Super-Charged Natural Killer Cells: A Promising Immunotherapeutic Strategy for Oral Cancer

Abstract

NK cells have traditionally been classified as effectors of innate immunity, even though they also exhibit some features of adaptive immunity such as memory. NK cells contribute to the lysis and growth inhibition of cancer, mediating direct cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) and regulating the functions of other immune cells, respectively. NK cells regulate the function of other immune cells via the release of inflammatory cytokines and chemokines. Currently, NK cell therapeutics in oral cancer have been less efficient due to several limitations, as follows: (a) lower percentages of NK cells in peripheral blood immune cells; (b) limited survival and decreased function of NK cells, especially in the tumor microenvironment; and (c) a lack of tools or methodologies to expand and activate NK cells to the levels that are required for the effective targeting of oral cancer. To overcome these limitations, we established and demonstrated a novel technology for activating and expanding highly functional NK cells coined as supercharged NK (sNK) cells. This review summarizes the characteristics of sNK cells and highlights their superior anti-cancer activity when compared to primary activated NK cells.

1. Introduction and Background: Oral Cancer and Natural Killer Cells

Oral cancer, which represents approximately three percent of all cancers diagnosed or about 54,000 new cases annually in the United States, is the most prevalent subset of head and neck cancer; it has a 50% overall survival rate at 5 years and there are no effective therapeutic modalities [1,2,3,4,5,6,7]. Oral cancer progression consists of a series of histopathological changes, including hyperplasia, dysplasia, carcinoma in situ, and, lastly, oral cancer [8]. Oral cancer has an epithelial origin and is found to be associated with several etiological factors, including genetic, epigenetic, microbial, habitual, geographical location, or ethical groups [9,10]. Many factors contribute to the onset of oral cancer, including low maintenance of oral hygiene, oral ulcers, human papillomavirus (HPV), mucosal leukoplakia, alcoholism, and the long-term use of tobacco [11,12,13]. Oral cancers are more prevalent in men due to their increased use of tobacco and alcohol [14]. There are very few treatment options for stage IV oral cancer, especially those associated with metastasis [15]. Also, oral cancer stem-like cells (CSCs) have downmodulated MHC-class I and are difficult to target with chemotherapeutic, radiotherapeutic, and T cell-based immunotherapeutic strategies; these CSCs also correlate negatively with oral cancer inhibition and therapeutic response [16,17]. Therefore, there is a need for effective treatment. In this regard, NK cells are known to target these aggressive tumors and can be used in patients to eliminate these tumors. This is a clear advantage of this therapy for hard-to-cure oral tumors. In T cell therapies, a graft vs. host disease can complicate the therapy. Fortunately, such a complication does not exist with NK cells, since they do not mediate graft vs. host disease [18,19].

Natural killer (NK) cells were found to play a crucial role in cancer inhibition due to their effector functions, including direct cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), as well as by regulating or activating the anti-cancer function of other immune effectors via NK cell-secreted inflammatory cytokines and chemokines [20,21,22]. NK cells induce greater lysis in CSCs expressing a low surface expression of MHC-class I, CD54, and PD-L1 compared to differentiation tumors with higher surface expression levels of MHC-class I, CD54, and PD-L1 [23,24]. Several in vitro studies have classified oral tumor cell lines as stem-like and differentiated tumors. Among these cell lines, we have extensively studied OSCSCs (oral squamous carcinoma stem-like cells) and OSCCs (oral squamous cell carcinomas) [25,26]. Stem-like OSCSC oral cancer cell lines were found to express high levels of CD44, EpCAM, CD26, and CD338, as well as expressing low levels of CD166, MHC-class I, CD54, and B7H1; however, the differentiated OSCC oral cancer cell lines were found to express higher levels of B7H1, CD54, MHC-class I, and EGF-R, and low levels of CD44 and CD133 on their surface [25,27,28,29]. We have found OSCSCs to be excellent targets of NK cell-mediated cytotoxicity, whereas OSCCs are significantly resistant to NK cell-mediated cytotoxicity [25,28,29]. Cytokines secreted from NK cells, particularly IFN-γ and TNF-α, induce differentiation in CSCs. NK cell-secreted cytokines induced significant differentiation in OSCSCs, resulting in their growth inhibition [16,24,28,30,31]. Upon differentiation, tumors proliferate and metastasize at a minimal rate. To sum up, NK cell-mediated lysis directly kills oral tumors, and NK cell-mediated oral tumor differentiation inhibits tumor growth and spread. Also, NK cell-induced differentiation in oral tumors enhances the efficacy of therapeutics such as CD8+ T immunotherapies, radiation, chemotherapies, checkpoint inhibitors, and antibodies that target differentiated tumor subsets. The efficacy of NK cell-based therapeutics has been shown in several solid tumors [24,32,33,34,35,36,37,38,39,40,41,42,43,44].

Thus, the optimal function of NK cells or NK cell-based therapies directly affects the prognosis of cancer patients and also reduces the chances of tumor relapse by targeting CSCs [16,30,31,43,44,45,46,47,48]. Increased numbers and the anti-cancer activity of peripheral blood-derived NK cells, as well as increased NK cell infiltration in the tumor tissues, play significant roles in improving cancer patients’ prognosis [45,46,47,48]. Studies from our laboratory and several other studies have reported reduced numbers and anti-cancer activities in NK cells from cancer patients [49,50,51,52]. Reduced percentages and the defective function of NK cells were found in both tumor tissue and peripheral blood-derived immune subsets of cancer patients [50,51]. Several technologies have been introduced to expand and activate NK cells, which allows for the production of a large number of NK cells as therapeutics for cancer patients [53,54,55,56,57]. We have previously demonstrated superior technology to induce significant cell expansion and anti-cancer functional activation in NK cells. Due to the superior anti-cancer function of these expanded NK cells, they were named supercharged NK (sNK) cells [52,58,59,60].

In this review, we discuss the characteristics of sNK cells, including their superior anti-cancer potential compared to primary peripheral blood-derived NK cells, particularly in inducing direct cytotoxicity or ADCC, as well as inducing differentiation in oral tumors. The increased anti-cancer activity of sNK cells was seen in both in vitro and in vivo studies. A single infusion of sNK cells in vivo stopped disease progression and induced the in vivo differentiation of oral tumors. Furthermore, we demonstrate that oral tumors differentiated by sNK cells become more susceptible to chemotherapeutic drugs as compared to those differentiated by primary activated NK cells. Finally, we demonstrate that sNK cells survive and retain their function in oral tumors, whereas primary NK cells lose their function and cannot survive in tumor tissue.

2. Supercharged NK (sNK) Cells

Supercharged NK (sNK) cell generation is performed by co-culturing peripheral blood-derived IL-2 and anti-CD16 mAb-activated NK cells with osteoclasts (OCs) and sonicated probiotic bacteria (sAJ2) (Figure 1). Osteoclasts were found to exhibit a positive surface expression of MICA/B, KLRG1, and ULBPs; these are ligands for NK cell-activating surface receptors. Osteoclasts also secrete a wide range of cytokines and chemokines, including IL-12, IL-15, IFN-γ, and IL-18, which are known to activate NK cells [61,62]. Thus, surface markers and the secreted factors of osteoclasts play a significant role in cell expansion and the functional activation of NK cells. Gram-positive probiotic bacteria Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus bulgaricus were selected based on their activation in NK cells. The combination of these probiotic treatments in NK cells was found to increase cytokine secretion by NK cells, including IFN-γ, which could facilitate the signals required for NK cell expansion [28,39,52,63]. Therefore, a combination treatment of both probiotics and OCs in NK cells results in the induction of signals participating in cell expansion and the functional activation of NK cells, generating sNK cells. sNK cells have demonstrated increased lifespan, cell expansion, cytotoxicity, and the secretion of cytokines; these sNK cell characteristics ultimately result in the increased differentiation and killing of oral cancer both in vivo and in vitro [52,58,59]. sNK cells’ characteristics and increased functions are discussed in the next sections of this review.

3. Superior Anti-Cancer Activity in Oral Tumors by sNK Cells

We have previously identified and classified primary oral tumor cell lines based on stem-like or differentiated phenotypes as OSCSCs (oral squamous carcinoma stem-like cells) and OSCCs (oral squamous cell carcinomas) [25]. The stem-like oral tumor OSCSCs were found to exhibit high expression levels of CD338, CD44, CD26, and EpCAM, as well as low expression levels of CD54, CD166, MHC-class I, and PD-L1 on their surface; they also expressed an increased sensitivity to NK cell-mediated killing [25]. In contrast, the differentiated counterpart OSCCs were found to express lower levels of CD133 and CD44 and higher levels of CD54, EGF-R, PD-L1, and MHC-class I on their surface, as well as being resistant to NK cell-mediated killing.

sNK cells induce cytotoxicity in oral CSCs as well in differentiated oral tumors. We observed significantly higher levels of cytotoxicity by sNK cells when compared with primary IL-2-activated NK cell-mediated cytotoxicity against OSCSCs. Increased levels of ADCC against oral tumors were also seen in sNK cells. It was found that differentiation in tumors results in their resistance to NK cell-mediated cytotoxicity [25]; however, significantly higher levels of cytotoxicity were seen when sNK cells were used as effectors against OSCCs. These sNK cell characteristics are critical to target the heterogenous population of tumors, especially tumors expressing higher MHC-class I, which are known to escape primary NK cell-mediated killing.

sNK cells induce higher levels of differentiation in oral tumors (Figure 1). Primary NK cell-secreted cytokines, such as IFN-γ and TNF-α, were found to play a key role in inducing differentiation in oral tumors. Significantly low levels of tumor growth and metastasis were seen in differentiated tumors compared to stem-like tumors; additionally, differentiation in tumors increases their sensitivity to chemotherapy drugs [24,64]. sNK cells were found to release significantly increased levels of cytokines compared to primary NK cells [52,60,65]. Primary NK cells express cytokine release or cell survival for 5–7 days, whereas sNKs survived and maintained cytokine secretion for 27–36 days [52,60,65]. sNK cell-secreted cytokines are superior at inducing differentiation in oral tumors compared to cytokines secreted by primary NK cells [52,60]. Since 90% of head and neck malignant neoplasms express differentiated phenotypes, sNK cells could effectively eradicate the majority of oral cancers [66]. Several studies have demonstrated that cancer therapeutics like CD8+ T cell immunotherapies, antibodies, checkpoint inhibitors, radiation, and chemotherapies have failed to treat CSC tumors, but are efficient against differentiated tumors [24,38,39,40,41]. This suggests that sNK cell-based immunotherapy alone or its combination with other therapeutics is the future of oral cancer therapeutics.

4. Infusion of sNK Cells Reversed Disease Progression in Oral Tumor-Bearing Humanized Mice

The sNK cells’ in vivo efficacy against oral tumors was investigated using humanized-BLT (hu-BLT) mice; this mouse model was found to be one of the best humanized pre-clinical models, representing a full repertoire of the human immune system [67,68]. Hu-BLT mice provide a platform to validate human therapeutics when interactions are involved between human immune cells and the tumor microenvironment. Oral CSC OSCSCs were implanted in the oral cavity of hu-BLT mice one week after tumor implantation (once the tumor was established); then, mice were infused with sNK cells. Oral tumor-bearing mice infused with sNK cells therapeutics expressed slight/no tumor burden and demonstrated an increased lifespan compared to those with no treatment. sNK cell therapeutics induced a significant reduction in oral tumors, whereby the tumors dissected from sNK-treated groups were significantly smaller in size compared to the untreated group [39].

sNK cells demonstrated the in vivo differentiation of oral tumors, as tumors dissected from the sNK therapy group grew at a very minimal rate ex vivo; expressed higher levels of PD-L1, CD54, and MHC-class I on the surface; and were resistant to primary NK cell-mediated cytotoxicity [24,25,39]. sNK therapeutics resulted in an up to ninefold increase in the percentages of immune cells in the tumor microenvironment of the infused group as compared to tumors from the untreated group [39]. sNK therapy also increased the percentages of CD3+CD8+ T cells in the immune cells of the spleen, peripheral blood, and bone marrow of sNK cell-treated mice [39].

It was found that immune function is reduced or defective in cancer patients [49,50,51,52]. Similarly, an inhibition in the immune cell function was seen in oral tumor hu-BLT mice. We observed a 60–90% restoration in the cytotoxic and cytokine secretion levels of immune cells in various tissue compartments of oral tumor hu-BLT mice treated with sNK cells [39]. These findings suggest that the mechanisms involved in sNK cell therapy are as follows: (a) selection/direct killing of the tumor, (b) differentiation of tumors, (c) recruiting immune cells or increasing immune infiltration in the tumor, and (d) activation in CD8+ T cells [39]. We observed a similar efficacy of sNK cell therapy irrespective of whether they were autologous or allogeneic sNK cells [60].

sNK cell therapy combined with chemotherapy against oral tumors in hu-BLT mice demonstrated a slightly better efficacy compared to sNK cells alone [60]. A further reduction in tumor load and a greater restoration of immune cell function was observed when sNK therapy was combined with chemotherapy [60]. When checkpoint inhibitor PD-1 antibody was combined with sNK cells, a reduced tumor burden and an increased immune cell function restoration was seen in tumor-bearing hu-BLT mice [60]. Combining sNK cells with chemo-drugs or check-point inhibitors could be highly effective in treating oral cancer, as sNK cells could directly kill CSCs and differentiated tumors in addition to inducing differentiation in tumors, allowing sNK and chemotherapy combined effects against those tumors [60]. This concludes the significant potential of sNK cells to treat oral cancer; combining sNK cell treatment with other therapies will provide successful therapeutics in oral cancer patients.

5. Increased Survival and Augmented Function of sNK Cells in Oral Tumor Microenvironments

Tumor microenvironment-induced suppressed function or cell death in primary NK cells was demonstrated, and reduced NK cell function was also seen when NK cells were co-cultured with tumors in vitro [69]. When primary NK cells and sNK cells were exposed to oral tumors dissected from hu-BLT mice or with OSCSCs, in both cases, primary NK cells expressed minimal survival and lost function, but sNK cells survived longer and retained their function (Figure 2). This indicates the persistence or continued anti-cancer effect of sNK cells in the tumor microenvironment, validating the fact that this approach is best for treating oral tumors.

6. Molecular Mechanism Underlying Superior Anti-Cancer Function of sNK Cells

The increased survival and retained function of sNK cells in the tumor microenvironment is due to higher expression levels of anti-apoptotic proteins, including BCL2, and decreased expression levels of pro-apoptotic proteins; this allows sNK cells to resist the induction of cell death and a loss of cytotoxicity within the tumor microenvironment (Figure 3). In addition to oral cancer, sNK cells induce significant levels of anti-cancer activity against hematological malignancies and pancreatic, hepatic, and glioblastoma tumors [39,52,60,61,65]. The increased cytotoxic function in sNK cells is due to the increased expression levels of cytotoxic-associated granules granzyme B and cathepsin C. Also, the level of Trail expression was found to be elevated in the sNK cells from the single-cell transcriptomic analysis (Figure 3). The increased regulatory function of sNK cells is due to the increased gene expression levels of STAT1, STAT2, IRF1, IRF7, IRF9, JUN, MYC, BHLHE40, and H1F1A (Figure 3). Increased sNK cell expansion is maintained because of increased proliferation-associated genes and proteins; the majority of cells are at the active cycling stage (Figure 3). sNK cells express higher levels of memory-associated genes. sNK cells demonstrated a higher expression of activating receptors CD16, CD56, Nkp30, Nkp44, Nkp46, NKG2D, and CD54, and a downmodulation of inhibitory receptor NKG2A ([52] and manuscript in press). Thus, the increased secretion of IFN-γ and TNF-α [60], as well as cytotoxic function, cell survival/expansion, regulatory function, and memory phenotype, contribute to the increased activity of sNK cells to lyse both stem-like and differentiated tumors [70]. A summary of mechanisms contributing to their superior anti-cancer activity, highlighting the enhanced characteristics of sNK cells and illustrating how these advanced traits make them more effective in targeting and eliminating tumor cells, is shown in Figure 3.

7. Conclusions

sNK cells induce significantly higher cytotoxicity against oral tumors compared to primary NK cells, as well as both CSCs and differentiated tumors. Also, sNK cells induce higher differentiation in oral tumors. The differentiation of oral tumors makes them sensitive to chemotherapeutic drugs. sNK cell efficacy as a cancer therapeutic was seen in oral tumor-bearing hu-BLT mice. Tumors resected from the group that received sNK cell therapy expressed a differentiated phenotype and grew minimal ex vivo tumors. Mice that received sNK cell therapy developed significantly small tumors. Our pre-clinical studies relating to sNK cell therapy in oral cancers have demonstrated the promising anti-cancer effects of this immunotherapy (Figure 4). sNK cells are very safe, as determined by the clinical trials of patients both in adult and pediatric populations. Indeed, the patients have reported an increase in energy, having a clear mind, and the loss of aches or pains after receiving sNK therapy. Because sNK cells can target both MHC-deficient and -competent tumors, they behave like CD8+ T cells [71,72]. The only disadvantage we can find for sNK therapy is the discomfort at the site of injection. The studies reviewed in this article suggest that sNK cell-based therapy alone or combined with other therapeutics is the future of oral cancer therapeutics.