Automated tracking of cell migration in phase contrast images with CellTraxx.

The ability of cells to move and migrate is required during development, but also in the adult in processes such as wound healing and immune responses. In addition, cancer cells exploit the cells' ability to migrate and invade to spread into nearby tissue and eventually metastasize. The majority of cancer deaths are caused by metastasis and the process of cell migration is therefore intensively studied. A common way to study cell migration is to observe cells through an optical microscope and record their movements over time. However, segmenting and tracking moving cells in phase contrast time-lapse video sequences is a challenging task. Several tools to track the velocity of migrating cells have been developed. Unfortunately, most of the automated tools are made for fluorescence images even though unlabelled cells are often preferred to avoid phototoxicity. Consequently, researchers are constrained with laborious manual tracking tools using ImageJ or similar software. We have therefore developed a freely available, user-friendly, automated tracking tool called CellTraxx. This software makes it easy to measure the velocity and directness of migrating cells in phase contrast images. Here, we demonstrate that our tool efficiently recognizes and tracks unlabelled cells of different morphologies and sizes (HeLa, RPE1, MDA-MB-231, HT1080, U2OS, PC-3) in several types of cell migration assays (random migration, wound healing and cells embedded in collagen). We also provide a detailed protocol and download instructions for CellTraxx.

Measuring Immune Cell Movement Toward the Soluble Microenvironment of Human Tissues Using a Boyden Chamber-Based Migration Assay.

Migration assays are used to measure cell movement toward a variety of chemoattractants in a controlled environment. Here we describe a method for a Boyden chamber-based migration assay using conditioned media generated from the tumor, liver, and visceral adipose tissue of cancer patients.

Regulatory mechanism of androgen receptor on NCAPD3 gene expression in prostate cancer.

BACKGROUND: Androgen receptor (AR) is an essential transcriptional factor that contributes to the development and progression of prostate cancer (PCa). NCAPD3 is a component of the condensin II complex and plays a critical role in cell mitosis by regulating chromosome condensation; however, the relationship between NCAPD3 and AR remains unknown. METHODS: Transcriptome sequencing assay is carried out to analyze the expression of the NCAP family in clinic samples. Chromatin immunoprecipitation (ChIP) sequencing, ChIP assay, and dual-luciferase assay are used to identify the androgen-responsive element in NCAPD3 enhancer. Immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, and western-blot assay are employed to check the expression of genes in PCa tissues and in PCa cells. Confocal immunofluorescence microscopy analysis is used for identifying the regulation of AR on NCAPD3-mediated chromosome condensation. Colony formation, cell cycle assay, wound healing assay, and transwell experiments are used to explore the regulation of AR on the functions of NCAPD3. In vivo experiment is employed to identify in vitro experimental results. RESULTS: NCAPD3 is an androgen/AR axis-targeted gene and is involved in AR-induced PCa cell proliferation, migration, and invasion in vitro and in vivo. Androgen treatment and AR overexpression increase the expression of NCAPD3 in PCa cell lines. The canonical exist in the enhancer region of NCAPD3. Androgen/AR axis regulates NCAPD3-invovled chromosome condensation during cell mitosis. CONCLUSIONS: Our report demonstrated that NCAPD3 is an androgen-responsive gene and upregulated by androgen/AR axis and involved in AR-promoted progression of PCa, suggesting a potential role of NCAPD3 in the PCa development.

GBP2 Is a Favorable Prognostic Marker of Skin Cutaneous Melanoma and Affects Its Progression via the Wnt/ß-catenin Pathway.

OBJECTIVE: Skin cutaneous melanoma (SKCM) is a common cutaneous malignant tumour. This study explored the expression and the downstream regulation mechanism of guanylate binding protein 2 (GBP2), an interferon (IFN)-induced protein, in SKCM. METHODS: Western blot was employed to verify the expression of SBP2 and its downstream Wnt/ß-catenin pathway-related proteins. We studied the relationship between GBP2 and the SKCM prognosis through database analysis. In vitro, gain-and-loss-of function experiments were conducted in SKCM cells. Cell viability was monitored by the cell counting kit-8 (CCK8) assay, the colony formation assay detected cell proliferation, and apoptosis was verified by flow cytometry. Transwell assay was conducted to test cell invasion and migration, while Western blot was employed to monitor the epithelial-mesenchymal transition (EMT) of SKCM cells. RESULTS: The GBP2 expression in SKCM cells and tissues was lower than normal cells and tissues. GBP2 overexpression inhibited SKCM cell proliferation, migration, invasion, and EMT and promoted cell apoptosis. In contrast, the GBP2 knockdown had the reverse effect. Mechanically, Wnt/ß-catenin was inactivated by GBP2 overexpression and was enhanced by GBP2 knockdown. Drug activation of Wnt/ß-catenin significantly attenuated the malignant phenotypic inhibition induced by GBP2 up-regulation in SKCM cells. CONCLUSION: GBP2 exerts anti-tumour effects by inhibiting the Wnt/ß-catenin pathway in SKCM and is related to a favourable prognosis.

Leukocyte kinetics during the early stage acts as a prognostic marker in patients with septic shock in intensive care unit.

ABSTRACT: The leukocytes play an important role in immune function during sepsis. We performed a retrospective study to investigate if leukocytes kinetics was associated with survival in critically ill patients with septic shock in intensive care unit (ICU).Patients with septic shock from January 1, 2014 to June 30, 2018 in our ICU were included. We extracted the demographic, clinical and laboratory data, comorbidities from our clinical database. The number of white blood cell, neutrophil and lymphocyte on day 1 and day 3 after diagnosis were collected and neutrophil to lymphocyte ratios (NLR) were calculated. Our primary outcome was 28-day mortality. Univariate and multivariate logistic regression models and cox proportional risk model were used to analyze the association between the leukocytes kinetics during first 3 days after ICU admission and the day-28 mortality.A total of 1245 septic shock patients with a 28-day mortality of 35.02% were included into analysis. There were no significant difference of lymphocyte number (0.83 ±â€Š0.02 vs 0.80 ±â€Š0.04, P = .552) between survival and non-survivals on day 1. However, the lymphocyte counts was significantly lower (0.95 ±â€Š0.03 vs 0.85 ±â€Š0.04, P = .024) on the third day. Both multivariate logistic and Cox regression analysis showed that lymphocyte counts on day 3 were associated with day-28 mortality. Moreover, Kaplan-Meier survival analysis revealed that increasing in lymphocyte counts and decreasing WBC, neutrophils and NLR during the first 3 days after diagnosis were associated with longer survival.Leukocytes kinetics during the first 3 days is a valuable prognostic marker in patients with septic shock in the ICU.

The role of lncRNA ANRIL in the progression of hepatocellular carcinoma.

OBJECTIVES: The aim of the current study was to identify the long noncoding RNAs (lncRNAs) ANRIL function and molecular pathways underlying hepatocellular carcinoma progression. METHODS: ANRIL knockdown with specific siRNA, and transfected into HepG2 cells according to the protocol of Lipofectamine 2000. Cell proliferation, apoptosis, migration and metastasis were assessed with MTT assay, flow cytometry and wound healing assay, respectively. Moreover, the expression level of ANRIL, apoptosis-related genes, and the Wnt pathway-associated genes were assessed by real time-PCR and Western blot assay. KEY FINDINGS: Knocking down of ANRIL led to alleviated cell growth and increased cell apoptosis of HepG2 cells through markedly increased expression levels of Bax and Bad. In contrast, dramatically diminished the expressions of anti-apoptotic factors including Bid and Bcl-2 in comparison to the scrambled control group (si-NC). Furthermore, ANRIL silencing resulted in an inactivated Wnt/ß-catenin pathway by suppressing key genes associated with this pathway. CONCLUSIONS: Taken together, these findings imply new insights into the regulatory network of the Wnt pathway through lncRNA ANRIL that indicate ANRIL may be a therapeutic factor potential for hepatocellular carcinoma.

Silencing Ribosomal Protein L22 Promotes Proliferation and Migration, and Inhibits Apoptosis of Gastric Cancer Cells by Regulating the Murine Double Minute 2-Protein 53 (MDM2-p53) Signaling Pathway.

BACKGROUND The aim of this study was to investigate the effect of ribosomal protein L22 (RPL22) on gastric cancer (GC) cell proliferation, migration, and apoptosis, and its correlation with the murine double minute 2-protein 53 (MDM2-p53) signaling pathway. MATERIAL AND METHODS The RPL22 expression in GC tissues and cells was detected by quantitative reverse transcription-polymerase chain reaction and western blotting. RPL22 was overexpressed in the MKN-45 cells by the transfection of a vector, pcDNA3.1 (pcDNA)-RPL22, whereas it was silenced in the MGC-803 cells by the transfection of short interfering (si) RNA (si-RPL22). Flow cytometric analysis, cell viability assays, wound healing assays, and transwell assays were utilized to explore the influences of RPL22 on the apoptosis, proliferation, migration, and invasion. Nutlin-3 (an MDM2-p53 inhibitor) was used to inhibit MDM2-p53 signaling. RESULTS The RPL22 expression was downregulated in GC tissues and cells. It was significantly lower in the advanced GC tissues than in the early GC tissues, and was significantly lower in the lymphatic metastatic tissues than in the non-lymphatic metastatic tissues. The transfection of si-RPL22 accelerated the ability of GC cells to proliferate and metastasize, whereas apoptosis was dampened. The transfection of pcDNA-RPL22 exerted the opposite effect on the GC cells; MDM2 expression was upregulated in RPL22-silenced GC cells, while the expression of p53 was downregulated. In vitro, treatment with nutlin-3 reversed the promoting effects of si-RPL22 on GC progression. CONCLUSIONS In vitro, the silencing of RPL22 aggravates GC by regulating the MDM2-p53 signaling pathway.

Motility disorders and disintegration into separate cells of Trichoplax sp. H2 in the presence of Zn2+ ions and L-cysteine molecules: A systems approach.

Placozoa remain an ancient multicellular system with a dynamic body structure where calcium ions carry out a primary role in maintaining the integrity of the entire animal. Zinc ions can compete with calcium ions adsorption. We studied the effect of zinc ions and l-cysteine molecules on the interaction of Trichoplax sp. H2 cells. The regularity of formless motion was diminished in the presence of 20-25 µM of Zn2+ ions leading to the formation of branching animal forms. Locomotor ciliated cells moved chaotically and independently of each other leaving the Trichoplax body and opening a network of fiber cells. Application of 100 µM cysteine resulted in dissociation of the plate into separate cells. The combined chemical treatment shifted the effect in a random sample of animals toward disintegration, i.e. initially leading to disorder of collective cell movement and then to total body fragmentation. Two dissociation patterns of Trichoplax plate as "expanding ring" and "bicycle wheel" were revealed. Analysis of the interaction of Ca2+ and Zn2+ ions with cadherin showed that more than half (54%) of the amino acid residues with which Ca2+ and Zn2+ ions bind are common. The contact interaction of cells covered by the cadherin molecules is important for the coordinated movements of Trichoplax organism, while zinc ions are capable to break junctions between the cells. The involvement of other players, for example, l-cysteine in the regulation of Ca2+-dependent adhesion may be critical leading to the typical dissociation of Trichoplax body like in a calcium-free environment. A hypothesis about the essential role of calcium ions in the emergence of Metazoa ancestor is proposed.

Analyzing the Roles of Rho GTPases in Cancer Cell Adhesion to Endothelial Cells Under Flow Conditions.

Adhesion between cancer cells and endothelial cells, lining the blood vessels, is an important event in tumor progression and metastasis formation. The expression of Rho GTPases is frequently altered in cancers, and they are known to regulate cell migration through their effects on adhesion and cytoskeletal dynamics. Several different types of assays are used to investigate how cancer cells attach to and cross the endothelium. Here, we describe an in vitro technique to study the effects of Rho GTPases on human cancer cell adhesion to endothelial cells under shear stress coupled to live cell imaging.

Probing Intravascular Adhesion and Extravasation of Tumor Cells with Microfluidics.

Cancer metastasis is a multistep process during which tumor cells leave the primary tumor mass and form distant secondary colonies that are lethal. Circulating tumor cells (CTCs) are transported by body fluids to reach distant organs, where they will extravasate and either remain dormant or form new tumor foci. Development of methods to study the behavior of CTCs at the late stages of the intravascular journey is thus required to dissect the molecular mechanisms at play. Using recently developed microfluidics approaches, we have demonstrated that CTCs arrest intravascularly, through a two-step process: (a) CTCs stop using low energy and rapidly activated adhesion receptors to form transient metastable adhesions and (b) CTCs stabilize their adhesions to the endothelial layer with high energy and slowly activated adhesion receptors. In this methods chapter, we describe these easy-to-implement quantitative methods using commercially available microfluidic channels. We detail the use of fast live imaging combined to fine-tuned perfusion to measure the adhesion potential of CTC depending on flow velocities. We document how rapidly engaged early metastable adhesion can be discriminated from slower activated stable adhesion using microfluidics. Finally, CTC extravasation potential can be assessed within this setup using long-term cell culture under flow. Altogether, this experimental pipeline can be adapted to probe the adhesion (to the endothelial layer) and extravasation potential of any circulating cell.

Real-Time Cell Migration Monitoring to Analyze Drug Synergism in the Scratch Assay Using the IncuCyte System.

Drug-mediated interference with metastasis represents a key approach to improve cancer therapy. In this regard, appropriate in vitro assays are needed to identify drugs, which inhibit cell migration as one feature for metastatic potential of cancer cells. One such migration assay is the wound healing or scratch assay, designed to allow cells for closure of an artificially generated gap (wound/scratch) in the monolayer. To identify possibly effective anti-migratory drugs as monotherapy or as synergistic drug combination, novel screening tools besides viability measurements at the experimental endpoint are needed. In this context, particularly drug combinations allow to increase treatment efficacy paralleled by lowered side effects. Here, a protocol for real-time monitoring cellular motility and its inhibition by anti-migratory drugs and combinations by the IncuCyte system and a 96-well scratch assay is described. A pipetting scheme allowing data collection for synergy calculation using one plate per replicate is provided. Using the IncuCyte System 2, drug combinations built of three biological replicates each using three technical replicates can be tested in parallel within hours to few days to accelerate identification of efficient antimetastatic drugs.

Measurement of Metabolites from Migrating Cells.

Metastasis is a multistep process that involves responses to extrinsic and intrinsic signals at every step. It is thus only truly appreciated in the context of a whole organism. Nevertheless, in vitro studies can be used to facilitate understanding of the possible factors contributing to any phenotype that is associated with metastatic competence. The use of migration assays-where monolayers of cells migrate to cover gaps or "wounds"-has been described for decades to identify signaling pathways that regulate motile competence and to screen for ways of interfering with this ability. Here we depict the combination of such an assay with assessment of indicators of carbon metabolism using commercially available assays. This enables identification of changes in cellular metabolism associated with actively migrating cells.

Matrix Degradation Assay to Measure the Ability of Tumor Cells to Degrade Extracellular Matrix.

During the metastatic process, carcinoma cells form invadopodia, F-actin enriched protrusive structures, to degrade the extracellular matrix (ECM) in order to invade the surrounding stroma and intravasate into the circulatory system. In this chapter, we describe the 2D-fluorescent matrix degradation assay, a highly sensitive and reproducible in vitro method used to measure invadopodia-mediated ECM degradation. We provide a detailed protocol on how to prepare the glass coverslips with fluorescent gelatin matrix and a standardized method to quantify gelatin degradation and invadopodia formation in order to evaluate cell invasion.

Evaluation of Real-Time In Vitro Invasive Phenotypes.

The methods described here provide a standardized process for assessing in vitro tumor cell migration and invasion in real time. The kinetic data generated under these standardized conditions are reproducible and characteristic of individual tumor cell lines. The complex kinetic features of the data can be analyzed using parameters modeled after pharmacokinetic data processing. Application of the method to the array of tumor types included in the National Cancer Institute's sixty cell line panel (NCI60) revealed distinct modes of invasion with some tumor cell lines utilizing a mesenchymal mode and generating information-rich kinetic profiles. Other cell lines utilized an amoeboid mode not suitable for detection with this method. The method described will be useful as a guide for tumor cell line selection and as a starting point in designing experiments probing migration and invasion.

Observation and Quantification of Eosinophil Motility.

Eosinophils are important for tissue homeostasis and host responses to pathogens and allergens. The impact of eosinophils within tissues depends in part on whether cytotoxic proteins in crystalloid granules are released. Determinants of eosinophil motility and loss of granule contents are incompletely understood. The goal of this chapter is to present methods to study the effects of potential mediators on purified human blood eosinophils interacting with adhesive proteins found in extracellular matrix. We show that differential interference contrast video-enhanced microscopy and a bead-clearing assay provide complementary information about how different mediator-adhesive protein combinations direct eosinophil motility and granule fate. The former method is rich in information about cell shape, pattern of movement, and state of granules whereas the latter method lends itself to quantification and interrogation of multiple conditions in replicate.

Cell adhesion molecule L1 like plays a role in the pathogenesis of idiopathic hypogonadotropic hypogonadism.

PURPOSE: The pathogenesis of idiopathic hypogonadotropic hypogonadism (IHH) is genetically complex. The aims of this study were to investigate the genetic profile and clinical manifestation of IHH in a Chinese pedigree and to discover new IHH-associated genes. METHODS: The first step was to follow up the clinical phenotype and therapeutic outcomes of the pedigree in university hospital. The second step was that mutation screening was performed in this pedigree and 100 healthy controls. The third step was to further verify the pathogenicity of the discovered rare sequencing variant (RSV) by functional experiments. Whole exome sequencing, Sanger sequencing, testicular volume (TV), semen analysis, assessment of cell migration and necroptosis were performed. RESULTS: One heterozygous RSV (p.G517E) in CHL1 was identified in two male IHH patients and their mother in the pedigree, but not in healthy controls. All the three individuals exhibited olfactory impairment. hCG/hMG treatment significantly improved TV, serum testosterone and/or semen parameters of the two male patients. Functional analysis indicated that CHL1 significantly regulated GnRH neuronal cell line (GN11 cells) migration and necroptosis, with alteration of ERK1/2 activation, calcium loading, and transcription of RIPK3 and MLKL. However, the above processes were negatively influenced by the CHL1 RSV. CONCLUSIONS: Our study reports the genetic relevance of CHL1 in IHH, and characterizes the phenotypic and therapeutic profiles in patients carrying the CHL1 RSV. CHL1 may act as a new IHH-associated gene, and should be taken into consideration in future investigations for this field.

In Vitro Models for Studying Tumor Progression.

Beyond cell proliferation, one of the most outstanding characteristics of the cancerous cells that promotes the tumoral progression is their high capacity to migrate and invade the surrounding healthy tissue. These cellular processes (migration and invasion) are critical steps to metastasis. Metastatic progression of the tumors is often the leading cause of morbidity and mortality in cancer patients. Critical genes and cell signaling pathways involved in cell migration and invasion of tumor cells have been identified, and several clinical efforts to alleviate cancer are focused on them; however, once the tumor has metastasized, it is extremely difficult to stop the progression of very aggressive forms of cancer such as glioblastomas. Therefore, it is crucial to elucidate the specific molecular mechanisms underlying tumor progression. In this chapter, we describe some methods to study tumor progression by assessing migration and cell invasion in 2D and 3D cell culture conditions.

Low-density lipoprotein from active SLE patients is more atherogenic to endothelial cells than low-density lipoprotein from the same patients during remission.

OBJECTIVES: SLE patients have an enhanced risk of atherosclerosis and cardiovascular disease. However, the increased prevalence of cardiovascular disease is not fully explained by traditional Framingham cardiovascular risk factors. Specific features of low-density lipoprotein (LDL) particles, other than plasma concentration, may induce accelerated atherosclerosis at early stages in these patients. Thus, we aimed to explore the impact of LDL from both active and inactive SLE patients on human aortic endothelial cells. METHODS: Human aortic endothelial cells were stimulated with the same concentration of LDL particles isolated from pooled serum that was collected from 13 SLE patients during both active and inactive states. Gene expression and cell migration assays were performed. RESULTS: Circulating LDL particles obtained from healthy volunteers and SLE patients in both remission and flare states were comparable in terms of number, cholesterol and triglyceride content, and net electric charge. Stimulation of cells with LDL from active SLE patients induced the expression of vascular cell adhesion molecule 1 (∼2.0-fold, P < 0.05), monocyte chemoattractant protein 1 (∼2.0-fold, P < 0.05) and matrix metallopeptidase 2 (∼1.6-fold, P < 0.01) compared with cells stimulated with LDL from inactive SLE patients. Additionally, LDL extracted from active patients increased cell migration in a wound-healing assay (1.4-fold, P < 0.05). CONCLUSION: Our data show that, at the same LDL concentration, LDL from active SLE patients had increased proatherogenic effects on endothelial cells compared with LDL from the same patients when in an inactive or remission state.

Ribonuclease 7-driven activation of ROS1 is a potential therapeutic target in hepatocellular carcinoma.

BACKGROUND & AIMS: There are currently limited therapeutic options for hepatocellular carcinoma (HCC), particularly when it is diagnosed at advanced stages. Herein, we examined the pathophysiological role of ROS1 and assessed the utility of ROS1-targeted therapy for the treatment of HCC. METHODS: Recombinant ribonucleases (RNases) were purified, and the ligand-receptor relationship between RNase7 and ROS1 was validated in HCC cell lines by Duolink, immunofluorescence, and immunoprecipitation assays. Potential interacting residues between ROS1 and RNase7 were predicted using a protein-protein docking approach. The oncogenic function of RNase7 was analyzed by cell proliferation, migration and invasion assays, and a xenograft mouse model. The efficacy of anti-ROS1 inhibitor treatment was evaluated in patient-derived xenograft (PDX) and orthotopic models. Two independent patient cohorts were analyzed to evaluate the pathological relevance of RNase7/ROS1. RESULTS: RNase7 associated with ROS1's N3-P2 domain and promoted ROS1-mediated oncogenic transformation. Patients with HCC exhibited elevated plasma RNase7 levels compared with healthy individuals. High ROS1 and RNase7 expression were strongly associated with poor prognosis in patients with HCC. In both HCC PDX and orthotopic mouse models, ROS1 inhibitor treatment markedly suppressed RNase7-induced tumorigenesis, leading to decreased plasma RNase7 levels and tumor shrinkage in mice. CONCLUSIONS: RNase7 serves as a high-affinity ligand for ROS1. Plasma RNase7 could be used as a biomarker to identify patients with HCC who may benefit from anti-ROS1 treatment. LAY SUMMARY: Receptor tyrosine kinases are known to be involved in tumorigenesis and have been targeted therapeutically for a number of cancers, including hepatocellular carcinoma. ROS1 is the only such receptor with kinase activity whose ligand has not been identified. Herein, we show that RNase7 acts as a ligand to activate ROS1 signaling. This has important pathophysiological and therapeutic implications. Anti-ROS1 inhibitors could be used to treatment patients with hepatocellular carcinoma and high RNase7 levels.

Cellular Migration Assay: An In Vitro Technique to Simulate the Wound Repair Mechanism.

Wound regeneration is a complex process, which necessitates proper coordination among the inflammatory response, vascularization, matrix formation, and reformation of epithelial tissue. It is a unique process, where healing and regeneration take place simultaneously. Matrix formation is the first critical stage that starts the communication between the keratinocytes, fibroblasts, and integrins. This, in turn, stimulates the differentiation of monocytes into macrophages, to produce cytokines for fibroblasts. This phenomenon is the crucial part for the keratinocyte migration and epithelialization to fill the wound. To understand the complex procedure of wound regeneration, there is a need for easy, convenient, and low-cost approaches that will simulate the wound-repairing process. Scratch assay or cellular migration assay is one of the most convenient and affordable approaches, commonly used by the scientific community. In this chapter, we present the fundamental principles of the experimental procedures required for the Scratch assay.