Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting

Background On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exoc...

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Autores principales:	Torsten Tonn, Winfried Wels, Jiri Eitler, Natalie Wotschel, Nicole Miller, Laurent Boissel, Hans G Klingemann
Formato:	article
Lenguaje:	EN
Publicado:	BMJ Publishing Group 2021
Materias:	Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
Acceso en línea:	https://doaj.org/article/42e0931eb06e42e8be5816160bc2b736
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spelling	oai:doaj.org-article:42e0931eb06e42e8be5816160bc2b7362021-11-15T22:30:05ZInability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting10.1136/jitc-2020-0013342051-1426https://doaj.org/article/42e0931eb06e42e8be5816160bc2b7362021-01-01T00:00:00Zhttps://jitc.bmj.com/content/9/1/e001334.fullhttps://doaj.org/toc/2051-1426Background On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors.Methods Here, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92.Results Unmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals.Conclusions These observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance.Keywords: NK cells, NK-92, haNK, ADCC, Chimeric Antigen Receptor (CAR), breast cancer, cancer immunotherapy, live-cell imaging, granule polarizationTorsten TonnWinfried WelsJiri EitlerNatalie WotschelNicole MillerLaurent BoisselHans G KlingemannBMJ Publishing GrouparticleNeoplasms. Tumors. Oncology. Including cancer and carcinogensRC254-282ENJournal for ImmunoTherapy of Cancer, Vol 9, Iss 1 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
spellingShingle	Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282 Torsten Tonn Winfried Wels Jiri Eitler Natalie Wotschel Nicole Miller Laurent Boissel Hans G Klingemann Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
description	Background On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors.Methods Here, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92.Results Unmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals.Conclusions These observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance.Keywords: NK cells, NK-92, haNK, ADCC, Chimeric Antigen Receptor (CAR), breast cancer, cancer immunotherapy, live-cell imaging, granule polarization
format	article
author	Torsten Tonn Winfried Wels Jiri Eitler Natalie Wotschel Nicole Miller Laurent Boissel Hans G Klingemann
author_facet	Torsten Tonn Winfried Wels Jiri Eitler Natalie Wotschel Nicole Miller Laurent Boissel Hans G Klingemann
author_sort	Torsten Tonn
title	Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_short	Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_full	Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_fullStr	Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_full_unstemmed	Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_sort	inability of granule polarization by nk cells defines tumor resistance and can be overcome by car or adcc mediated targeting
publisher	BMJ Publishing Group
publishDate	2021
url	https://doaj.org/article/42e0931eb06e42e8be5816160bc2b736
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Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting

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