Electrotaxis of Glioblastoma and Medulloblastoma Spheroidal Aggregates.
ABSTRACT: Treatment of neuroepithelial cancers remains a daunting clinical challenge, particularly due to an inability to address rampant invasion deep into eloquent regions of the brain. Given the lack of access, and the dispersed nature of brain tumor cells, we explore the possibility of electric fields inducing directed tumor cell migration. In this study we investigate the properties of populations of brain cancer undergoing electrotaxis, a phenomenon whereby cells are directed to migrate under control of an electrical field. We investigate two cell lines for glioblastoma and medulloblastoma (U87mg & DAOY, respectively), plated as spheroidal aggregates in Matrigel-filled electrotaxis channels, and report opposing electrotactic responses. To further understand electrotactic migration of tumor cells, we performed RNA-sequencing for pathway discovery to identify signaling that is differentially affected by the exposure of direct-current electrical fields. Further, using selective pharmacological inhibition assays, focused on the PI3K/mTOR/AKT signaling axis, we validate whether there is a causal relationship to electrotaxis and these mechanisms of action. We find that U87?mg electrotaxis is abolished under pharmacological inhibition of PI3K?, mTOR, AKT and ErbB2 signaling, whereas DAOY cell electrotaxis was not attenuated by these or other pathways evaluated.
Project description:Treatment of neuroepithelial cancers remains a daunting clinical challenge, particularly due to an inability to address rampant invasion deep into eloquent regions of the brain. Given the lack of access, and the dispersed nature of brain tumor cells, we explore the possibility of electric fields inducing directed tumor cell migration. In this study we investigate the properties of populations of brain cancer undergoing electrotaxis, a phenomenon whereby cells are directed to migrate under control of an electrical field. We investigate two cell lines for glioblastoma and medulloblastoma (U87mg & DAOY, respectively), plated as spheroidal aggregates in matrigel-filled electrotaxis channels, and report opposing electrotactic responses. To further understand electrotactic migration of tumor cells, we performed RNA-sequencing for pathway discovery to identify signaling that is differentially affected by the exposure of direct-current electrical fields.
Project description:Migration of cancer cells leads to the invasion of distant organs by primary tumors. Further, endogenous electric fields (EFs) in the tumor microenvironment direct the migration of lung cancer cells by a process referred to as electrotaxis - although the precise mechanism remains unclear. Caveolin-1 (Cav-1) is a multifunctional scaffolding protein that is associated with directional cell migration and lung cancer invasion; however, its precise role in lung cancer electrotaxis is unknown. In the present study, we first detected outward electric currents on the tumor body surface in lung cancer xenografts using a highly-sensitive vibrating probe. Next, we found that highly-metastatic H1650-M3 cells migrated directionally to the cathode. In addition, reversal of the EF polarity reversed the direction of migration. Mechanistically, EFs activated Cav-1 and the downstream signaling molecule STAT3. RNA interference of Cav-1 reduced directional cell migration, which was accompanied by dampened STAT3 activation. Furthermore, pharmacological inhibition of STAT3 significantly reduced the electrotactic response, while rescue of STAT3 activation in Cav-1 knock-down cells restored electrotaxis. Taken together, these results suggest that endogenous EFs in the tumor micro-environment might play an important role in lung cancer metastasis by guiding cell migration through a Cav-1/STAT3-mediated signaling pathway.
Project description:Collective cell migration plays an important role in wound healing, organogenesis, and the progression of metastatic disease. Analysis of collective migration typically involves laborious and time-consuming manual tracking of individual cells within cell clusters over several dozen or hundreds of frames. Herein, we develop a label-free, automated algorithm to identify and track individual epithelial cells within a free-moving cluster. We use this algorithm to analyze the effects of partial E-cadherin knockdown on collective migration of MCF-10A breast epithelial cells directed by an electric field. Our data show that E-cadherin knockdown in free-moving cell clusters diminishes electrotactic potential, with empty vector MCF-10A cells showing 16% higher directedness than cells with E-cadherin knockdown. Decreased electrotaxis is also observed in isolated cells at intermediate electric fields, suggesting an adhesion-independent role of E-cadherin in regulating electrotaxis. In additional support of an adhesion-independent role of E-cadherin, isolated cells with reduced E-cadherin expression reoriented within an applied electric field 60% more quickly than control. These results have implications for the role of E-cadherin expression in electrotaxis and demonstrate proof-of-concept of an automated algorithm that is broadly applicable to the analysis of collective migration in a wide range of physiological and pathophysiological contexts.
Project description:Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including penetrating injury to epithelial barriers. An applied electric field with strength within the physiologic range can induce directional cell migration (i.e., electrotaxis) of epithelial cells, endothelial cells, fibroblasts, and neutrophils suggesting a potential role in cell positioning during wound healing. In the present study, we investigated the ability of lymphocytes to respond to applied direct current (DC) electric fields. Using a modified Transwell assay and a simple microfluidic device, we show that human PBLs migrate toward the cathode in physiologically relevant DC electric fields. Additionally, electrical stimulation activates intracellular kinase signaling pathways shared with chemotactic stimuli. Finally, video microscopic tracing of GFP-tagged immunocytes in the skin of mouse ears reveals that motile cutaneous T cells actively migrate toward the cathode of an applied DC electric field. Lymphocyte positioning within tissues can thus be manipulated by externally applied electric fields, and may be influenced by endogenous electrical potential gradients as well.
Project description:BACKGROUND:The limited regenerative capacity of cardiac tissue has long been an obstacle to treating damaged myocardium. Cell-based therapy offers an enormous potential to the current treatment paradigms. However, the efficacy of regenerative therapies remains limited by inefficient delivery and engraftment. Electrotaxis (electrically guided cell movement) has been clinically used to improve recovery in a number of tissues but has not been investigated for treating myocardial damage. OBJECTIVE:The purpose of this study was to test the electrotactic behaviors of several types of cardiac cells. METHODS:Cardiac progenitor cells (CPCs), cardiac fibroblasts (CFs), and human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-CPCs) were used. RESULTS:CPCs and CFs electrotax toward the anode of a direct current electric field, whereas hiPSC-CPCs electrotax toward the cathode. The voltage-dependent electrotaxis of CPCs and CFs requires the presence of serum in the media. Addition of soluble vascular cell adhesion molecule to serum-free media restores directed migration. We provide evidence that CPC and CF electrotaxis is mediated through phosphatidylinositide 3-kinase signaling. In addition, very late antigen-4, an integrin and growth factor receptor, is required for electrotaxis and localizes to the anodal edge of CPCs in response to direct current electric field. The hiPSC-derived CPCs do not express very late antigen-4, migrate toward the cathode in a voltage-dependent manner, and, similar to CPCs and CFs, require media serum and phosphatidylinositide 3-kinase activity for electrotaxis. CONCLUSION:The electrotactic behaviors of these therapeutic cardiac cells may be used to improve cell-based therapy for recovering function in damaged myocardium.
Project description:The nematode C. elegans is a leading model to investigate the mechanisms of stress-induced behavioral changes coupled with biochemical mechanisms. Our group has previously characterized C. elegans behavior using a microfluidic-based electrotaxis device, and showed that worms display directional motion in the presence of a mild electric field. In this study, we describe the effects of various forms of genetic and environmental stress on the electrotactic movement of animals. Using exposure to chemicals, such as paraquat and tunicamycin, as well as mitochondrial and endoplasmic reticulum (ER) unfolded protein response (UPR) mutants, we demonstrate that chronic stress causes abnormal movement. Additionally, we report that pqe-1 (human RNA exonuclease 1 homolog) is necessary for the maintenance of multiple stress response signaling and electrotaxis behavior of animals. Further, exposure of C. elegans to several environmental stress-inducing conditions revealed that while chronic heat and dietary restriction caused electrotaxis speed deficits due to prolonged stress, daily exercise had a beneficial effect on the animals, likely due to improved muscle health and transient activation of UPR. Overall, these data demonstrate that the electrotaxis behavior of worms is susceptible to cytosolic, mitochondrial, and ER stress, and that multiple stress response pathways contribute to its preservation in the face of stressful stimuli.
Project description:Kindler syndrome (KS) is an autosomal recessive blistering skin disease resulting from pathogenic mutations in FERMT1. This gene encodes kindlin-1, a focal adhesion protein involved in activation of the integrin family of extracellular matrix receptors. Most cases of KS show a marked reduction or complete absence of the kindlin-1 protein in keratinocytes, resulting in defective cell adhesion and migration. Electric fields also act as intrinsic regulators of adhesion and migration in the skin, but the molecular mechanisms by which this occurs are poorly understood. Here we show that keratinocytes derived from KS patients are unable to undergo electrotaxis, and this defect is restored by overexpression of wild-type kindlin-1 but not a W612A mutation that prevents kindlin-integrin binding. Moreover, deletion of the pleckstrin homology domain of kindlin-1 also failed to rescue electrotaxis in KS cells, indicating that both integrin and lipid binding are required for this function. Kindlin-1 was also required for the maintenance of lamellipodial protrusions during electrotaxis via electric field-activated ?1 integrin. Indeed, inhibition of ?1 integrins also leads to loss of electrotaxis in keratinocytes. Our data suggest that loss of kindlin-1 function may therefore result in epithelial insensitivity to electric fields and contribute to KS disease pathology.
Project description:Electric fields are generated <i>in vivo</i> in a variety of physiologic and pathologic settings, including wound healing and immune response to injuries to epithelial barriers (e.g. lung pneumocytes). Immune cells are known to migrate towards both chemical (chemotaxis), physical (mechanotaxis) and electric stimuli (electrotaxis). Electrotaxis is the guided migration of cells along electric fields, and has previously been reported in T-cells and cancer cells. However, there remains a need for engineering tools with high spatial and temporal resolution to quantify EF guided migration. Here we report the development of an electrotaxis-on-chip (ETOC) platform that enables the quantification of dHL-60 cell, a model neutrophil-like cell line, migration toward both electrical and chemoattractant gradients. Neutrophils are the most abundant white blood cells and set the stage for the magnitude of the immune response. Therefore, developing engineering tools to direct neutrophil migration patterns has applications in both infectious disease and inflammatory disorders. The ETOC developed in this study has embedded electrodes and four migration zones connected to a central cell-loading chamber with migration channels [10 µm X 10 µm]. This device enables both parallel and competing chemoattractant and electric fields. We use our novel ETOC platform to investigate dHL-60 cell migration in three biologically relevant conditions: 1) in a DC electric field; 2) parallel chemical gradient and electric fields; and 3) perpendicular chemical gradient and electric field. In this study we used differentiated leukemia cancer cells (dHL60 cells), an accepted model for human peripheral blood neutrophils. We first quantified effects of electric field intensities (0.4V/cm-1V/cm) on dHL-60 cell electrotaxis. Our results show optimal migration at 0.6 V/cm. In the second scenario, we tested whether it was possible to increase dHL-60 cell migration to a bacterial signal [N-formylated peptides (fMLP)] by adding a parallel electric field. Our results show that there was significant increase (6-fold increase) in dHL60 migration toward fMLP and cathode of DC electric field (0.6V/cm, n=4, p-value<0.005) <i>vs</i>. fMLP alone. Finally, we evaluated whether we could decrease or re-direct dHL-60 cell migration away from an inflammatory signal [leukotriene B<sub>4</sub> (LTB<sub>4</sub>)]. The perpendicular electric field significantly decreased migration (2.9-fold decrease) of dHL60s toward LTB<sub>4</sub> <i>vs</i>. LTB<sub>4</sub> alone. Our microfluidic device enabled us to quantify single-cell electrotaxis velocity (7.9 µm/min ± 3.6). The magnitude and direction of the electric field can be more precisely and quickly changed than most other guidance cues such as chemical cues in clinical investigation. A better understanding of EF guided cell migration will enable the development of new EF-based treatments to precisely direct immune cell migration for wound care, infection, and other inflammatory disorders.
Project description:Cell migration is involved in physiological processes such as wound healing, host defense, and cancer metastasis. The movement of various cell types can be directed by chemical gradients (i.e., chemotaxis). In addition to chemotaxis, many cell types can respond to direct current electric fields (dcEF) by migrating to either the cathode or the anode of the field (i.e., electrotaxis). In tissues, physiological chemical gradients and dcEF can potentially co-exist and the two guiding mechanisms may direct cell migration in a coordinated manner. Recently, microfluidic devices that can precisely configure chemical gradients or dcEF have been increasingly developed and used for chemotaxis and electrotaxis studies. However, a microfluidic device that can configure controlled co-existing chemical gradients and dcEF that would allow quantitative cell migration analysis in complex electrochemical guiding environments is not available. In this study, we developed a polydimethylsiloxane-based microfluidic device that can generate better controlled single or co-existing chemical gradients and dcEF. Using this device, we showed chemotactic migration of T cells toward a chemokine CCL19 gradient or electrotactic migration toward the cathode of an applied dcEF. Furthermore, T cells migrated more strongly toward the cathode of a dcEF in the presence of a competing CCL19 gradient, suggesting the higher electrotactic attraction. Taken together, the developed microfluidic device offers a new experimental tool for studying chemical and electrical guidance for cell migration, and our current results with T cells provide interesting new insights of immune cell migration in complex guiding environments.
Project description:The nematode C. elegans displays complex dynamical behaviors that are commonly used to identify relevant phenotypes. Although its maintenance is straightforward, sorting large populations of worms when looking for a behavioral phenotype is difficult, time consuming and hardly quantitative when done manually. Interestingly, when submitted to a moderate electric field, worms move steadily along straight trajectories. Here, we report an inexpensive method to measure worms crawling velocities and sort them within a few minutes by taking advantage of their electrotactic skills. This method allows to quantitatively measure the effect of mutations and aging on worm's crawling velocity. We also show that worms with different locomotory phenotypes can be spatially sorted, fast worms traveling away from slow ones. Group of nematodes with comparable locomotory fitness could then be isolated for further analysis. C. elegans is a growing model for neurodegenerative diseases and using electrotaxis for self-sorting can improve the high-throughput search of therapeutic bio-molecules.