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Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated
Jiri Eitler1,2, Natalie Wotschel1,2, Nicole Miller1,2
1Experimental Transfusion Medicine, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Germany.
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.
Insights
Retargeting natural killer (NK) cells with chimeric antigen receptors (CARs) or antibody-dependent cell-mediated cytotoxicity (ADCC) overcomes tumor resistance by enabling lytic granule polarization. This targeted approach activates crucial signaling pathways, leading to effective cancer cell killing.
Area of Science:
- Immunology
- Cell Biology
- Cancer Research
Background:
- Natural killer (NK) cells are crucial for innate immunity, mediating cytotoxicity via directed degranulation.
- NK cell retargeting strategies like CARs and mAbs show promise against resistant tumors.
- Lytic granule dynamics in retargeted NK cells against resistant tumors remain poorly understood.
Purpose of the Study:
- To investigate the lytic granule dynamics of retargeted NK cells against resistant cancer cells.
- To elucidate the signaling pathways involved in NK cell-mediated cytotoxicity upon retargeting.
- To determine if retargeting can overcome NK-cell resistance in specific cancer types.
Main Methods:
- Live-cell imaging using spinning disk confocal microscopy.
- Analysis of ErbB2-targeted CAR-expressing NK-92 cells and FcR transgenic NK-92 cells with Herceptin.
- Coculture with ErbB2-positive, NK-resistant breast cancer cells (MDA-MB-453).
Main Results:
- Unmodified NK-92 cells formed conjugates but failed to polarize granules to the immunological synapse (IS).
- Retargeted NK cells (CAR or FcR+Herceptin) successfully polarized granules to the IS, leading to effective cytotoxicity.
- Retargeting by CAR or ADCC provided essential PLCγ and MEK/ERK signaling, which were absent in unmodified NK-92 cells interacting with resistant cancer cells.
Conclusions:
- NK cells require specific activation signals for granule polarization and effective cytotoxicity against resistant tumors.
- CAR and FcR/mAb (ADCC) retargeting provide these necessary signals, overcoming tumor resistance.
- Targeted NK cell therapies can be optimized by understanding and manipulating these signaling pathways.
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