differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) has been extensively studied. However, the alternations of the cell-surface ultrastructures and the membrane tension/reservoir of the cells during this differentiation process are poorly understood. Therefore, atomic force microscopy (AFM) was utilized in this study to observe the cell-surface ultrastructural changes among rat bone marrow-derived mesenchymal stem cells (rBMMSCs), partially differentiated cells, and fully differentiated neuron-like cells. By analyzing the stiffness of plasma membranes, lamellipodial extensions, average heights of small membrane protrusions and relatively larger uplifted structures, and peak-peak spacing among protrusions and/or uplifted structures, we found that the membrane reservoir may potentially decrease upon the differentiation from rBMMSCs to partially differentiated cells and to fully differentiated neuron-like cells. The results may help to better understanding the membrane tension of various types of cells and related biological processes, such as membrane traffic, cell adhesion, motility, differentiation, among others. The data also implies that AFM may be a useful tool for evaluating membrane reservoir by imaging cell-surface ultrastructures.     

Dissociated cells of the calcareous sponge clathrina: a model for investigating cell adhesion and cell motility in vitro

The study of cell-cell and cell-substratum adhesion in vitro is useful for understanding cell behavior in a three-dimensional pattern. We have used dissociated cells (choanocytes represent the main fraction) from the calcareous sponge Clathrina, namely C. cerebrum and C. clathrus, to illustrate our present understanding on three main aspects of cell-cell and cell-substratum adhesion in vitro: (1) cytoskeletal protrusions; (2) cell behaviours on organic substrata; and (3) paths of locomotory sponge cell. Cell locomotion occurs by the extensions of scleropodial and lamellipodial protrusions, by way of actin polymerization. The extent to which cells produce these cytoplasmic processes varies according to the substratum (e.g., collagen, fibronectin, laminin, polylysine). It was found that more cell extensions were produced on collagen substrata, and this led to greater cell movement. Advancing choanocytes are not polarized. Their paths are particularly complicated, showing linear segments, which produce a more efficent cellular translocation, and winding tracts with frequent turns or loops. Small amoeboid cells describe more linear paths with a wide range of speed variation than larger cells. The presence of cell-derived substratum reduces the progressive dispersion of cells and allows cells to encounter one another in such a way that the initial random walking later turns into non-random displacement. Even though cAMP-treated cells exhibit different aggregative tactics, cAMP 10(-8) M remarkably enhances cell encounters and supports the existing information that this cyclic nucleotide represents a signal that affects cell morphology and locomotion. The bulk of data on sponge cell-cell and cell-substratum adhesion has been evaluated by mentioning the significant advances and references concerning studies of other cell systems.     

Surface activity of cancer cells: The fusion of two cell aggregates

A key feature that distinguishes cancer cells from all other cells is their capability to spread throughout the body. Although how cancer cells collectively migrate by following molecular rules which influence the state of cell-cell adhesion contacts has been comprehensively formulated, the impact of physical interactions on cell spreading remains less understood. Cumulative effects of physical interactions exist as the interplay between various physical parameters such as (1) tissue surface tension, (2) viscoelasticity caused by collective cell migration, and (3) solid stress accumulated in the cell aggregate core region. This review aims to point out the role of these physical parameters in cancer cell spreading by considering and comparing the rearrangement of various mono-cultured cancer and epithelial model systems such as the fusion of two cell aggregates. While epithelial cells undergo volumetric cell rearrangement driven by the tissue surface tension, which directs cell movement from the surface to the core region of two-aggregate systems, cancer cells rather perform surface cell rearrangement. Cancer cells migrate toward the surface of the two-aggregate system driven by the solid stress while the surface tension is significantly reduced. The solid stress, accumulated in the core region of the two-aggregate system, is capable of suppressing the movement of epithelial cells that can undergo the jamming state transition; however, this stress enhances the movement of cancer cells. We have focused here on the multi-scale rheological modeling approaches that aimed at reproducing and understanding these biological systems.     

Three-dimensional reconstruction and motion analysis of living, crawling cells

Cell behavior is three-dimensional (3-D), even when it takes place on a flat surface. Migrating cells form pseudopods on and off the substratum, and the cell body undergoes height changes associated with a 1 min behavior cycle. Inside the cell, the nucleus has a 3-D migratory cycle, and vesicles move up and down in the z-axis as a cell locomotes. For these reasons, the two-dimensional (2-D) analysis of cellular and subcellular behavior is, in many cases, inadequate. We have, therefore, developed 3-D motion analysis systems that reconstruct the cell surface, nucleus, pseudopods, and vesicles of living, crawling cells in 3-D at time intervals as short as 1 s, and compute more than 100 parameters of motility and dynamics morphology at 1-s intervals. We are now in the process of developing a multimode reconstruction system that will allow us to reconstruct and analyze fluorescently tagged molecular complexes within the differential interference contrast-imaged subcellular architecture of a crawling cell. These evolving technologies should find wide application for a host of biomedical problems.     

Using the atomic force microscope to observe and study the ultrastructure of the living BIU-87 cells of the human bladder cancer

In this study, the ultrastructure of living BIU-87 cells of human bladder cancer was mapped using atomic force microscopy to reveal the dynamic change of single cancerous cell division. Simultaneously, the feasibility and functional reliability of the atomic force microscope (AFM) were established and a laboratory model using AFM to study living cancerous cells was created. In this experiment, BIU-87 cells of human bladder cancer were cultured by conventional methods and grown in gelatin-treated dishes. A thermostat was used for preserving the cell's living temperature. Scanning of these cells using AFM was carried out in physiologic condition. The AFM images of the ultrastructure of living BIU-87 cells as well as those of the cell's membrane and cytoskeleton were very clear. The dynamic phenomenon of single cell division was observed. It was concluded that the AFM was able to observe and depict the ultrastructure of living cells of human bladder cancer directly and in real time. This experimental model is expected to play an important role in elucidating the cancerous mechanism of bladder normal cells at the atomic or nanometer level.     

Automatic detection and analysis of cell motility in phase‐contrast time‐lapse images using a combination of maximally stable extremal regions and Kalman filter approaches

Phase‐contrast illumination is simple and most commonly used microscopic method to observe nonstained living cells. Automatic cell segmentation and motion analysis provide tools to analyze single cell motility in large cell populations. However, the challenge is to find a sophisticated method that is sufficiently accurate to generate reliable results, robust to function under the wide range of illumination conditions encountered in phase‐contrast microscopy, and also computationally light for efficient analysis of large number of cells and image frames. To develop better automatic tools for analysis of low magnification phase‐contrast images in time‐lapse cell migration movies, we investigated the performance of cell segmentation method that is based on the intrinsic properties of maximally stable extremal regions (MSER). MSER was found to be reliable and effective in a wide range of experimental conditions. When compared to the commonly used segmentation approaches, MSER required negligible preoptimization steps thus dramatically reducing the computation time. To analyze cell migration characteristics in time‐lapse movies, the MSER‐based automatic cell detection was accompanied by a Kalman filter multiobject tracker that efficiently tracked individual cells even in confluent cell populations. This allowed quantitative cell motion analysis resulting in accurate measurements of the migration magnitude and direction of individual cells, as well as characteristics of collective migration of cell groups. Our results demonstrate that MSER accompanied by temporal data association is a powerful tool for accurate and reliable analysis of the dynamic behaviour of cells in phase‐contrast image sequences. These techniques tolerate varying and nonoptimal imaging conditions and due to their relatively light computational requirements they should help to resolve problems in computationally demanding and often time‐consuming large‐scale dynamical analysis of cultured cells.     

基于PCA和SVM算法的肝癌细胞显微后向散射光谱分类

为了实现对肝癌的早期实时和在体探测,基于前期搭建的光纤共聚焦后向散射(FCBS)光谱仪获取肝癌细胞的显微后向散射光谱,分别使用主成分分析(PCA)和支持向量机(SVM)两种算法,对获得的正常肝细胞株(L02)、低转移潜能肝癌细胞株(MHCC97-L)和高转移潜能肝癌细胞株(HCCLM3)三种细胞的后向散射光谱进行分类。使用PCA对获得的三种细胞光谱数据进行降维分析,得到的前两个主成分综合了全部信息的95.4%,由主成分1和主成分2的得分图可以观察到,三种细胞在直观上有明显的区分;对同一数据集选取69例对象通过SVM机器学习算法训练分类模型,随机抽取50例作为训练集,19例作为预测集,最终分类的准确度达到了94.7%。实验结果表明:使用光纤共聚焦后向散射(FCBS)光谱仪获取的细胞显微后向散射光谱可以分别通过PCA和SVM对不同转移潜能的肝癌细胞进行自动分类,这将为研究活检提供必要的检测手段。     

多尺度区域生长与去粘连模型的乳腺细胞分割

乳腺癌已经成为女性最常见的恶性肿瘤,组织切片显微图像的病理分析是诊断的主要手段,细胞的准确分割是病理分析的重要环节。该文提出了一种新的乳腺细胞显微图像的自动分割算法:首先结合小波分解和多尺度区域生长算法分离细胞和背景,实现对细胞的精确定位;然后采用改进的数学形态学对粘连细胞进行一次细分割;接着再采用基于曲率尺度空间(CSS)的角点检测分割算法对粘连细胞进行二次细分割;两次细分割方法构成了一个双策略去粘连模型,保证了去粘连的准确性和鲁棒性。将算法应用到22幅乳腺细胞显微图像上,可以对不同类型的乳腺细胞图像进行全自动分割,有较高的分割灵敏度(0.944±0.024)和特异度(0.937±0.038),且具有较好的普适性。     

MicroRNA-181a is elevated by 10-hydroxycamptothecin and represses lung carcinoma progression by downregulating FOXP1

Tumor progression is usually characterized by proliferation, migration, and angiogenesis, which is essential for supplying both nutrients and oxygen to the tumor cells. Therefore, targeting angiogenesis has been considered a promising therapeutic strategy for cancer prevention and treatment. In the present study, we demonstrated that in addition to suppressing lung cancer cell proliferation and migration in vitro, 10-hydroxycamptothecin (10-HCPT) is also capable of inhibiting angiogenesis in vivo with a miR-181a-dependent manner. Mechanistically, by upregulating miR-181a, which in turn downregulating FOXP1, 10-HCPT can inhibit the PI3K/Akt/ERK signaling pathwaymediated angiogenesis. Furthermore, reduced levels of miR-181a have been found in both lung cancer cell lines and xenograft with concurrently elevated levels of FOXP1, VEGF, bFGF, and HDGF. Consistent with the findings from the in vitro experiments, miR-181a impairs neovascularization in our xenograft model. In summary, our findings have not only established the anti-oncogenic role of miR-181a in lung cancer angiogenesis but also suggest that 10-HCPT could be a potential therapeutic reagent for lung cancer treatment.     

A thermal study of cellular motility by optical time-resolved correlation

The study of motor properties of cells under appropriate physical-chemical conditions is a significant problem nowadays. The standard techniques presently used do not allow to evaluate neither large samples nor to control their thermodynamic conditions. In this work, we report a cell motility sensor based on an optical technique with a time-resolved correlation, adapted in a system able to study several samples simultaneously. Image correlation analysis is used to follow their temporal behavior. A wide variety of motile cells, such as archaea, bacteria, spermatozoa, and even contractile cells, can be studied using this technique. Here, we tested our technique with the study of sperm motility. In particular, both the sperm motility and its prevalence are studied under a temperature range from 0 to 37 °C. We found that incubation at 10 °C presents the lengthiest prevalence in motility and observed, for the first time, an interesting thermal reversibility behavior.     

17‐beta‐estradiol analog inhibits cell proliferation by induction of apoptosis in breast cell lines

Microtubules are important targets when studying potential anticancer agents since disturbance of these microtubule dynamics results in cell cycle arrest and cell death. 2‐Methoxyestradiol is a naturally occurring metabolite that exerts antiproliferative activity and induces apoptosis. Due to limited biological accessibly and rapid metabolic degradation, several analogs were synthesized. This study investigated the antiproliferative influence of an 2‐methoxyestradiol analog, (8R, 13S, 14S, 17S)‐2‐Ethyl‐13‐methyl‐7, 8, 9, 11, 12,13, 14, 15, 16, 17‐decahydro‐6H‐cyclopenta[a]phenanthrane‐3, 17‐diyl bis(sulfamate) (EMBS) on cell proliferation, morphology and apoptosis induction in a estrogen receptor‐positive breast adenocarcinoma cells line (MCF‐7), estrogen receptor‐negative highly metastatic breast cell line (MDA‐MB‐231) and a non‐tumorigenic breast epithelial cell line (MCF‐12A). Spectrophotometry results indicated that EMBS exerted differential antiproliferative activity in the three cell lines. Cell growth of the breast adenocarcinoma and highly metastatic breast cell line reached a plateau effect at 0.4 μM after 24 h of exposure. Light microscopy and polarization‐optical transmitted light differential interference contrast demonstrated compromised cell density, cells blocked in metaphase and the presence of apoptotic characteristics after EMBS exposure for 24 h in all three cell lines. Transmission electron microscopy and scanning electron microscopy revealed hallmarks of apoptosis namely the presence of apoptotic bodies, shrunken cells and cell debris in EMBS‐exposed cells. This investigation demonstrated that EMBS does exert antimitotic activity and induces apoptosis contributing to elucidating the signal transduction of EMBS in tumorigenic and non‐tumorigenic breast cell lines. Findings warrant in‐depth analysis of specific targets in vitro and subsequent in vivo investigation for anticancer therapy. Microsc. Res. Tech. 77:236–242, 2014. © 2014 Wiley Periodicals, Inc.     

Berberine inhibits the proliferation of pancreatic cancer cells by targeting pancreatic cancer stem cells through regulating EMT signaling pathway

Pancreatic ductal adenocarcinoma (PDAC) is universally acknowledged as the cancer with the highest mortality rate. Berberine has high medicinal value and has been used as an anti-cancer agent. Hence the purpose of this study was to investigate the anti-cancer effect of berberine in PDAC. Berberine was shown to have a selective anti-cancer effect on PDAC by MTT assay in vitro. Pancreatic cancer stem cells (PCSCs), regulated by epithelial–mesenchymal transition (EMT), could promote the proliferation of PDAC cells. However, berberine suppressed the proliferation and stemness of PCSCs through immunofluorescence staining, stem cell sphere assays and so forth in vitro. In vivo, berberine reduced tumor size and decreased the expression levels of Ki67, a marker of cellular proliferation, in orthotopic pancreatic tumors. In addition, berberine inhibited the EMT signaling pathway by RT-PCR and Western blotting methods both in vitro and in vivo. Our study indicates that berberine inhibits the proliferation of PDAC cells both in vivo and in vitro. The mechanism of the anti-cancer effect of berberine likely involves the inhibition of EMT. Therefore, berberine may be a novel antineoplastic drug with clinical efficacy in PDAC.