NKcell-based Cancer Immunotherapy Research Articles

Abstract 5519: Enlarged metastasis-free tumor-draining lymph node is a marker of tumor invasion in local tissues with an active NK cell-mediated immunity

Abstract Background: Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for about 10% of all new cancer cases. The tumor-draining lymph node (TDLN) is a key site for the development of anti-tumor immunity that has been reported to be involved in both CD8+ T and NK cells. As a major role of innate immunity, NK cells can produce IFN-γ, perforin and granzyme to eliminate tumor cells in the early stages of cancer development. This study aimed to investigate the difference of clinicopathological and immunological profiles in local tissues between CRC patients with enlarged and non-enlarged metastasis-free TDLNs. Methods: The patient cohort was obtained from the NEPDC cohort (No. 2015CB554000). A total of 45 patients with pathologically confirmed stage I-II colorectal cancer were included in this study. Based on the MRI, these patients were divided into enlarged and non-enlarged TDLN groups (lymph nodes >/= 5mm and lymph nodes < 5mm). We collected the tumor tissue and tumor-adjacent normal tissue that was 2 cm away from the tumor lesion. The genomic DNA of the tumor and tumor-adjacent normal tissues was treated with bisulfite and analyzed with the EPIC methylation array. The MeTMEC algorithm based on the methylation profile estimated subsequent cell abundance in these patient tissues. Results: The study sample was comprised of 22 enlarged TDLN and 23 non-enlarged TDLN patients. There were 4 T1-T2 and 18 T3-T4 patients in the enlarged TDLN group, with 12 T1-T2 and 11 T3-T4 patients in the non-enlarged TDLN group. The proportion of T3-T4 patients in the enlarged TDLN group was higher than that in the non-enlarged TDLN group (p=0.017, Chi-squared test), suggesting an advanced invasion in the local tissue of patients with enlarged TDLN. Then, the abundance of monocytes, dendritic cells, macrophages, neutrophils, eosinophils, Treg cells, naive T cells, memory T cells, CD8+ T cells, NK cells, and B cells in the tumor and tumor-adjacent normal tissues were inferred with the MeTMEC algorithm by using DNA methylation profile. In the T1-T2 subset, we found that the infiltration of NK cells in the tumor-adjacent normal tissues was significantly more abundant in the enlarged TDLN group compared with the non-enlarged TDLN group (p=0.021, t-test), suggesting an active immune response of NK cells for potential local tumor control. No significant difference in other immune cells was observed between the two groups. Conclusion: Enlarged TDLN indicates an advanced invasion of tumors in local tissues with an active NK cell-mediated immunity. Our findings suggest that enlarged metastasis-free TDLN might be a promising marker for NK cell-based cancer immunotherapy. Key words: Colorectal Cancer, Lymph Nodes, Natural Killer Cells Citation Format: Zixiao Wan, Huichuan Yu. Enlarged metastasis-free tumor-draining lymph node is a marker of tumor invasion in local tissues with an active NK cell-mediated immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5519.

Tanshinone IIA enhances susceptibility of non-small cell lung cancer cells to NK cell-mediated lysis by up-regulating ULBP1 and DR5.

Natural killer (NK) cells have a great potential in cancer immunotherapy. However, their therapeutic efficacy is clinically limited owing to cancer cell immune escape. Therefore, it is urgently necessary to develop novel method to improve the antitumor immunity of NK cells. In the present study, it was found that the natural product tanshinone IIA (TIIA) enhanced NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells. TIIA in combination with adoptive transfer of NK cells synergistically suppressed the tumor growth of NSCLC cells in an immune-incompetent mouse model. Furthermore, TIIA significantly inhibited the tumor growth of Lewis lung cancer (LLC) in an immune-competent syngeneic mouse model, and such inhibitory effect was reversed by the depletion of NK cells. Moreover, TIIA increased expressions of ULBP1 and DR5 in NSCLC cells, and inhibition of DR5 and ULBP1 reduced the enhancement of NK cell-mediated lysis by TIIA. Besides, TIIA increased the levels of p-PERK, ATF4 and CHOP. Knockdown of ATF4 completely reversed the up-regulation of ULBP1 and DR5 by TIIA in all detected NSCLC cells, while knockdown of CHOP only partly reduced these enhanced expressions in small parts of NSCLC cells. These results demonstrated that TIIA could increase the susceptibility of NSCLC cells to NK cell-mediated lysis by up-regulating ULBP1 and DR5, suggesting that TIIA had a promising potential in cancer immunotherapy, especially in NK cell-based cancer immunotherapy.

MiRNAs in NK Cell-Based Immune Responses and Cancer Immunotherapy.

The incidence of certain forms of tumors has increased progressively in recent years and is expected to continue growing as life expectancy continues to increase. Tumor-infiltrating NK cells may contribute to develop an anti-tumor response. Optimized combinations of different cancer therapies, including NK cell-based approaches for targeting tumor cells, have the potential to open new avenues in cancer immunotherapy. Functional inhibitory receptors on NK cells are needed to prevent their attack on healthy cells. Nevertheless, disruption of inhibitory receptors function on NK cells increases the cytotoxic capacity of NK cells against cancer cells. MicroRNAs (miRNAs) are small non-coding RNA molecules that target mRNA and thus regulate the expression of genes involved in the development, maturation, and effector functions of NK cells. Therapeutic strategies that target the regulatory effects of miRNAs have the potential to improve the efficiency of cancer immunotherapy. Interestingly, emerging evidence points out that some miRNAs can, directly and indirectly, control the surface expression of immune checkpoints on NK cells or that of their ligands on tumor cells. This suggests a possible use of miRNAs in the context of anti-tumor therapy. This review provides the current overview of the connections between miRNAs and regulation of NK cell functions and discusses the potential of these miRNAs as innovative biomarkers/targets for cancer immunotherapy.

The role of oxidative stress in NK-cell based cancer immunotherapy

Immunooncology gained great attention in cancer treatment over the last few years. Adoptive cell therapy is a type of immunotherapy that uses cytotoxic lymphocytes or NK cells. NK cells precisely recognize and kill cancer cells without affecting healthy cells. These properties make NK cells an attractive strategy with potential therapeutic applications. NK cells can be further modified with chimeric antigen receptors (CAR). Currently, several dozen clinical trials with NK cells are registered for cancer treatment, including several CAR-NK cells or NK-92 cell line modified with CAR. Despite the undeniable success of immunotherapy in recent years, many concerns still remain unresolved. One of the challenges for the effective immunotherapy is the immunosuppressive tumor environment. Numerous studies indicate that persistent oxidative stress, an important element associated with cancer, inhibits NK cell activity. However, the exact mechanisms responsible for protecting NK cells against suppressive oxidative stress are not identified. Therefore, in my doctoral dissertation co-financed by the Scientific Polpharma Foundation doctoral scholarship I focused on investigating and identifying mechanisms protecting NK cells against oxidative stress. The obtained results indicate that NK cells are the most sensitive to the oxidative stress, when compared withother immune cells. Moreover, inhibition of PRDX1-TXN1-TXNRD1 system impairs NK cell effector functions, while the increase of PRDX1 significantly improves the survival of NK cells and CAR-NK cells in the presence of H<sub>2</sub>O<sub>2</sub>, resulting in improved effectiveness of NK cells under the oxidative stress conditions.

Enhancement of NK cell-mediated lysis of non-small lung cancer cells by nPKC activator, ingenol 3,20 dibenzoate.

The IFN-γ production is crucial for NK cell-mediated lysis of cancer cells. Thus increasing the IFN-γ production by NK cells may be an ideal strategy to improve their tumoricidal effect. Since the focus on new drug development has shifted towards natural products, limited information is out there about natural products that enhance the IFN-γ production by NK cells. In this study, through a high-throughput screening, we have identified a natural product ingenol 3,20 dibenzoate (IDB), an activator of tumor suppressor protein kinase C (PKC) isozymes, could increase the IFN-γ production and degranulation by NK cells, especially when NK cells were stimulated by non-small lung cancer (NSCLC) cells. IDB also significantly enhanced the NK cell-mediated lysis of NSCLC cells. Furthermore, PKC inhibitor, sotrastaurin abrogated IDB-induced IFN-γ production, degranulation and cytotoxicity, but did not affect IFN-γ production by NK cells without IDB treatment and NSCLC cell stimulation. The IFN-γ neutralization reversed the IDB-induced enhancement of NK cell mediated killing. In conclusion, our study indicated that IDB enhanced NK cell-mediated lysis of NSCLC cells is dependent on specific PKC mediated IFN-γ production and degranulation. Thus, IDB may have a promising application in clinic for NK cell-based cancer immunotherapy.

Perturbation of NK cell peripheral homeostasis accelerates prostate carcinoma metastasis

The activating receptor NK cell group 2 member D (NKG2D) mediates antitumor immunity in experimental animal models. However, whether NKG2D ligands contribute to tumor suppression or progression clinically remains controversial. Here, we have described 2 novel lines of "humanized" bi-transgenic (bi-Tg) mice in which native human NKG2D ligand MHC class I polypeptide-related sequence B (MICB) or the engineered membrane-restricted MICB (MICB.A2) was expressed in the prostate of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous carcinogenesis. Bi-Tg TRAMP/MICB mice exhibited a markedly increased incidence of progressed carcinomas and metastasis, whereas TRAMP/MICB.A2 mice enjoyed long-term tumor-free survival conferred by sustained NKG2D-mediated antitumor immunity. Mechanistically, we found that cancer progression in TRAMP/MICB mice was associated with loss of the peripheral NK cell pool owing to high serum levels of tumor-derived soluble MICB (sMICB). Prostate cancer patients also displayed reduction of peripheral NK cells and high sMIC levels. Our study has not only provided direct evidence in "humanized" mouse models that soluble and membrane-restricted NKG2D ligands pose opposite impacts on cancer progression, but also uncovered a mechanism of sMIC-induced impairment of NK cell antitumor immunity. Our findings suggest that the impact of soluble NKG2D ligands should be considered in NK cell-based cancer immunotherapy and that our unique mouse models should be valuable for therapy optimization.

Comparison of Level of NKG2D Ligands between Normal and Tumor Tissue Using Multiplex RT-PCR

The abilities of NKG2D ligands to specifically mark stressed or transformed cells and activate NK cells suggest the possibility that the expression levels of NKG2D ligands in cancers may be helpful to predict the efficacy of NK cell-based cancer immunotherapy. Therefore, a multiplex RT-PCR was developed and used for rapid and simultaneous analysis of the expression level of NKG2D ligands in cancer cells and tissues. With total RNAs isolated from various cancer cell lines, the multiplex RT-PCR revealed various expression patterns of NKG2D ligands. With total tissue RNAs, the gastrointestinal tumors showed consistently increased NKG2D ligands, compared with adjacent normal tissues. However, NKG2D ligands were not always consistently increased in tumor tissues and expression patterns of NKG2D ligands were heterogeneous between patients, especially in breast and lung cancers. In addition, expression patterns of NKG2D ligands were very similar between various paired primary and their multidrug-resistant/metastatic cells, except MCF-7 sublines. These results demonstrated that the multiplex RT-PCR might be a useful diagnostics to detect the expression levels of NKG2D ligands in tissues as well as cells, and suggested that the gastrointestinal tumors might be good candidates for NK cell-based cancer immunotherapy, since it showed significantly higher levels of NKG2D ligands than adjacent normal tissues.