Natural killer (NK) cells are a type of cytotoxic lymphocyte that play a critical role in the innate immune system, primarily by recognizing and destroying virus-infected cells. NK cells express a number of activation and inhibitory receptors that work in concert to distinguish infected or diseased cells from normal cells. In close proximity to a target cell slated for killing, NK cells are activated and secrete a membrane permeabilizing protein (perforin) and proteases (granzymes) which collectively cause target cell death via apoptosis or osmotic lysis. These mechanisms employed by NK cells to recognize and destroy infected cells are also critical to killing cancer cells.
Unlike T cells which must be educated by antigen-presenting cells before they recognize tumors, NK cells spontaneously lyse certain types of tumor cells in vivo and in vitro without requiring immunization or pre-activation. Similar to virally infected cells, tumor cells also down-regulate their MHC-1 expression. Recognizing this change in expression, NK cells destroy cancer cells through perforin/granzyme mediated lysis. Owing to this capacity, NK cells are being investigated for the purposes of immunotherapy. As such, quantifying and dynamically monitoring NK cell-mediated cytolysis, in the context of immunotherapy, can easily be done on the xCELLigence RTCA system without labeling the target cells.
Key Benefits Of Using xCELLigence RTCA For Studying NK Cell-mediated Cytolysis:
xCELLigence RTCA was used to quantitatively measure the cytolytic activity of NK cells in real-time. After growing adherent breast cancer MCF7 cells in the bottom of E-Plate wells, NK-92 cells were added at different effector to target (E:T) ratios. The data clearly demonstrate NK-92 cell-mediated lysis of the MCF7 cells in a dose- and time-dependent manner as shown by normalized cell index and % cytolysis.
Importantly, real-time impedance monitoring by the xCELLigence system is sensitive enough to detect target cell killing even at low E:T ratios. For plotting purposes, the percentage of cytolysis is readily calculated using a simple formula: Percentage of cytolysis = ((Cell Index no effector – Cell Index effector)/Cell Index no effector) X 100. Real-time monitoring of NK-92 cell-mediated cytolysis of MCF7 breast cancer cells. Adherent MCF7 target cells were grown in multiple wells of an E-Plate. Different quantities of NK-92 cells were added to each well and impedance was monitored continuously for the next ~20 hours (left panel). The time-dependent cytolytic activity of NK-92 cells at different E:T ratios (right panel) was calculated as described above. Figures adapted from Agilent’s Application Note entitled “Label-Free Assay for NK Cell-Mediated Cytolysis”.
NK92 shows a broad spectrum of cytolytic activity on cancer cell lines. The cytolysis mediated by NK92 occurred quickly and reached the maximum killing activity in less than eight hours. Among seven cell lines tested, four cell lines (H460, HepG2, MCF7, and MDA-MB-231) reached 90% cytolysis.
In this application note, we will summarize a series of experiments to demonstrate how the xCELLigence RTCA system can be applied to quantitatively, dynamically monitor NK cell-mediated cytolysis without labeling the target cells
In this application note, a series of experiments demonstrate how the xCELLigence RTCA system can be applied to quantitatively, dynamically monitor NK cell-mediated cytolysis without labeling the target cells.
Using a combination of Trastuzumab and PBMCs as a model system, this study demonstrates differences in the impedance- and image-based data for HER2-negative versus HER2‑positive target cells.
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Dynamic and label-free monitoring of natural killer cell cytotoxic activity using electronic cell sensor arrays. Zhu J, Wang X, Xu X, Abassi YA. J Immunol Methods. 2006 Feb 20;309(1-2):25-33. (ACEA Biosciences, USA)
Unique functional status of natural killer cells in metastatic stage IV melanoma patients and its modulation by chemotherapy. Fregni G, Perier A, Pittari G, Jacobelli S, Sastre X, Gervois N, Allard M, Bercovici N, Avril MF, Caignard A. Clin Cancer Res. 2011 May 1;17(9):2628-37. (INSERM, France)
Real-time profiling of NK cell killing of human astrocytes using xCELLigence technology. Moodley K, Angel CE, Glass M, Graham ES. J Neurosci Methods. 2011 Sep 15;200(2):173-80. (University of Auckland, Australia)
Mutations of the von Hippel-Lindau gene confer increased susceptibility to natural killer cells of clear-cell renal cell carcinoma. Perier A, Fregni G, Wittnebel S, Gad S, Allard M, Gervois N, Escudier B, Azzarone B, Caignard A. Oncogene. 2011 Jun 9;30(23):2622-32. (INSERM, France)
Evaluation of NK cell function by flowcytometric measurement and impedance based assay using real-time cell electronic sensing system. Park KH, Park H, Kim M, Kim Y, Han K, Oh EJ. Biomed Res Int. 2013;2013:210726. (Catholic University of Korea, South Korea)
Mature cytotoxic CD56(bright)/CD16(+) natural killer cells can infiltrate lymph nodes adjacent to metastatic melanoma. Messaoudene M, Fregni G, Fourmentraux-Neves E, Chanal J, Maubec E, Mazouz-Dorval S, Couturaud B, Girod A, Sastre-Garau X, Albert S, Guédon C, Deschamps L, Mitilian D, Cremer I, Jacquelot N, Rusakiewicz S, Zitvogel L, Avril MF, Caignard A. Cancer Res. 2014 Jan 1;74(1):81-92. (Institut Cochin, France)
Phenotypic and functional characteristics of blood natural killer cells from melanoma patients at different clinical stages. Fregni G, Messaoudene M, Fourmentraux-Neves E, Mazouz-Dorval S, Chanal J, Maubec E, Marinho E, Scheer-Senyarich I, Cremer I, Avril MF, Caignard A. PLoS One. 2013 Oct 18;8(10):e76928. (University of Lausanne, Switzerland)
Inhibition of mesenchymal stromal cells by pre-activated lymphocytes and their culture media. Valencic E, Loganes C, Cesana S, Piscianz E, Gaipa G, Biagi E, Tommasini A. Stem Cell Res Ther. 2014 Jan 9;5(1):3. (Institute of Maternal and Child Health IRCCS Burlo Garofolo, Italy)
Cetuximab-induced natural killer cell cytotoxicity in head and neck squamous cell carcinoma cell lines: investigation of the role of cetuximab sensitivity and HPV status. Baysal, H., De Pauw, I., Zaryouh, H. et. Al. Br J Cancer (2020).
Boosting Natural Killer Cell-Mediated Targeting of Sarcoma Through DNAM-1 and NKG2D. Sayitoglu EC, Georgoudaki A-M, Chrobok M, Ozkazanc D, Josey BJ, Arif M, Kusser K, Hartman M, Chinn TM, Potens R, Pamukcu C, Krueger R, Zhang C, Mardinoglu A, Alici E, Temple HT, Sutlu T and Duru AD (2020) . Front. Immunol. 11:40. doi: 10.3389/fimmu.2020.00040
Enhanced SLAMF7 Homotypic Interactions by Elotuzumab Improves NK Cell Killing of Multiple Myeloma. Pazina T, James AM, Colby KB, et al. Cancer Immunol Res. 2019;7(10):1633-1646. doi:10.1158/2326-6066.CIR-18-057
Biological treatment of pediatric sarcomas by combined virotherapy and NK cell therapy. Klose, C., Berchtold, S., Schmidt, M. et al. BMC Cancer 19, 1172 (2019). https://doi.org/10.1186/s12885-019-6387-5
Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies. Friedman J, Morisada M, Sun L, et al. J Immunother Cancer. 2018;6(1):59. Published 2018 Jun 21. doi:10.1186/s40425-018-0374-2
For Research Use Only. Not for use in diagnostic procedures.
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