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.