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We extended our previous observations with other tumor models to study seven ovarian tumor cell lines—OVCAR3, OVCAR4, OVCAR8, SKOV3, Kuramochi, OAW28, and CaOV3. We found that NK cells targeted and killed poorly differentiated OVCAR8 and CAOV3; these two tumor lines express lower MHC-class I and higher CD44 surface receptors. OVCAR3 and OVCAR4 were more resistant to NK cell-mediated cytotoxicity, and SKOV3, Kuramochi and OAW28 had intermediate sensitivity to NK cell-mediated cytotoxicity, likely representing well-differentiated and moderately differentiated ovarian tumor cell lines, respectively. Similar trends were observed for secretion of IFN-γ by the NK cells when co-cultured with different ovarian tumor cell lines. Treatment with both IFN-γ and TNF-α upregulated MHC-class I in all ovarian tumor cell lines and resulted in tumor resistance to NK cell-mediated cytotoxicity and decreased secretion of IFN-γ in co-cultures of NK cells with tumors cells with the exception of OVCAR8 and CAOV3 which did not upregulate MHC-class I and remained sensitive to NK cell-mediated cytotoxicity and increased secretion of IFN-γ when co-cultured with NK cells. Similarly, treatment with NK cell supernatants induced resistance to NK cell-mediated cytotoxicity in OVCAR4 but not in OVCAR8, and the resistance to killing was correlated with the increased surface expression of MHC-class I in OVCAR4 but not in OVCAR8. In addition, OVCAR4 was found to be carboplatin sensitive before and after treatment with IFN-γ and NK cell supernatants, whereas OVCAR8 remained carboplatin resistant with and without treatment with IFN-γ and NK cell supernatants. Overall, sensitivity to NK cell-mediated killing correlated with the levels of tumor differentiation and aggressiveness, and more importantly, poorly differentiated ovarian tumors were unable to upregulate MHC-class I under the activating conditions for MHC-class I, a feature that was not seen in other tumor models and may likely be specific to ovarian tumors. Such tumors may also pose a significant challenge in elimination by the T cells; however, NK cells are capable of targeting such tumors and can be exploited to eliminate these tumors in immunotherapeutic strategies. [ABSTRACT FROM AUTHOR]

The majority of the previous reports on NK cells use cross-sectional studies to establish the status of patient NK cell function, however such studies fail to evaluate the immune status of the patients on a continuous basis from the disease-free stage to progression of cancer. In this study, we performed a prospective study of the immune function by continuously monitoring the NK numbers, expansion and function of a pancreatic cancer patient from 1/6/2016 to 2/14/2019. The results indicated that at initial stages of the disease where no overt disease was identified, the patient had consistently higher percentages of NK and B cells and lower percentages of CD3 + T cells in the peripheral blood. The percentages of CD14 + monocytes were similar at the initial stages of the disease, and at the later stages of the disease, it increased and remained higher in the patient when compared to those from healthy donors. The numbers of expanded NK cells and the cytotoxic function, as well as secretion of IFN-γ from primary and osteoclast expanded patient NK cells remained consistently low throughout the years of follow up. Similarly, the majority of cytokines in patient's serum remained lower with the exception of IL-6 which was higher. The IFN-γ secreted from the patients' NK cells had much lower ability to differentiate the poorly differentiated oral tumors as assessed by their lack of ability to upregulate differentiation antigens. Overall, before any evidence of overt disease, patient NK cells exhibited significant dysfunction. Intervention at the stage of no disease or minimal disease may be important for the prevention of pancreatic cancer progression. [ABSTRACT FROM AUTHOR]

Cysteine cathepsins are lysosomal peptidases involved in the regulation of innate and adaptive immune responses. Among the diverse processes, regulation of granule-dependent cytotoxicity of cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells during cancer progression has recently gained significant attention. The function of cysteine cathepsins is regulated by endogenous cysteine protease inhibitors—cystatins. Whereas other cystatins are generally cytosolic or extracellular proteins, cystatin F is present in endosomes and lysosomes and is thus able to regulate the activity of its target directly. It is delivered to endosomal/lysosomal vesicles as an inactive, disulphide-linked dimer. Proteolytic cleavage of its N-terminal part leads to the monomer, the only form that is a potent inhibitor of cathepsins C, H and L, involved in the activation of granzymes and perforin. In NK cells and CTLs the levels of active cathepsin C and of granzyme B are dependent on the concentration of monomeric, active cystatin F. In tumour microenvironment, inactive dimeric cystatin F can be secreted from tumour cells or immune cells and further taken up by the cytotoxic cells. Subsequent monomerization and inhibition of cysteine cathepsins within the endosomal/lysosomal vesicles impairs granzyme and perforin activation, and provokes cell anergy. Further, the glycosylation pattern has been shown to be important in controlling secretion of cystatin F from target cells, as well as internalization by cytotoxic cells and trafficking to endosomal/lysosomal vesicles. Cystatin F is therefore an important mediator used by bystander cells to reduce NK and T-cell cytotoxicity. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are functionally suppressed in the glioblastoma multiforme (GBM) tumor microenvironment. We have recently shown that survival and differentiation of cancer stem-like cells (CSCs)/poorly differentiated tumors are controlled through two distinct phenotypes of cytotoxic and non-cytotoxic/split anergized NK cells, respectively. In this paper, we studied the function of NK cells against brain CSCs/poorly differentiated GBM and their NK cell-differentiated counterparts. Brain CSCs/poorly differentiated GBM, differentiated by split anergized NK supernatants (supernatants from NK cells treated with IL-2 + anti-CD16mAb) expressed higher levels of CD54, B7H1 and MHC-I and were killed less by the NK cells, whereas their CSCs/poorly differentiated counterparts were highly susceptible to NK cell lysis. Resistance to NK cells and differentiation of brain CSCs/poorly differentiated GBM by split anergized NK cells were mediated by interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Brain CSCs/poorly differentiated GBM expressed low levels of TNFRs and IFN-γRs, and when differentiated and cultured with IL-2-treated NK cells, they induced increased secretion of pro-inflammatory cytokine interleukin (IL)-6 and chemokine IL-8 in the presence of decreased IFN-γ secretion. NK-induced differentiation of brain CSCs/poorly differentiated GBM cells was independent of the function of IL-6 and/or IL-8. The inability of NK cells to lyse GBM tumors and the presence of a sustained release of pro-inflammatory cytokines IL-6 and chemokine IL-8 in the presence of a decreased IFN-γ secretion may lead to the inadequacy of NK cells to differentiate GBM CSCs/poorly differentiated tumors, thus failing to control tumor growth. [ABSTRACT FROM AUTHOR]

Based on data obtained from oral, pancreatic and lung cancers, glioblastoma, and melanoma, we have established that natural killer (NK) cells target cancer stem-like cells (CSCs). CSCs displaying low MHC class I, CD54, and PD-L1 are killed by cytotoxic NK cells and are differentiated by split anergized NK cells through both membrane bound and secreted forms of TNF-α and IFN-γ. NK cells select and differentiate both healthy and transformed stem-like cells, resulting in target cell maturation and shaping of their microenvironment. In our recent studies, we have observed that oral, pancreatic, and melanoma CSCs were capable of forming large tumors in humanized bone marrow, liver, thymus (hu-BLT) mice with fully reconstituted human immune system. In addition, major human immune subsets including NK cells, T cells, B cells, and monocytes were present in the spleen, bone marrow, peripheral blood, and tumor microenvironment. Similar to our previously published in vitro data, CSCs differentiated with split anergized NK cells prior to implantation in mice formed smaller tumors. Intravenous injection of functionally potent osteoclast-expanded NK cells inhibited tumor growth through differentiation of CSCs in humanized mice. In this review, we present current approaches, advances, and existing limitations in studying interactions of the immune system with the tumor, in particular NK cells with CSCs, using in vivo preclinical hu-BLT mouse model. In addition, we discuss the use of osteoclast-expanded NK cells in targeting cancer stem-like tumors in humanized mice-a strategy that provides a much-needed platform to develop effective cancer immunotherapies. [ABSTRACT FROM AUTHOR]