17, 97109. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). We assessed the individual cell trajectories in M3 melanoma cells after 20 h treatment with Mibefradil (5 M). No use, distribution or reproduction is permitted which does not comply with these terms. Nat. Our in vitro step-by-step protocols offer, to a wide range of scientists the capacity to determine and measure an extensive variety of cell motility parameters related to the migration process, such as wound area, velocity, healing speed, front cell velocity, traveled distance, invasion, and spreading rate. Rev. Depending on the aim of your research, it may be interesting to make use of some other modifications. Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. Mutat. Supplementary Figure 1. Supplementary Data 1. Spreading assay is schematically described (Figure 5A), where cells were treated for 24 h with Mibefradil (10 M), were trypsinized and seeded in fibronectin-coated plates. (2018). 31, 484495. Cell migration: integrating signals from front to back. Physiol. Supplementary Figure 3. Cell 147, 9921009. Science 302, 17041709. Rev. 7:107. doi: 10.3389/fcell.2019.00107. The in vitro individual cell-tracking assay is a method to analyze the migration distance and velocity of single cells independently from cell-cell interactions, either after a drug treatment, in mutant cells, or in siRNA libraries. Mechanoreciprocity in cell migration. Contrarily, when there is rich media (with 10% FBS) in the upper and lower chamber, there is not significant decrease in migration by Mibefradil treatment (p = 0.6358) because there is no chemoattractant gradient to favor migration directionality (Figure 4D). 32, 491526. doi: 10.1038/nrm3897, De Pascalis, C., and Etienne-Manneville, S. (2017). doi: 10.1038/nrc1075, Icha, J., Weber, M., Waters, J. C., and Norden, C. (2017). Immunol. Step-by-step proceeding used in order to count the number of unattached and spread cells in ImageJ/Fiji. (B) The same fluorescent image, as in (A), followed by a manual cell recount using the Cell counter application in ImageJ/Fiji (right panel). doi: 10.1016/j.cell.2011.11.016, Friedl, P., and Weigelin, B. doi: 10.1371/journal.pone.0134714, Stuelten, C. H., Parent, C. A., and Montell, D. J. (B) Brightfield images of the fixed cells are background subtracted, edges are found and the edged image is thresholded (using the Triangle method), and total cell number is counted after a few binary operations, including fill holes, and watershed. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Compr. Supplementary Figure 4. 58, 25282532. Scale bars, 100 m. The migration-related phenotypes determined by these methods may provide useful information about the metastatic potential of the type of cancer studied in vivo for the prognosis of the disease. ), but it will also increase image segmentation efficiency for its further analysis. To evaluate the effect of TTCC blockers treatment, we compared the untreated M3 cells with 10% FBS (67.22 10.37% cells migrated) with M3 cells treated with Mibefradil and 10% FBS (49.32 6.23% cells migrated). Mol. 25, 407429. doi: 10.1128/MCB.25.3.1025-1040.2005, Charras, G., and Sahai, E. (2014). Regarding the wound healing assay, in initial stages of your research you may perform a scratch assay with pipette tip just taking images at initial and end time (in order to screen for several drugs and/or doses). Biol. Received: 28 January 2019; Accepted: 29 May 2019; Published: 14 June 2019. Phototoxicity in live fluorescence microscopy, and how to avoid it. Nat. doi: 10.1146/annurev.cellbio.042308.113231, Salvatierra, E., Alvarez, M. J., Leishman, C. C., Rivas Baquero, E., Lutzky, V. P., Chuluyan, H. E., et al. Plasticity of cell migration in vivo and in silico. Cell motility in cancer invasion and metastasis: insights from simple model organisms. (2013). In melanoma cells, adhesion, cytoskeleton structure, and cell size can be compromised (Salvatierra et al., 2015). Step-by-step of different settings to determine the measurable area by applying Find edges, Gaussian blur (of radius 5 pixels), and Threshold adjust. (2015). Pigment Cell Melanoma Res. These automated methods are not as accurate as the manual ones, but the analysis is done much faster, which enables to quantify a higher amount of images and get more statistically robust data in a fraction of time. JP, AM, and AP wrote the first draft of the manuscript. Res. Nat. These results suggest that Chloroquine reduce the collective migration rate, velocity and healing speed compared to untreated cells (Maiques et al., 2018). (2015). (2003). Farina, K. L., Wyckoff, J. We analyzed M3 cell line spreading capacity either treated or not with TTCC blocker Mibefradil. 16, 185204. Interstitial leukocyte migration and immune function. Sci Rep. 5:10293. doi: 10.1038/srep10293, Xu, L., Gordon, R., Farmer, R., Pattanayak, A., Binkowski, A., Huang, X., et al. We are convinced that the inclusion of these analyses with the corresponding quantitative methods will be a powerful tool for the characterization of cancer metastatic prognosis and for preclinical screening of novel therapeutic drugs designed to impair metastatic progression. Cell. Rev. Quantification of in vitro migration processes in cancer cell lines using time-lapse microscopy can be a crucial tool to study new potential therapeutic anti-cancer drugs, and also to understand basic principles of novel molecular metastatic pathways. Quantitative analysis shows that only 17 5.04% of untreated cells migrated without FBS, whereas 10% FBS chemoattraction induced a significant increase in cell migration percentage (48.01 4.98%). doi: 10.1038/s41571-018-0134-8, Boya, P., Gonzalez-Polo, R.-A., Casares, N., Perfettini, J.-L., Dessen, P., Larochette, N., et al. Mol. Nevertheless, we have to be cautious when obtaining fluorescence images, since excitation and/or emitted light could generate photo-toxicity and photo-sensitization in live imaging experiments, thus altering cell behavior and even its morphology (Icha et al., 2017). The representative cell migration trajectories were plotted with a common origin in untreated (green) and Mibefradil treated cells (red) (Figure 3B). doi: 10.1126/science.1092053, Rrth, P. (2009). Collective cell migration. Roughly, round and refringent cells were annotated as unspread, while darker cells with visible cytoplasm surrounding the nuclei were considered as spread cells. Copyright 2019 Pijuan, Barcel, Moreno, Maiques, Sis, Marti, Maci and Panosa. Cell Biol. JP, CB, DM, OM, PS, AM, and AP performed the experiments and analyzed the results. After treatment, fixation and image acquisition, the spreading capacity was quantified using the macro NotSpread&All_.ijm in ImageJ/Fiji (Supplementary Figure 4 and provided in Supplementary Data 3). Nuclei are stained with Hoechst (blue fluorescence). Still, we have to accept that these methodologies have some limitations, which may be mitigated with specific adjustments. 28, 18331846. doi: 10.1146/annurev-cellbio-111315-125201, Trepat, X., Chen, Z., and Jacobson, K. (2012). doi: 10.1038/nrm.2015.14, Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy, G., et al. Biomed. Consequently, we quantified the accumulated distance average (m) (Figure 3C) and velocity (m/h) (Figure 3D). 15, 813824. After applying the Find maxima parameter (e.g., noise tolerance 5) in ImageJ/Fiji, an automatically cell recount was applied (right panel). We observed that untreated cells present a mean of 280.57 42.74 m and 15.30 2.33 m/h in accumulated distances and velocity, respectively whereas the treated cells showed a significant reduction (p = 0.0074; p = 0.0158) in both parameters (148.83 15.42 m and 8.70 1.61 m/h, respectively). Screenshots to set step-by-step the Manual Tracking and Chemotaxis tool plugins by ImageJ/Fiji. In vitro cell migration and invasion assays. We next investigated melanoma cells migration capacity under the effect of 10% FBS as a chemoattractant and Mibefradil (5 M) treatment. (A) Time-lapse sequence and main window of Manual Tracking with the different parameters to set time interval, x/y calibration and track. Rev. Oncol. (2008). (E) Final step and results (area values) of analysis with Wound_healing macro. Tumour-cell invasion and migration: diversity and escape mechanisms. Single and collective cell migration: the mechanics of adhesions. BioEssays. After this initial screen, we can proceed to study exhaustively the wound closure time and cell front healing speed using the insert and recording the complete time-lapse. doi: 10.1091/mbc.e17-03-0134. 9:2454. doi: 10.1038/s41467-018-04465-5, Zhu, Y., Ye, T., Yu, X., Lei, Q., Yang, F., Xia, Y., et al. We studied the effect of T-type calcium channel (TTCC) blocker (Mibefradil) in melanoma individual cell migration. Aman, A., and Piotrowski, T. (2010). Cell Dev. Total melanoma cells (input, left panels in Figure 4C) and migrated ones (right panels in Figure 4C) were stained with Hoechst, and cell number in each side of the transwell was counted. Nat. All these results suggest that FBS chemoattractant increases cell migration capacity, but this effect can be attenuated with Mibefradil treatment, which reduces migration rate by blocking the autophagic flux in M3 melanoma cells (Maci et al., 2015; Maiques et al., 2018). Dev. doi: 10.1038/ni.f.212, Friedl, P., and Wolf, K. (2003). Calcium channel expression and applicability as targeted therapies in melanoma. 2, 23692392. 9, 960969. These findings revealed that Mibefradil significantly impaired M3 melanoma cells motility in a single cell scale (Maiques et al., 2018). 25, 10251040. Biol. Annu. In this assay, we can evaluate the migration or invasive capacity in cancerous cells, depending on the use of matrigel or not (see STEPWISE PROCEDURES). 104 m2/h) (Figure 2E). doi: 10.1002/cphy.c110012, van Helvert, S., Storm, C., and Friedl, P. (2018). doi: 10.1038/s41556-017-0012-0, Wang, X., Lao, Y., Xu, N., Xi, Z., Wu, M., Wang, H., et al. JP and DM developed the macros. Res. Further, these protocols explain an accurate and robust procedure to quantify these parameters using ImageJ/Fiji software. (C,D) Main window of Chemotaxis tool plugin with time interval, x/y calibration and plot size and import dataset settings. Phase-contrast representative images of Mibefradil treated cells (right panel) and untreated cells (left panel) before (0 h) and after (20 h) live cell imaging analysis are shown (Figure 3A). PLoS ONE 10:e0134714. This analysis is simple and gives valuable initial information about cell front migration, but does not provide dynamic information. Rev. In order to accelerate this step, we wrote two novel macros for running in ImageJ/Fiji program for the quantification of wound healing and spreading assays. Cancer Res. (B) Results window after tracking cells at different time-points by Manual Tracking, track n, slice n, X, Y, distance, velocity and pixel value are shown. The main advantages of these in vitro assays are that they are relatively easy handling, fast, accurate and with high reproducibility, non-expensive and do not require particularly special equipment. Res. CB holds predoctoral fellowship from UdL-IRBLleida. Nat Commun. doi: 10.1016/j.ydbio.2009.11.014, PubMed Abstract | CrossRef Full Text | Google Scholar, Anderson, R. L., Balasas, T., Callaghan, J., Coombes, R. C., Evans, J., Hall, J. Step-by-step setting of Wound_healing macro for ImageJ/Fiji. Cell Dev. Cell Dev. B., Rivera, J., Lee, H., Segall, J. E., Condeelis, J. S., et al. Wound_healing.ijm macro code. Cell Biol. Moreover, we specified the ImageJ/Fiji built-in functions that provide an accurate image analyses in single cell and transwell assays. Setting to count cell number in ImageJ/Fiji. Oblongifolin C inhibits metastasis by up-regulating keratin 18 and tubulins. Biol. Annu. Nat. For example, the use of fluorescent-labeled cells (either genetically modified to express fluorescent proteins such as GFP fusions or stained with fluorescent cell tracker dyes) will provide an extra layer of relevant biological information by itself (e.g., protein localization, expression levels, metabolic state of certain organelles, etc. 20, 820. JP, RM, AM, and AP conceived and designed this project. Front. Cell Biol. AM holds postdoctoral fellowship from AECC. Besides, the methods described in this protocol may be used as a basis, but in order to deal with fluorescence images some steps should be modified. M3 melanoma cells were seeded in the upper part of the membrane (see scheme in Figure 4A) and treated either with or without FBS (used as chemoattractant) at the upper chamber and/or treated with FBS and Mibefradil at the bottom chambers (see scheme in Figure 4B). The quantitative analysis of spread cells and the effect of the treatment were analyzed, indicating a significant decrease in percentage (p > 0.001) of spread cells after Mibefradil treatment (36.25 3.10%) compared to untreated cells (65.68 4.43%) (Figure 5C). (2019). Cancer invasion and the microenvironment: plasticity and reciprocity. In order to analyze the ability of melanoma single cells to directionally respond to various chemoattractants and treatments (TTCC blockers) we have performed a transwell assay. *Correspondence: Anna Maci, amacia@irblleida.cat; Anas Panosa, anais.panosa@udl.cat, Present Address: Jordi Pijuan, Laboratory of Neurogenetics and Molecular Medicine, Institut de Recerca Sant Joan de Du, Barcelona, Spain Oscar Maiques, Barts Cancer Institute, Queen Mary University of London, John Vane Science Building, London, United Kingdom, https://imagej.nih.gov/ij/plugins/track/track.html, http://ibidi.com/fileadmin/products/software/chemotaxis_tool/chemotaxis_tool.jar, http://ibidi.com/software/chemotaxis_and_migration_tool/?x504f5=f74e46e10dfbebe758db0683f9de1303, http://rsbweb.nih.gov/ij/plugins/cell-counter.html, https://www.frontiersin.org/articles/10.3389/fcell.2019.00107/full#supplementary-material, Creative Commons Attribution License (CC BY). Cancer 3, 362374. This work was supported by grants from ISCIII/FEDER Una manera de hacer Europa (PI12/00260, PI15/00711 to RM and PI18/00573 to RM and AM) and CIBERONC. Another phenotype to take into account is whether cells are able to grow vertically; if that is not the case they should not be used in this kind of invasion assays. Cell migration during morphogenesis. T-type calcium channels drive migration/invasion in BRAFV600E melanoma cells through Snail1. We observed a significant decrease in migration rate when cells are treated with TTCC blockers (p = 0.0009). Cell Biol. Related to the invasion analysis using the transwell assay, it is essential to check previously if cells are capable to invade the membrane and the Matrigel coating, as there exist cell types which can migrate horizontally very fast but they cannot invade a pore membrane (Trepat et al., 2012). A framework for the development of effective anti-metastatic agents. Supplementary Figure 2. Moreover, they often allow the examination and phenotypic analysis during single cell assay. Sci Rep. 6:20253. doi: 10.1038/srep20253, Keywords: cell migration/invasion, wound healing assay, scratch assay, transwell assay, spreading assay, live cell imaging, data analysis, ImageJ/Fiji, Citation: Pijuan J, Barcel C, Moreno DF, Maiques O, Sis P, Marti RM, Maci A and Panosa A (2019) In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis. 39:1700003. doi: 10.1002/bies.201700003, Kramer, N., Walzl, A., Unger, C., Rosner, M., Krupitza, G., Hengstschlger, M., et al. Rev. 341, 2033. Biol. Int. Biol. Cancer 18, 296312. This result indicates that Mibefradil impairs the spreading capacity in M3 melanoma cells. A., et al. Wound_healing_Choose_Threshold.ijm macro code. The high throughput image data generated by these methods and specially its downstream quantification, represent one of the major limitations in its clinical application. doi: 10.1016/j.mrrev.2012.08.001, Maci, A., Herreros, J., Mart, R. M., and Cant, C. (2015). Representative phase-contrast images with spread and unspread untreated M3 melanoma cells (left panel) and Mibefradil-treated cells (right panel) are shown (Figure 5B). Supplementary Data 2. (2005). Supplementary Data 3. Determining migratory, adhesion and invasion phenotype of tumor cells and understanding molecular mechanisms is fundamental for novel clinical strategies in cancer diagnosis, prognosis, drug development, and treatment. (2016). We thank Dr. Petya Valcheva for critical review of the manuscript. Another characteristic aspect in melanoma cells behavior studies is the spreading capacity. (1998). Cell migration. The front and rear of collective cell migration. Nat. Mol. NotSpread&All.ijm macro code. 752, 1024. Nat. (A) Set scale and Set measurements dialog boxes allow adjusting the scale (pixel/m) and the parameters to measure (BD). Rev. Precision therapeutic targeting of human cancer cell motility. doi: 10.1038/nrc.2018.15, te Boekhorst, V., Preziosi, L., and Friedl, P. (2016). (A) Brightfield images of the fixed cells are background subtracted and thresholded (using the Yen method), and non-attached cell number is counted after a few binary operations, including fill holes and watershed. doi: 10.1111/pcmr.12690, Mayor, R., and Etienne-Manneville, S. (2016). Metastasis is the main cause of cancer lethality, 90% of deaths from solid tumors can be ascribed to metastatic dissemination (Kramer et al., 2013), and understanding the multi-step migration, adhesion and invasion progress, represents an enormous challenge in cancer treatment (Anderson et al., 2019). Mol. Clin. Individual cell migration analysis should be used after researchers have evaluated their cell type velocity and have stablished whether they move independently or not, since they exist cell lines that need to form a group to activate migration mechanism (De Pascalis and Etienne-Manneville, 2017). All authors contributed to manuscript revision, read and approved the submitted version. Physical influences of the extracellular environment on cell migration. Altogether, these varieties of assays generate quantitative metrics capable of describing accurately the migratory behavior of cancer cells from different perspectives. 2015, 17. (A) Fluorescent image with migrated cells after 20 h in transwell insert (left panel). Inhibition of macroautophagy triggers apoptosis. Mutat. doi: 10.1155/2015/587135, Maiques, O., Barcel, C., Panosa, A., Pijuan, J., Orgaz, J. L., Rodriguez-Hernandez, I., et al. (2018). Friedl, P., and Alexander, S. (2011). Nifuroxazide exerts potent anti-tumor and anti-metastasis activity in melanoma. (2018). The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2019.00107/full#supplementary-material. SPARC controls melanoma cell plasticity through Rac1.
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