UCLA

OBJECTIVES: To demonstrate that similar to differentiated oral tumors, differentiated pancreatic tumors are more resistant whereas their undifferentiated counterparts, or cancer stem cells, are more sensitive to Natural Killer (NK) cell mediated lysis. Furthermore, to reveal that anergized NK cells induce tumor differentiation and to examine the mechanism by which these differentiated cells resist NK cell mediated cytotoxicity. Lastly, to demonstrate that NAC protects tumor cells from lysis by Cisplatin chemotherapeutic treatments, while conversely, when used in conjunction with Paclitaxel, NAC causes a synergistic effect significantly increasing tumor cell lysis versus Paclitaxel treatments alone.

METHODS: Differentiated and undifferentiated, or cancer stem cells, for pancreatic cancer was used to determine their resistance and sensitivity to NK cell mediated

cytotoxicity and secretion of cytokines. The function of NK cells was assessed using 51Cr release assay, and the secretion of cytokines by cytokine arrays. Flow cytometry was used for surface analysis. Also, tumor cells were cultured with NK supernatants prior to undergoing cytotoxic 51Cr release assays and flow cytometry surface analysis. In addition, tumor cells were treated with Cisplatin, Paclitaxel, and NAC, and cell death was determined using propidium iodide (PI) staining or ethanol permeabilization, followed by flow cytometry analysis.

RESULTS: Similar to differentiated oral tumors, differentiated tumors of the pancreas are resistant to NK cell mediated cytotoxicity whereas their undifferentiated counterparts or cancer stem cells are significantly more sensitive. Differentiated tumors exhibited lower CD44 and CD54 and higher B7H1 and MICA when compared to undifferentiated or cancer stem cells. Differentiated tumors triggered no or very low levels of IFN-g secretion by the NK cells. Anergized NK cells release factors that cause tumor cell resistance to NK cell mediated lysis and an increase in B7H1 surface expression on the target cells. Use of anti-B7H1 treatments on these differentiated tumor cells does not restore their sensitivity to NK cell lysis. Furthermore, NAC protects target tumor cells against Cisplatin-mediated killing, yet enhances cell death when used together with Paclitaxel.

CONCLUSIONS: Sensitivity to NK cell mediated lysis is dependent on the stage of differentiation and it is irrespective of the type of cancer. Therefore, NK cells are primary cells to eliminate cancer stem cells. Known inactivation of NK cell cytotoxic function in many cancers including oral tumors may be a major underlying mechanism for the

survival and expansion of cancer stem cells. Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells. There is inactivation of NK cell cytotoxicity function in many cancers, and here we show that anergized NK cytokines gives tumor cells resistance to NK cell mediated lysis and up regulates surface expression of B7H1 on the tumor cells. This marker may be partially responsible for conferring the resistance to NK cell mediated lysis. Knowledge of tumor cell responses to NK cytokines will aid in determining more effective treatments for cancer patients. In addition, the known function of NAC to protect cells is not seen when used with Paclitaxel to lyse tumor cells, therefore patients with cancer may benefit from this synergistic drug combination. NAC may be a factor that could enhance the effects of Paclitaxel chemotherapeutic treatments.