Cancer Immunotherapy: NK Cell-Mediated Cytolysis

ACEA Biosciences xCELLigence

What are NK Cells?

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.

Application Highlight: NK-92 Cell-Mediated Cytolysis of MCF7 Breast Cancer Cells

In the experiment shown below, 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 (left and right panels).

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.

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 ACEA’s Application Note entitled “Label-Free Assay for NK Cell-Mediated Cytolysis”.

Key Benefits Of Using xCELLigence For Studying NK Cell-mediated Cytolysis:
  1. Label-Free: Allowing for more physiological assay conditions; labeling or secondary assays aren’t required.
  2. Real-Time: Quantitative monitoring of both fast (hours) and slow (days) killing kinetics.
  3. Sensitive: Capable of evaluating low effector cell to target cell ratios that are physiologically relevant.
  4. Simple Workflow: Requires only the addition of effector cells to target cells (in the presence or absence of antibodies); homogeneous assay without additional sample handling.
  5. Automatic Data Plotting: RTCA software enables facile data display and objective analysis, precluding the subjective data vetting that is common to imaging-based assays.

NK Cell-mediated Cytolysis – Supporting Information:

  • Adherent cell lines tested:
    HT1080, H460, HepG2, MCF-7, A549, HeLa, MDA-MB-231, NIH3T3, MelC, MelS, astrocyte-like cell (NT2A), RCC6, RCC4, mesenchymal stromal cells (MSCs)
  1. 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)
  2. 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)
  3. 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)
  4. 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)
  5. 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)
  6. 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)
  7. 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)
  8. 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)