UCLA

NK cells are large granular lymphocytes known to mediate direct and antibody dependent cellular cytotoxicity (ADCC) against tumors as well as to regulate the function of other cells through the secretion of cytokines and chemokines. In previous experiments our lab has shown that NK cells are significantly more cytotoxic towards stem cells than their differentiated counterparts, as was shown with oral squamous carcinoma stem cells (OSCSCs) versus the differentiated oral squamous carcinoma cells (OSCCs), thus, demonstrating that their stage of differentiation is predictive of sensitivity to NK cell mediated cytotoxicity. The same was seen in human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) in which they were lysed more significantly than their differentiated counterparts. Moreover, interaction of NK cells with stem cells leads to the suppression of NK cell cytotoxicity and in an increase in cytokine secretion termed split anergy, however, the interaction of NK cells with resistant tumors does not lead to suppression of NK cytotoxicity or increase in cytokine secretion. NK cells may play an important role in the differentiation of cells.

In this study we examined the role of NK cells in selection and differentiation of lung cancer cells. Our objective was to characterize the lung tumor cell line hA549 and to demonstrate induction of split anergy in NK cells decreases cytotoxicity against hA549, but increases secretion of cytokines, and that anergized NK cells support the differentiation of lung cancer stem cells through secreted cytokines. Furthermore, we explored the role of MHC 1 and intracellular signaling pathways in differentiation of lung tumors. In addition, we wanted to demonstrate that undifferentiated or poorly differentiated lung tumors are more resistant to chemotherapy drugs than differentiated lung tumors

hA549 and H292 lung tumor cells, as well as, healthy SCAP cells were used. Human NK cells where purified using negative selection and activated with IL-2 and IL-2 + anti-CD16mAb. NK cell cytotoxicity was assessed using Cr51 release assay. Lung tumor cells were differentiated using recombinant TNF-alpha, IFN-gamma, a combination of TNF-alpha, and IFN-gamma, or NK IL2 + anti-CD16mAb (anergized) supernatants. Differentiation was assessed by using surface marker analysis by flow cytometry. Lung tumors were treated with Cisplatin, Paclitaxel, NAC, Cisplatin +NAC, and Paclitaxel + NAC. Cells were stained with Propidium Iodide and cell death was assessed by flow cytometry analysis.

The experimental findings of the project demonstrated that hA549 were sensitive to NK cell mediated cytotoxicity. In addition, based on our accumulated results interaction of NK cells with poorly differentiated tumor cells induced split anergy in NK cells resulting in increased secretion of TNF-alpha and IFN-gamma which induced differentiation and resistance of tumor cells. Furthermore, increased expression of MHC class 1 and activation of JNK, AKT, and STAT3 intracellular signaling pathways was correlated with differentiation of hA549 by anergized NK supernatants. Thus, NK cells may inhibit or significantly slow the tumor progression 1-by elimination of cancer stem cells or poorly differentiated tumors and 2- by inducing differentiation of tumors thereby limiting tumor induced inflammation which could further fuel the tumors. In addition, we found that hA549 were more resistant to the chemotherapy drugs, Cisplatin and Paclitaxel, than the more differentiated H292.