Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo : Role in osteoclast-mediated NK cell activation Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: Role in osteoclast-mediated NK cell activation

Natural killer (NK) cells are known to target cancer stem cells and undifferentiated tumors. In this paper, we provide a novel strategy for expanding large numbers of super-charged NK cells with significant potential to lyse and differentiate cancer stem cells, and demonstrate the differences in the dynamics of NK cell expansion between healthy and cancer patients. Decline in cytotoxicity and lower IFN-γ secretion by osteoclast-expanded NK cells from cancer patients correlates with faster expansion of residual contaminating T cells within purified NK cells, whereas healthy donors’ osteoclasts continue expanding super-charged NK cells while limiting T cell expansion for up to 60 days. Similar to patient NK cells, NK cells from tumor-bearing BLT humanized mice promote faster expansion of residual T cells resulting in decreased numbers and function of NK cells, while NK cells from mice with no tumor continue expanding NK cells and retain their cytotoxicity. In addition, dendritic cells in contrast to osteoclasts are found to promote faster expansion of residual T cells within purified NK cells resulting in the decline in NK cell numbers from healthy individuals. Addition of anti-CD3 mAb inhibits T cell proliferation while enhancing NK cell expansion, however, expanding NK cells have lower cytotoxicity but higher secretion of IFN-. Expansion and functional activation of super-charged NK cells by osteoclasts is dependent on IL-12 and IL-15. Thus, in this report we not only provide a novel strategy to expand super-charged NK cells, but also demonstrate that rapid and sustained expansion of residual T cells within the purified NK cells during expansion with dendritic cells or osteoclasts could be a potential mechanism by which the numbers and function of NK cells decline in cancer patients and in BLT humanized mice.

Natural Killer (NK) cells target oral, pancreatic, lung, breast, glioblastoma and melanoma stem-like/poorly differentiated tumors. Differentiation of the abovementioned tumors with supernatants from split-anergized NK cells decreases their susceptibility to NK cells, but increases their sensitivity to cisplatin (CDDP)-mediated cell death. Breast and melanoma tumor cells with CD44 knockdown display enhanced susceptibility to NK cell-mediated lysis, potentially due to decreased differentiation. We also demonstrate that sulindac, a non-steroidal anti-inflammatory drug and a chemopreventive agent, not only limits the growth of oral tumor cells, but also aids in cancer cell elimination by NK cells. Treatment of oral tumors with sulindac, but not adriamycin inversely modulates the expression and function of NFκB and JNK, resulting in a significant down-regulation of IL-6, and VEGF secretion by oral tumor cells. In addition, increased secretion of IL-6 and VEGF is blocked by sulindac during interaction of oral tumors with NK cells. Sulindac treatment prevents synergistic induction of VEGF secretion by the tumor cells after their co-culture with untreated NK cells since non-activated NK cells lack the ability to efficiently kill tumor cells. Moreover, sulindac is able to profoundly reduce VEGF secretion by the tumor cells cultured with IL-2 activated NK cells, which are able to significantly lyse the tumor cells. Based on the data presented in this study, we propose the following combinatorial approach for the treatment of stem-like/ poorly differentiated tumors in cancer patients with metastatic disease. Stem-like/ poorly differentiated tumor cells may in part undergo lysis or differentiation after NK cell immunotherapy, followed by treatment of differentiated tumors with chemotherapy and chemopreventive agents to eliminate the bulk of the tumor. This dual approach should limit tumor growth and prevent metastasis.

Background and Aims: We have previously demonstrated that the stage of differentiation of tumors has profound effect on the function of NK cells, and that stem-like/poorly differentiated tumors were preferentially targeted by the NK cells. Therefore, in this study we determined the role of super-charged NK cells in immune mobilization, lysis, and differentiation of stem-like/undifferentiated tumors implanted in the pancreas of humanized-BLT (hu-BLT) mice fed with or without AJ2 probiotics. The phenotype, growth rate and metastatic potential of pancreatic tumors differentiated by the NK cells (NK-differentiated) or patient derived differentiated or stem-like/undifferentiated pancreatic tumors were investigated. Methods: Pancreatic tumor implantation was performed in NSG and hu-BLT mice. Stage of differentiation of tumors was determined using our published criteria for well-differentiated tumors exhibiting higher surface expression of MHC- class I, CD54, and PD-L1 (B7H1) and lower expression of CD44 receptors. The inverse was seen for poorly-differentiated tumors. Results: Stem-like/undifferentiated pancreatic tumors grew rapidly and formed large tumors and exhibited lower expression of above-mentioned differentiation antigens in the pancreas of NSG and hu-BLT mice. Unlike stem-like/undifferentiated tumors, NK-differentiated MP2 (MiaPaCa-2) tumors or patient-derived differentiated tumors were not able to grow or grew smaller tumors, and were unable to metastasize in NSG or hu-BLT mice, and they were susceptible to chemotherapeutic drugs. Stem-like/undifferentiated pancreatic tumors implanted in the pancreas of hu-BLT mice and injected with super-charged NK cells formed much smaller tumors, proliferated less, and exhibited differentiated phenotype. When differentiation of stem-like tumors by the NK cells was prevented by the addition of antibodies to IFN-γ and TNF-α, tumors grew rapidly and metastasized, and they remained resistant to chemotherapeutic drugs. Greater numbers of immune cells infiltrated the tumors of NK-injected and AJ2-probiotic bacteria-fed mice. Moreover, increased IFN-γ secretion in the presence of decreased IL-6 was seen in tumors resected and cultured from NK-injected and AJ2 fed mice. Tumor-induced decreases in NK cytotoxicity and IFN-γ secretion were restored/increased within PBMCs, spleen, and bone marrow when mice received NK cells and were fed with AJ2. Conclusion: NK cells prevent growth of pancreatic tumors through lysis and differentiation, thereby curtailing the growth and metastatic potential of stem-like/undifferentiated-tumors.

Natural killer (NK) cells are the key immune effectors with the ability to mediate selection and differentiation of a number of different cancer stem cells/undifferentiated tumors via lysis, and secreted or membrane-bound interferon (IFN)-γ and tumor necrosis factor (TNF)-α, respectively, leading to curtailment of tumor growth and metastasis. In this review, we present an overview of our recent findings on the biology and significance of NK cells in selection and differentiation of stem-like tumors using in vitro and in vivo studies conducted in humanized-BLT mice and in cancer patients. In addition, we present current advances in NK cell expansion and therapeutic delivery, and discuss the utility of allogeneic supercharged NK cells in the treatment of cancer patients. Moreover, we discuss the potential loss of NK cell numbers and function at the neoplastic and pre-neoplastic stages of tumorigenesis in induction and progression of pancreatic cancer. Therefore, because of their indispensable role in targeting cancer stem-like/undifferentiated tumors, NK cells should be placed high in the armamentarium of tumor immunotherapy. A combination of allogeneic supercharged NK cells with other immunotherapeutic strategies such as oncolytic viruses, antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies, checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, CAR NK cells, and chemotherapeutic and radiotherapeutic strategies can be used for the ultimate goal of tumor eradication.