Caveolin-1-mediated STAT3 activation determines electrotaxis of human lung cancer cells.
ABSTRACT: 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:The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement-specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1-3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca2+ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca2+ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways.
Project description:Small direct current (DC) electric fields (EFs) guide neurite growth and migration of rodent neural stem cells (NSCs). However, this could be species dependent. Therefore, it is critical to investigate how human NSCs (hNSCs) respond to EF before any possible clinical attempt. Aiming to characterize the EF-stimulated and guided migration of hNSCs, we derived hNSCs from a well-established human embryonic stem cell line H9. Small applied DC EFs, as low as 16 mV/mm, induced significant directional migration toward the cathode. Reversal of the field polarity reversed migration of hNSCs. The galvanotactic/electrotactic response was both time and voltage dependent. The migration directedness and distance to the cathode increased with the increase of field strength. (Rho-kinase) inhibitor Y27632 is used to enhance viability of stem cells and has previously been reported to inhibit EF-guided directional migration in induced pluripotent stem cells and neurons. However, its presence did not significantly affect the directionality of hNSC migration in an EF. Cytokine receptor [C-X-C chemokine receptor type 4 (CXCR4)] is important for chemotaxis of NSCs in the brain. The blockage of CXCR4 did not affect the electrotaxis of hNSCs. We conclude that hNSCs respond to a small EF by directional migration. Applied EFs could potentially be further exploited to guide hNSCs to injured sites in the central nervous system to improve the outcome of various diseases.
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. 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:BACKGROUND:Electrotaxis is the movement of adherent living cells in response to a direct current (dc) electric field (EF) of physiological strength. Highly metastatic human lung cancer cells, CL1-5, exhibit directional migration and orientation under dcEFs. To understand the transcriptional response of CL1-5 cells to a dcEF, microarray analysis was performed in this study. METHODOLOGY/PRINCIPAL FINDINGS:A large electric-field chip (LEFC) was designed, fabricated, and used in this study. CL1-5 cells were treated with the EF strength of 0 mV/mm (the control group) and 300 mV/mm (the EF-treated group) for two hours. Signaling pathways involving the genes that expressed differently between the two groups were revealed. It was shown that the EF-regulated genes highly correlated to adherens junction, telomerase RNA component gene regulation, and tight junction. Some up-regulated genes such as ACVR1B and CTTN, and some down-regulated genes such as PTEN, are known to be positively and negatively correlated to cell migration, respectively. The protein-protein interactions of adherens junction-associated EF-regulated genes suggested that platelet-derived growth factor (PDGF) receptors and ephrin receptors may participate in sensing extracellular electrical stimuli. We further observed a high percentage of significantly regulated genes which encode cell membrane proteins, suggesting that dcEF may directly influence the activity of cell membrane proteins in signal transduction. CONCLUSIONS/SIGNIFICANCE:In this study, some of the EF-regulated genes have been reported to be essential whereas others are novel for electrotaxis. Our result confirms that the regulation of gene expression is involved in the mechanism of electrotactic response.
Project description:Proper control of cell migration is critically important in many biologic processes, such as wound healing, immune surveillance, and development. Much progress has been made in the initiation of cell migration; however, little is known about termination and sometimes directional reversal. During active cell migration, as in wound healing, development, and immune surveillance, the integrin expression profile undergoes drastic changes. Here, we uncovered the extensive regulatory and even opposing roles of integrins in directional cell migration in electric fields (EFs), a potentially important endogenous guidance mechanism. We established cell lines that stably express specific integrins and determined their responses to applied EFs with a high throughput screen. Expression of specific integrins drove cells to migrate to the cathode or to the anode or to lose migration direction. Cells expressing ?M?2, ?1, ?2, ?IIb?3, and ?5 migrated to the cathode, whereas cells expressing ?3, ?6, and ?9 migrated to the anode. Cells expressing ?4, ?V, and ?6?4 lost directional electrotaxis. Manipulation of ?9 molecules, one of the molecular directional switches, suggested that the intracellular domain is critical for the directional reversal. These data revealed an unreported role for integrins in controlling stop, go, and reversal activity of directional migration of mammalian cells in EFs, which might ensure that cells reach their final destination with well-controlled speed and direction.-Zhu, K., Takada, Y., Nakajima, K., Sun, Y., Jiang, J., Zhang, Y., Zeng, Q., Takada, Y., Zhao, M. Expression of integrins to control migration direction of electrotaxis.
Project description:In this paper, we report a new method to incorporate 3D scaffold with electrotaxis measurement in the microfluidic device. The electrotactic response of lung cancer cells in the 3D foam scaffolds which resemble the in vivo pulmonary alveoli may give more insight on cellular behaviors in vivo. The 3D scaffold consists of ordered arrays of uniform spherical pores in gelatin. We found that cell morphology in the 3D scaffold was different from that in 2D substrate. Next, we applied a direct current electric field (EF) of 338 mV/mm through the scaffold for the study of cells' migration within. We measured the migration directedness and speed of different lung cancer cell lines, CL1-0, CL1-5, and A549, and compared with those examined in 2D gelatin-coated and bare substrates. The migration direction is the same for all conditions but there are clear differences in cell morphology, directedness, and migration speed under EF. Our results demonstrate cell migration under EF is different in 2D and 3D environments and possibly due to different cell morphology and/or substrate stiffness.
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: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: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:Correct guidance of the migration of neural progenitor cells (NPCs) is essential for the development and repair of the central nervous system (CNS). Electric field (EF)-guided migration, electrotaxis, has been observed in many cell types. We report here that, in applied EFs of physiological magnitude, embryonic and adult NPCs show marked electrotaxis, which is dependent on the PI3K/Akt pathway. The electrotaxis was also evidenced by ex vivo investigation that transplanted NPCs migrated directionally towards cathode in organotypic spinal cord slice model when treated with EFs. Genetic disruption or pharmacological inhibition of phosphoinositide 3-kinase (PI3K) impaired electrotaxis, whereas EF exposure increased Akt phosphorylation in a growth factor-dependent manner and increased phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels. EF treatments also induced asymmetric redistribution of PIP3, growth factor receptors, and actin cytoskeleton. Electrotaxis in both embryonic and adult NPCs requires epidermal growth factor (EGF) and fibroblast growth factor (FGF). Our results demonstrate the importance of the PI3K/Akt pathway in directed migration of NPCs driven by EFs and growth factors and highlight the potential of EFs to enhance the guidance of various NPC populations in CNS repair therapies.