Osteonecrosis of jaw, is a bone disorder affecting the jaw bone. Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a condition found in patients who have received intravenous and oral forms of bisphosphonate therapy for various bone-related conditions. Bisphosphonates (BPs) are a class of drugs used to treat osteoporosis and malignant bone metastasis. Osteoclast's are the bone cells capable of resorbing bone, are the potential target of BP therapy. The objective of this study is to demonstrate the role of osteoclasts in ONJ, effect of BP on the differentiation and function of osteoclasts, inflammatory mediators secreted by osteoclasts involved in ONJ

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model, interaction of osteoclasts with other immune cells especially NK cells, as well as role of

IL-10 in osteoclasts and BP interaction. This study demonstrate that, i.v nitrogen-containing

bisphosphonates Zolendronic Acid and oral nitrogen-containing bisphosphonates Alendronate and oral non-nitrogen containing Etidronate modulate the pro-inflammatory functions of osteoclasts. Nitrogen containing Bisphosphonates mediated significant dose dependent release of pro-inflammatory cytokines IL-6, TNF-α, IL-1b and they inhibited anti-inflammatory IL-10 secretion by osteoclasts. Zolendronic Acid, Alendronate and Etidronate each had a different effect on the secretion of cytokines, with Zolendronic Acid having the highest and Etidronate the lowest ability to up-regulate the cytokine secretion. Zolendronic Acid, Alendronate and Etidronate each had a different effect on cell viability, Zolendronic Acid caused highest reduction in osteoclasts numbers, followed by Alendronate and Etidronate almost cause no cell death. The surface expression of CD14, CD33, CD54, CD44, CD11b, MHC class I and II and B7H1 were significantly increased when osteoclasts were treated with Zolendronic Acid, effect was higher at 500nM conc. and down-modulated at higher concentration of Zolendronic Acid, but still higher than untreated OC. All three bisphosphonates were capable of decreasing pit formation by osteoclasts. Osteoclasts act as key immune effectors capable of modulating the function of Natural Killer (NK) cells. Treatment of osteoclasts with Zolendronic Acid and much less with Alendronate was capable of inhibiting NK cell cytotoxicity whereas it induced significant secretion of cytokines, IFN-γ, IL-6, IL-10 and IL-18 and chemokines in the cultures of NK cells with osteoclasts. Resistance to NK cell cytotoxicity was higher at low concentration of Zolendronic Acid, comparative to higher concentration. NK cells were able to lyse osteoclasts much more than their precursor cell monocytes and this correlated with the decreased

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expression of MHC class I and CD54 expression on osteoclasts. These results suggest that Zolendronic Acid treated osteoclasts may remain viable in the microenvironment for a prolonged period of time during interaction with NK cells providing continuous secretion of pro-inflammatory cytokines and chemokines in the absence of anti-inflammatory cytokine IL-10 resulting in the chronicity of inflammation.

Zolendronic Acid injections could similarly increase IFN-g and IL-6 secretion and Cytotoxicity against the tumor cells, in the bone marrows but decrease the secretion of IFN-g in gingival, pancreatic and adipose tissues. These observations are of significant value since they may provide potential mechanisms for the pathogenesis seen in Osteonecrosis of the Jaw (ONJ). It is possible that an increase in activation of Bone Marrow derived cells by Zolendronic Acid result in the eventual loss or inhibition of IFN-g secreting cells when they reach to the tissues. Since gingival derived immune cells are of activated phenotype when compared to bone marrow derived immune cells in healthy subjects, prior activation of bone marrow derived cells when mobilized to the gingival tissues and are exposed to additional activation signals from antigens in gingiva may undergo activation induced cell death, or simply become exhausted, and result in the lower secretion of IFN-g by the immune cells as seen in our in vivo model system. These possibilities are under investigation in our laboratory. The phenotype and nature of immune cells, including NK cells, one of the main contributors of IFN-g secretion in bone marrow and tissues are currently being investigated in our laboratory.

Natural Killer cells (NK) cells are known to limit growth and expansion of cancer stem cells by direct cytotoxicity, providing key cytokines such as IFN-γ and TNF-α, which drive differentiation of stem-like/poorly differentiated cancer stem cells and through antibody-dependent cell cytotoxicity (ADCC). One of the important surface antigens on transformed cells is Major Histocompatibility Complex class I chain-related protein A and B (MICA/MICB). Induction of NK cell-mediated cytotoxicity resistance and differentiation in the stem cells correlate with the increased expression of CD54, B7H1, and MHCI, and MICA/MICB and antibodies specific to MICA/MICB increased NK cell-mediated ADCC against differentiated oral and pancreatic tumors, while the stem-like/poorly differentiated cancer stem cells were not targeted. This antibody also increased IFN-γ secretion by NK cells when cultured with differentiated oral tumors expressing MICA/MICB. This study also showed that Expanded NK cells target both undifferentiated and differentiated tumor cells while primary NK cells preferentially target undifferentiated/Stem-like population. In addition, it showed that the combination of IL-2 and antiCD16 treatment induces split anergy in primary NK cells but not in super-charged NK cells and Expanded NK cells were found to mediate lower levels of ADCC than primary NK cells. Knowing the key role of IFN-γ production in differentiation of oral and pancreatic cancer cells, then aimed to determine strategies to increase NK cell-mediated production of IFN-γ and we showed that AJ2 probiotic bacteria and fucoidan extracted from Mekabu could individually increase NK cells’ IFN-γ secretion ability. The combination of AJ2 probiotic bacteria and fucoidan extracted from Mekabu resulted in synergic secretion of IFN-γ by NK cells. In conclusion, differentiated and stem-like/ poorly differentiated oral and pancreatic tumors have different expression level of MICA/MICB and can be targeted differentially through ADCC. Primary and Expanded NK cells have very different characteristics and biological functions and all the diverse functions of different subsets of NK cells should be considered in NK cell- immunotherapeutic approaches.

NK cells are known to limit growth and expansion of cancer stem cells and oral tumors by providing key cytokines such as IFN-γ and TNF-α, which drive differentiation of stem-like/poorly differentiated cancer stem cells. The detailed understanding of how and why their function is affected by the cancer microenvironment is very important for outlining the most efficient NK cell-based immunotherapies for cancer patients. In this regard, we first characterized NK cells from the cancer patients for their IFN-γ secretion, cytotoxic function against the cancer stem cells, and their surface receptors. In previous studies, we demonstrated that IFN-γ plays a dominant role in up-regulation of B7H1, CD54 and MHC1 surface receptor expression and inhibition of tumor growth by differentiating the cancer stem cells, and via inflammatory cytokine release when they are in contact with cancer stem cells. We also showed that induction of NK cell-mediated cytotoxicity resistance and differentiation in the stem cells correlated with the increased expression of CD54, B7H1, and MHCI, and mediated by the combination of membrane-bound or secreted IFN-γ. In this study, we demonstrated that NK media adjusted based cells from cancer patients secreted lower IFN-γ. When we used the NK cell condition media from the cancer patient and healthy NK cells to treat the cancer stem cells, the condition media from cancer patient NK cells was less capable of differentiating the cancer stem cells. The cancer stem cells treated with patient NK cells condition media, were more susceptible to NK cell-mediated cytotoxicity and displayed lower surface expression of CD54, MHC1, and B7H1 when compared to cancer stem cells treated with healthy IFN-γ.

We also demonstrated that among all the cells we characterized to design a novel strategy to expand highly potent NK cells, osteoclasts fit best based on the NK receptors ligand expression and secreted cytokines required for NK cells expansion and activation. In this regard, we tested the cancer patient osteoclasts (OCs) for their surface expression of MHC1 and MICA/B, both were showed lower expression levels on cancer patient OCs. So, not just the NK cells receptors required for tumor recognition are lower on cancer patient NK cells, but the ligands required to increase the expression of those receptors are also down-modulated in cancer patient OCs. Next, we cultured the cancer patient NK cells with healthy donor OCs, and we were able to increase the surface expression of NKG2D on patient NK cells but, again the cytotoxic activity and IFN-γ were lower in patient NK cells. We confirmed our finding by analyzing the OCs from KRAS mutated mice and hu-BLT mice injected with tumor.

Based on our data, we were not convinced in the use of autologous NK cells to treat the cancer patients, our strategy was to use expanded allogenic NK cells as cancer cell immunotherapy. Next, we tested the expanded allogeneic NK cells in tumor implanted hu-BLT mice. With just one NK cell injection (1.5 million cells), tumor growth was inhibited in the mice. On analyzing the immune cell compartments, NK cell-mediated cytotoxicity and IFN-γ from immune cells was improved significantly. Tumors dissected from animals that received NK therapy showed differentiation profile, high MHC1, CD54 and B7H1 on their surface and lower growth in the culture when compared to tumor dissected from hu-BLT mice without NK cell immunotherapy. It has been known that mature alloreactive NK cells can be safely infused into patients with no increased incidence of graft versus host disease (GvDH). To avoid any kind of risk of GvHD, we can also consider isolating the contaminating T cells from the super-charged NK cells, and injecting the high purity NK cells.

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.

This is a novel report of the correlation and potential impact of the immune system, specifically NK cells and Interferon Gamma, in bone quality alteration related to pancreatic cancer in hu-BLT mice. For this purpose, we studied how MP2 tumors injected in hu-BLT mice affected the bone structure. Also, we analyzed how MP2 tumor-bearing mice injected with NK cells and fed with and without AJ2 influenced the bone structure.We demonstrated the suppression of NK cell cytotoxicity and decreased secretion of IFN‐γ in tumor‐bearing mice within all tissue compartments and restoration by super‐charged NK Cells. There was a remarkable correlation between the micro-CT analysis results, the induction and secretion of IFN‐γ, and bone morphology. The MP2 tumor-bearing mice injected with NK cells' cultures and fed with AJ2 presented increased bone formation with statistically significant higher trabecular bone volume compared to MP2 tumor-bearing mice group and MP2 tumor-bearing mice fed with AJ2 group, respectively. On the contrary, MP2 tumor-bearing mice showed decreased IFN‐γ and decreased bone formation. Consistent with the micro-CT findings, histological analysis of the AJ2 treatment group exhibited increased bone formation when compared to the Control and MP2 tumor groups. TRAP staining suggested more osteoclast activity and bone resorption in the MP2 tumor sample compared to the rest of the samples. Therefore, we can hypothesize that IFN‐γ induce secretion by NK cells, can inhibit tumor growth, and decreases skeletal complications of malignancy by directly acting on host cells to inhibits osteoclast formation and function. In our study, the enhanced osteolytic lesion formation in BLT tumor-bearing mice and NK cells' ability to secrete IFN‐γ to significantly reduce bone loss in tumor-bearing mice strongly supports a direct anti-osteoclastogenic role for IFN‐γ in the setting of cancer-induced bone disease. Furthermore, this report suggests that IFN‐γ can directly promote bone formation. Overall, our data demonstrated that the injection of NK cells into tumor‐bearing mice increased IFN‐γ secretion in hu-BLT mice. It also suggests that IFN‐γ has direct anti-tumor effects and potentially may suppress tumor-induced bone loss by directly targeting host osteoclasts to inhibit osteolysis. A better understanding of the bone marrow microenvironment and processes that influence tumor cell maintenance and bone growth should present opportunities to understand bone remodeling and osteolysis. To the best of our knowledge, the present work is a novel report of bone quality alteration related to pancreatic cancer in hu-BTL mice and the role of IFN‐γ secreted by NK cells in the suppression of tumor-induced bone loss and induction of bone formation.

Natural Killer (NK) cells are essential components of the immune system, responsible for combating tumors and virulent pathogens. This thesis explores the effects of supercharged NK (sNK) cells, expanded through osteoclast co-culture, on uterine cancer cell lines and in vivo tumor models. The study focuses on comparing the cytotoxicity and functionality of sNK cells expanded using live and sonicated osteoclasts, investigating their effectiveness against poorly differentiated uterine cancer cell lines, and assessing their impact on tumor progression in humanized BLT (huBLT) mouse models.In Chapter 1, we establish the functional differences between sNK cells expanded through live and sonicated osteoclasts. Our results show that live osteoclast-expanded sNK cells exhibit superior cytotoxicity and higher IFN-γ secretion, indicating a more potent immune response. Chapter 2 investigates the cytotoxicity of sNK cells against the poorly differentiated AN3CA and well-differentiated HEC-1B uterine cancer cell lines. Our findings reveal that sNK cells exhibit higher cytotoxicity against AN3CA cells, comparable to their effect on stem-like cancer cells (OSCSC), while showing lower cytotoxicity against HEC-1B cells. Real-time cytotoxicity monitoring further supports these results, highlighting the dynamic and enhanced killing capability of sNK cells. Additionally, sNK cells demonstrate superior performance compared to primary NK (pNK) cells treated with IL-2 or IL-2+sAJ4, underscoring the enhanced efficacy of osteoclast-expanded sNK cells. In Chapter 3, we assess the in vivo efficacy of sNK cells in targeting AN3CA tumor progression in huBLT mice. The results indicate a significant reduction in tumor burden in sNK-treated mice, with decreased tumor weight and volume compared to controls. Furthermore, increased IFN-γ secretion levels in sNK-treated mice suggest a reinvigoration of the immune system's capacity to combat cancer, potentially reversing tumor-induced immunosuppression. Overall, this thesis demonstrates the enhanced cytotoxic capabilities and therapeutic potential of sNK cells in targeting poorly differentiated and aggressive cancer cells. The findings support the development of sNK cell-based immunotherapies as a promising approach for treating aggressive cancers and improving patient outcomes.

Our previous research has shown that natural killer (NK) cells play a vital role as immune effectors in the selection and differentiation of various cancer stem cells and undifferentiated tumors. They achieve this through the direct killing and the release of IFN-γ and TNF-α, which inhibit tumor growth and curtail metastasis. Mechanistically, our studies have revealed that NK cells expanded by Osteoclasts display the ability to lyse CD4+ T cells while sparing CD8+ T cells, suggesting a potential selective effect of OC-activated NK cells on CD8+ T cell expansion. Consistent with these findings, we observed increased IFN-γ secretion, NK cell-mediated cytotoxicity, and higher proportions of CD8+ T cells in various tissue compartments of oral tumor-bearing hu-BLT mice following immunotherapy with OC-expanded NK cells. In this study, we focused on investigating the lytic and differentiation capabilities of supercharged NK (sNK) cells in ovarian cancer cell lines (OVCAR 4 and OVCAR 8), oral cancer cell lines (OSCC and OSCSC), and a pancreatic cancer cell line (MP 2). Our results demonstrated that sNK cells exhibited greater lysing activity against both poorly differentiated and well differentiated tumors compared to pNK cells treated with IL-2. Furthermore, the supernatants of sNK cells exhibited significantly higher levels of IFN-γ and induced differentiation by an increase in MHC class1 expressions in all the cell lines tested, except for OVCAR 8. Lastly, we observed a downmodulation of NKG2A and CD94 inhibitory receptors on sNK cells, suggesting potential alterations in their functional properties.

NK mediated expansion of T-cells preferentially expands CD8+ but not the CD4+ subsets in both healthy and cancer donors. This results in a lower CD4/CD8 ratio. While NK Cells target CD4+ helper T-cells they do not target their CD8+ cytotoxic counterparts. This study also reveals that Cancer patients NK cells have reduced cytotoxic ability and secrete much less IFN-γ than healthy donors and Osteoclast and sAJ2 expanded super-charged NKs intended for immunotherapeutic use from healthy donors expand more immune cells at a faster rate. NK immunotherapy was studied in combination with supplementation of probiotic bacteria in the humanized mouse model. NK immunotherapy increased CD8+ cytotoxic T cell population in immune tissue compartments and Increased immune infiltration in the tumor microenvironment, while probiotic supplementation further enhanced these effects, resulting in decreased tumor burden. This novel expansion technique can provide a non-invasive and highly effective treatment for the treatment of oral squamous cell carcinoma and other tumorigenic cancers.