Tumor-infiltrating Natural Killer Cells Research Articles

Developing natural killer cells producing hypoxia-inducible cytokines for effective immunotherapy against solid tumors.

Abstract Natural Killer (NK) cells are a promising means for adoptive immunotherapy for cancers. NK cell therapies against hematological cancers such as acute myeloid leukemia have demonstrated good efficacy. However, the poor effector function of tumor-infiltrating NK cells limits the potential of NK cells against solid tumors. Accumulating data suggests that hypoxia in the tumor microenvironment (TME) suppresses the effector function of NK cells. One of the immunosuppressive features of hypoxia is the generation of adenosine, a metabolite that highly suppresses NK cell cytotoxicity and proliferation. Although engineering NK cells to overexpress stimulatory cytokines before the adoptive immunotherapy has demonstrated promising potential for cancer therapy, it raises a concern that high systemic levels of cytokines may lead to cytokine-mediated pathology. Therefore, we hypothesize that driving overexpression of the potent stimulatory cytokines exclusively in the TME would circumvent systemic toxicity. An attractive strategy to achieve this goal is to take advantage of the hypoxic condition, a key characteristic of the TME. Hypoxia-inducible factor 1 alpha (HIF1-α) plays a pivotal role in expressing hypoxia-responsive genes by associating with promoters containing hypoxia response element (HRE). By mimicking the biological system, HRE-induced cytokine expression vectors were generated and tested in NK cells. The expression was inducible under hypoxic conditions and was reversible upon re-oxygenated conditions. Our results suggest that the hypoxia-inducible vector system can be beneficial to trigger TME specific cytokine expressions in NK cells and provide insight into efficient immunotherapy against solid tumors.

Abstract IA-024: Sirt2: A tumor suppressor or promotor?

Abstract SIRT2, an NAD+-dependent histone deacetylase, has been shown to play a pivotal role in various physiological processes, however, its role in cancer is currently controversial. In recent years, SIRT2 has been described as both a tumor suppressor and oncogene with divergent expression and function in various malignancies. Using murine xenograft melanoma models, our results suggest increased systemic expression of SIRT2 promotes tumor progression. In this study, SIRT2-overexpressing mice exhibited enhanced tumor growth and larger tumor volumes compared to their wild-type littermates. Mechanistically, systemic overexpression of SIRT2 reduces the number of tumor-infiltrating natural killer (NK) cells and suppresses NK cell activation and proliferation within tumor microenvironment (TME). Furthermore, despite the effect of increased tumor growth rate and tumor volume in wild-type littermate mice, NK cell depletion does not affect that in SIRT2-overexpressing mice. Lastly, pharmacological inhibition of SIRT2 increases NK cell tumor infiltration and suppresses melanoma tumor growth in mice. The findings of this study identify a dynamic functional interaction between systemic SIRT2 and NK cell activity, which promotes melanoma tumor progression. Given the recent renewed interest in NK-cell-mediated immunotherapy response, SIRT2 could present a new opportunity to mediate immunotherapy response and resistance. Citation Format: Fen Xia. Sirt2: A tumor suppressor or promotor? [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-024.

Abstract LT006: NK cells from prostate cancer patients acquire a pro-angiogenic phenotype and polarize macrophages towards a M2-like/TAM subset

Abstract Angiogenesis has been shown to play a crucial role in prostate cancer (PCa) progression in several preclinical models, in the clinic, pro-angiogenic factors have been shown to correlate with, Gleason score, metastasis and prognosis in PCa. Plasma levels of VEGF were shown to be higher in patients with metastatic PCa than those with localized disease. The natural killer (NK) cells have been found to be non cytolytic, immunosuppressed in solid cancers. Our group and others demonstrated that tumor infiltrating NK cells in lung and colorectal cancer acquire a pro-angiogenic phenotype. These cells are similar to NK cell within the decidua (dNK), and functionally support angiogenesis in a TGFb and Stat3-dependent manner. A similar subset is present in the peripheral blood of oncology patients. We characterized systemic peripheral blood NK cells (pNK) from PCa patients and their interaction with endothelial cells and macrophages. NK cell subset distribution was investigated by multicolor flow cytometry (FC) for surface antigens on peripheral blood samples PCa patients. Conditioned media (CM) of three different PCa cell lines were used to polarize the healthy donors pNK cells. CM from FACS-sorted PCa pNKs were used for functional studies of angiogenesis, on human umbilical-vein endothelial cells (HUVEC), studies for macrophage recruitment (migration assay on Boyden chambers) and polarization. Molecular studies were performed by real time PCR (qPCR) on HUCECs and macrophages exposed to CM of pNKs. Protein arrays were performed to characterize the secretome on FACS-sorted pNKs. We found that PCa pNKs acquire a pro-angiogenic/decidual-like CD56brightCD9+CD49a+CXCR4+ phenotype. The same phenotype was observed from healthy donors pNK cells exposed to CMs of three different PCa cell lines. These results were confirmed also exposing heathy-donor derived NK cells to conditioned media of 3 different prostate cancer cell lines (PC-3, DU-145, LNCaP), together with increased production of CXCL8, Angiogenin, Angiopoietin1 and reduced production of TNFa, IFNg and GranzymeA. CMs from pNK cells support the formation on capillary-like structures on HUVEC, together with increased expression of VEGF, VEGR-2, CXCL8, ICAM-1 and VCAM-1. Although in male patients, PCa NK cells resemble decidual-like ones. Finding these cells polarized in PB suggests that they could be used as non-invasive marker of inflammation in prostate cancer. Secretome analysis revealed the ability of pNK cells release pro-angiogenic factors (CLXL8, MMP-1, MMP-9; uPAR) and cytokines/chemokines involved in macrophage recruitment (CCL1, CCL2, CCL5, CCL7,CCL13, CXCL1, CXCL11) and M2-like polarization (IL-10). CMs from pNK can recruiting THP-1 monocyte and polarize THP-1 macrophage towards CD206, Arginase1, CXCL8-expressing M2-like/TAM phenotype. Our results place PCa pNK cells interaction with endothelial cells and via macrophage polarization able in supporting angiogenesis in PCa patients. Citation Format: Adriana Albini, Denisa Baci, Matteo Gallazzi, Federico Dehò, Angelo Naselli, Andrea Guernieri, Lorenzo Mortara, Douglas M. Noonan, Antonino Bruno. NK cells from prostate cancer patients acquire a pro-angiogenic phenotype and polarize macrophages towards a M2-like/TAM subset [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT006.

Prognostic Value of Natural Killer Cells Besides Tumor-Infiltrating Lymphocytes in Breast Cancer Tissues

BackgroundEach subgroup of immune cells has a different prognostic role in breast cancer; however, the prognostic impact of tumor-infiltrating natural killer cells (TINKs) is still not well established. Our aim was to assess the prognostic impact of natural killer (NK) cells in breast carcinomas. Materials and MethodsNK cells infiltration were assessed by immunohistochemistry (IHC). Statistical analyses were performed to evaluate the correlation of NK cells with clinical-pathological features and outcome. ResultsCD56 IHC was realized in 126 patients. NK cells infiltration showed significant and positive association with tumor high Scarff-Bloom-Richardson (SBR) grade. NK cells were significantly associated with HER2-positive breast cancer and triple-negative breast cancer subtypes. Analyses showed significant and inverse correlation with progesterone and estrogen receptors expression status. High NK cells were significantly related to high Ki-67 labeling index. Our data showed that high NK cells infiltrate was significantly associated with tumor-infiltrating lymphocytes in breast cancer tissues.At a median follow-up of 5.5 years, high CD56 expression (≥ 5 cells/10 high power field) was associated significantly with a good overall survival and with good disease-free survival. ConclusionIn this study, we assessed the important prognostic role of TINKs in breast carcinomas, which seems to be evident despite its association with aggressive pathological features. Thus evaluation of NK cells can be standardized and integrated in daily routine.

Tumor-Infiltrating Natural Killer Cells.

Because of their potent antitumor activity and their proinflammatory role, natural killer (NK) cells are at the forefront of efforts to develop immuno-oncologic treatments. NK cells participate in immune responses to tumors by killing target cells and producing cytokines. However, in the immunosuppressive tumor microenvironment, NK cells become dysfunctional through exposure to inhibitory molecules produced by cancer cells, leading to tumor escape. We provide an overview of what is known about NK tumor infiltration and surveillance and about the mechanisms by which NK cells become dysfunctional. SIGNIFICANCE: The functions of tumor-infiltrating NK cells may be impaired. This review aims to describe the various mechanisms by which tumors alter NK-cell functions.

Anacardic 6-pentadecyl salicylic acid induces apoptosis in breast cancer tumor cells, immunostimulation in the host and decreases blood toxic effects of taxol in an animal model

Many antineoplastic agents induce myelosuppression and leukopenia as secondary effects in patients. The development of anticancer agents that simultaneously provoke antitumor immune response represents an important therapeutic advance. The administration of 6-pentadecyl salicylic acid (6SA) contributes to the antitumor immunity using 4T1 breast cancer cells in Balb/c female mice, with Taxol as a positive control and in cotreatment with 6SA (6SA + Taxol; CoT).Our results show that 6SA reduces tumor volume and size by inducing caspase-8-mediated apoptosis without reducing tumor infiltrated lymphocytes. Also, 6SA reduced lung metastasis and increased the proportion of immune cells in blood, lymph nodes and bone marrow; more evidently, in the proportion of tumor-infiltrated natural killer (NK) cells and cytotoxic T lymphocytes. Taxol reduces helper and cytotoxic lymphocytes causing systemic immunosuppression and myelosuppression in bone marrow, whereas 6SA does not decrease any immune cell subpopulations in circulating blood and lymph nodes. More importantly, the CoT decreased the Taxol-induced cytotoxicity in circulating T cells and bone marrow. Treatment with 6SA increases the secretion of IL-2, IL-12, GM-CSF, TNF-α and IFN-γ and significantly reduces IL-10 and IL-17 secretion, suggesting that the reduction of regulatory T cells and tumor-associated macrophages contribute to the host control of tumor development.Finally, 6SA has an effective antineoplastic activity against breast cancer cells in an immunocompetent animal, reduces the myelosuppression and leukopenia that Taxol produces, improves the antitumoral immunological microenvironment and increases the overall survival of the animals improving the quality of life of patients with cancer.

IL15 Stimulation with TIGIT Blockade Reverses CD155-mediated NK-Cell Dysfunction in Melanoma

Natural killer (NK) cells play a critical role in tumor immunosurveillance. Multiple activating and inhibitory receptors (IR) regulate NK-cell-mediated tumor control. The IR T-cell immunoglobulin and ITIM domain (TIGIT) and its counter-receptor CD226 exert opposite effects on NK-cell-mediated tumor reactivity. We evaluated the frequency, phenotype, and functions of NK cells freshly isolated from healthy donors and patients with melanoma with multiparameter flow cytometry. We assessed TIGIT and CD226 cell surface expression and internalization upon binding to CD155. We evaluated the role of IL15 and TIGIT blockade in increasing NK-cell-mediated cytotoxicity in vitro and in two mouse models. NK cells are present at low frequencies in metastatic melanoma, are dysfunctional, and downregulate both TIGIT and CD226 expression. As compared with TIGIT- NK cells, TIGIT+ NK cells exhibit higher cytotoxic capacity and maturation, but paradoxically lower cytotoxicity against CD155+ MHC class I-deficient melanoma cells. Membrane bound CD155 triggers CD226 internalization and degradation, resulting in decreased NK-cell-mediated tumor reactivity. IL15 increases TIGIT and CD226 gene expression by tumor-infiltrating NK cells (TiNKs) and, together with TIGIT blockade, increases NK-cell-mediated melanoma cytotoxicity in vitro and decreases tumor metastasis in two mouse melanoma models. Specific deletion of TIGIT on transferred NK cells enhances the antimetastatic activity of IL15, while CD226 blockade decreases the effects of IL15 and TIGIT blockade. Our findings support the development of novel combinatorial immunotherapy with IL15 and TIGIT blockade to promote NK-cell-mediated destruction of MHC class I-deficient melanoma, which are refractory to CD8+ T-cell-mediated immunity.See related commentary by Pietra et al., p. 5274.

Modulating NK cell metabolism for cancer immunotherapy

Natural killer (NK) cells are lymphocytes with potent antitumor functions and, therefore, multiple NK cell-based cancer immunotherapies have been developed and are currently being tested. However, there is a necessity to find new means to improve these therapies, and immunometabolism represents an attractive target. NK cell effector functions are intricately linked to their metabolism, and modulating the latter could be the key to release their full potential. In this review, we have summarized how NK cell metabolism is regulated during some processes, such as maturation, viral infection, and cytokine stimulation. Additionally, we provide an overview of how NK cell metabolism is affected by current therapeutic approaches aimed to promote NK cell expansion and/or to increase their effector functions. We have also recapitulated several strategies that could help alleviating the metabolic impairment that characterizes tumor-infiltrating NK cells, and thus increase or restore their effector functions. Furthermore, we have reviewed several therapeutic approaches targeting cancer metabolism that could synergize with NK cell-based cancer immunotherapies, and thus enhance their efficacy.

Thioredoxin activity confers resistance against oxidative stress in tumor-infiltrating NK cells.

To improve the clinical outcome of adoptive NK cell therapy in patients with solid tumors, NK cells need to persist within the tumor microenvironment (TME) in which the abundance of ROS could dampen antitumor immune responses. In the present study, we demonstrated that IL-15-primed NK cells acquired resistance against oxidative stress through the thioredoxin system activated by mTOR. Mechanistically, the activation of thioredoxin showed dependence on localization of thioredoxin-interacting protein. We show that NK cells residing in the tumor core expressed higher thiol densities that could aid in protecting other lymphocytes against ROS within the TME. Furthermore, the prognostic value of IL15 and the NK cell gene signature in tumors may be influenced by tobacco smoking history in patients with non-small-cell lung cancer (NSCLC). Collectively, the levels of reducing antioxidants in NK cells may not only predict better tumor penetrance but potentially even the immune therapy response.

Abstract 1605: Extracellular vesicles from metastatic non-small cell lung cancer induce the angiogenic switch in natural killer cells

Abstract Lung cancer is the leading cause of cancer-related death worldwide and the only curative treatment remains surgery. Recurrence rates in resected patients remain high: from 30% for stage I to 80% for stage III. We previously demonstrated the role of 24 microRNAs in the pathogenesis and aggressiveness of lung tumors. Natural Killer (NK) cells, cellular mediators involved in tumor immunosurveillance are functionally compromised in cancer patients. We discovered that tumor infiltrating NK cells (TINKs) in non-small cell lung cancer (NSCLC) acquire the proangiogenic CD56brightCD16-VEGF+PlGF+ phenotype and functions and that TGFβ is involved the NK cell angiogenic switch. Tumor cells can release large amounts of extracellular vesicles (EVs), that have been reported to be endowed with potent immunosuppressive activities, also in NK cells. Here, we investigate the abilities of EVs from NSCLC cell lines and patients to induce a pro-angiogenic polarization in NK cells. EVs were isolated from conditioned media of NSCLC primary tumor (A549, LT73) and metastatic (H460) cell lines, and from plasma of NSCLC metastatic patients or heavy smoker individuals (EV-donor), by ultra-centrifugation and their purity were confirmed by transmission electron microscopy (TEM), Nanosight and western blot. NK cells, FACS sorted from healthy controls were exposed for 72 hours to 15 μg of EVs, then characterized for surface antigen expression, by multicolour flow cytometry. miRNA-profiling was performed on NK cells polarized for 72 hours with 15 μg of EVs isolated from A549 or LT73 NSCLC cells lines, or from plasma of patients with metastatic NSCLC or heavy smoker individuals. NanoSight analysis revealed that the EVs-Patients and EVs-Donors had a similar median size and morphology. We found that EVs from NSCLC cell lines and NSCLC patients induce the CD56brightCD9+CD49a+ NK cell phenotype, together with upregulation of CXCR4 expression and decreased levels of the NKG2D activation marker. Interestingly, the metastatic H460 cell line exhibited stronger NK cell polarization effects as compared to those from A549 and LT73 cells. Strong pro-angiogenic NK cell polarization was also observed in cytolytic NKs, exposed to EVs from metastatic NSCLC patients, as compared to those exposed to EVs-donors Finally, miRNA profiling on NK cells polarized with EVs, showed up-regulation of miR-133a and miR-221 suggesting a possible role of these miRNAs in NK angiogenic switch. Our study demonstrates that NSCLC EVs can support lung cancer progression acting on the NK cell angiogenic switch. Citation Format: Matteo Gallazzi, Orazio Fortunato, Ugo Pastorino, Gabriella Sozzi, Douglas Noonan, Adriana Albini, Antonino Bruno. Extracellular vesicles from metastatic non-small cell lung cancer induce the angiogenic switch in natural killer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1605.

The Effects of Mesenchymal Stem Cells on Antimelanoma Immunity Depend on the Timing of Their Administration.

There is still a lively debate about whether mesenchymal stem cells (MSCs) promote or suppress antitumor immune response. Although several possible explanations have been proposed, including different numbers of injected and engrafted MSCs, heterogeneity in phenotype, and function of tumor cells, the exact molecular mechanisms responsible for opposite effects of MSCs in modulation of antitumor immunity are still unknown. Herewith, we used a B16F10 murine melanoma model to investigate whether timing of MSC administration in tumor-bearing mice was crucially important for their effects on antitumor immunity. MSCs, intravenously injected 24 h after melanoma induction (B16F10+MSC1d-treated mice), significantly enhanced natural killer (NK) and T cell-driven antitumor immunity, suppressed tumor growth, and improved survival of melanoma-bearing animals. Significantly higher plasma levels of antitumorigenic cytokines (TNF-α and IFN-γ), remarkably lower plasma levels of immunosuppressive cytokines (TGF-β and IL-10), and a significantly higher number of tumor-infiltrating, IFN-γ-producing, FasL- and granzyme B-expressing NK cells, IL-17-producing CD4+Th17 cells, IFN-γ- and TNF-α-producing CD4+Th1 cells, and CD8+cytotoxic T lymphocytes (CTLs) were observed in B16F10+MSC1d-treated mice. On the contrary, MSCs, injected 14 days after melanoma induction (B16F10+MSC14d-treated mice), promoted tumor growth by suppressing antigen-presenting properties of tumor-infiltrating dendritic cells (DCs) and macrophages and by reducing tumoricidal capacity of NK cells and T lymphocytes. Significantly higher plasma levels of TGF-β and IL-10, remarkably lower plasma levels of TNF-α and IFN-γ, and significantly reduced number of tumor-infiltrating, I-A-expressing, and IL-12-producing macrophages, CD80- and I-A-expressing DCs, granzyme B-expressing CTLs and NK cells, IFN-γ- and IL-17-producing CTLs, CD4+Th1, and Th17 cells were observed in B16F10+MSC14d-treated animals. In summing up, the timing of MSC administration into the tumor microenvironment was crucially important for MSC-dependent modulation of antimelanoma immunity. MSCs transplanted during the initial phase of melanoma growth exerted tumor-suppressive effect, while MSCs injected during the progressive stage of melanoma development suppressed antitumor immunity and enhanced tumor expansion.

Prognostic Significance of Tumor-Infiltrating Natural Killer Cells in Solid Tumors: A Systematic Review and Meta-Analysis.

Background: Tumor-infiltrating natural killer (NK) cells (TINKs) are crucial immune cells in tumor defense, and might be related to tumor prognosis. However, the results were discrepant among different studies. The present meta-analysis was performed to comprehensively assess the prognostic value of NK cell markers in solid tumor tissues.Methods: PubMed, Web of Science, and EMBASE were searched to identify original researches reporting the prognostic significance of TINKs in solid tumors. NK cell markers CD56, CD57, NKp30, and NKp46 were included in the analysis. The hazard ratios (HRs) and 95% confidence intervals (CIs) of pooled overall survival (OS), disease-free survival (DFS), metastasis-free survival (MFS), progression-free survival (PFS), and recurrence-free survival (RFS) were calculated by STATA software 14.0 to assess the prognostic significance.Results : Of the 56 included studies, there were 18 studies on CD56, 31 studies on CD57, 1 study on NKp30, and 7 studies on NKp46. High levels of CD56, CD57, NKp30, and NKp46 were significantly correlated with better OS of patients with solid malignancies (HR = 0.473, 95%CI: 0.315–0.710, p < 0.001; HR = 0.484, 95%CI: 0.380–0.616, p < 0.001; HR = 0.34, 95%CI: 0.14–0.80, p = 0.014; HR = 0.622, 95%CI: 0.470–0.821, p < 0.001, respectively). Our results also revealed that CD56, CD57, and NKp46 could act as independent prognostic predictors for favorable OS (HR = 0.372, 95%CI: 0.261–0.531, p < 0.001; HR = 0.525, 95%CI: 0.346–0.797, p = 0.003; HR = 0.559, 95%CI: 0.385–0.812, p = 0.002, respectively).Conclusions : Our results indicated that high levels of NK cell markers in solid tumor tissues could predict favorable prognosis for solid tumor patients.

Renal cell tumors convert natural killer cells to a proangiogenic phenotype.

Natural killer (NK) cells are classically associated with immune surveillance and destruction of tumor cells. Inconsistent with this function, NK cells are found in advanced human tumors including renal cell carcinoma (RCC). NK cells with non-classical phenotypes (CD56+CD16dim/neg; termed decidua NK (dNK) cells) accumulate at the maternal-fetal interface during embryo implantation. These dNK cells are poorly cytotoxic, proangiogenic, and facilitate placenta development. As similarities between embryo implantation and tumor growth exist, we tested the hypothesis that an analogous shift in NK cell phenotype and function occurs in RCC tumors. Our results show that peripheral NK (pNK) cells of RCC patients were uniformly CD56+CD16bright, but lacked full cytotoxic ability. By comparison, RCC tumor-infiltrated NK (TiNK) cells were significantly enriched for CD56+CD16dim-neg cells, a phenotype of dNK cells. Gene expression analysis revealed that angiogenic and inflammatory genes were significantly increased for RCC TiNK versus RCC pNK populations, with enrichment of genes in the hypoxia inducible factor (HIF) 1α pathway. Consistent with this finding, NK cells cultured under hypoxia demonstrated limited cytotoxicity capacity, but augmented production of vascular endothelial growth factor (VEGF). Finally, comparison of gene expression data for RCC TiNK and dNK cells revealed a shared transcriptional signature of genes with known roles in angiogenesis and immunosuppression. These studies confirm conversion of pNK cells to a dNK-like phenotype in RCC tumors. These characteristics are conceivably beneficial for placentation, but likely exploited to support early tumor growth and promote metastasis.

Exosome-Containing Preparations From Postirradiated Mouse Melanoma Cells Delay Melanoma Growth In Vivo by a Natural Killer Cell–Dependent Mechanism

To investigate the ability of radiation to stimulate exosome release from melanoma cells and to characterize the resulting exosome-containing vesicle preparations for their ability to promote a host antitumor immune response. Cultured B16F10 murine melanoma cells or tumors were irradiated, and secreted extracellular vesicles were isolated and characterized. The exosome-containing vesicle preparations were injected into fresh tumors in syngeneic mice, and tumor growth and infiltrating T cells and natural killer (NK) cells were characterized. Irradiation stimulated exosome release from B16F10 murine melanoma cells. Exosome preparations from irradiated cell culture supernatants were biologically active, as demonstrated by uptake into recipient cells and by the ability to induce dendritic cell maturation and activation in vitro. Intratumoral injection significantly delayed tumor growth in vivo, whereas injection of similar preparations from non irradiated cells had no effect. The antitumor effect was correlated to an increase in interferon gamma-producing tumor-infiltrating NK cells. Pretreatment of the host mice with anti-NK cell antibodies abolished the effect, whereas pretreatment with anti-CD8+ T-cell antibodies did not. Exosomes from irradiated cells, or synthetic mimics, might provide an effective strategy for potentiation of NK cell-mediated host antitumor immunity.

PET Imaging of the Natural Killer Cell Activation Receptor NKp30.

Redirecting the immune system in cancer treatment has led to remarkable responses in a subset of patients. Natural killer (NK) cells are innate lymphoid cells being explored as they engage tumor cells in different mechanisms compared with T cells, which could be exploited for treatment of nonresponders to current immunotherapies. NK cell therapies are monitored through measuring peripheral NK cell concentrations or changes in tumor volume over time. The former does not detect NK cells at the tumor site, and the latter is inaccurate for immunotherapies because of pseudoprogression. Therefore, new imaging methods are required as companion diagnostics for optimizing immunotherapies. Methods: In this study, we developed and completed preclinical in vivo validation of 2 antibody-based PET probes specific for NKp30, an activation natural cytotoxicity receptor expressed by human NK cells. Quantitative, multicolor flow cytometry during a variety of NK cell activation conditions was completed on primary human NK cells and the NK92MI cell line. Human renal cell carcinoma (RCC) tumors were stained for the NK cell receptors CD56, NKp30, and NKp46 to determine expression on tumor-infiltrating NK cells. An NKp30 antibody was radiolabeled with 64Cu or 89Zr and evaluated in subcutaneous xenografts and adoptive cell transfer mouse models. Results: Quantitative flow cytometry showed consistent expression of the NKp30 receptor during different activation conditions. NKp30 and NKp46 costained in RCC samples, demonstrating the expression of these receptors on tumor-infiltrating NK cells in human tumors, whereas tumor cells in one RCC sample expressed the peripheral NK marker CD56. Both PET tracers showed high stability and specificity in vitro and in vivo. Notably, 89Zr-NKp30Ab had higher on-target contrast than 64Cu-NKp30Ab at their respective terminal time points. 64Cu-NKp30Ab delineated NK cell trafficking to the liver and spleen in an adoptive cell transfer model. Conclusion: The consistent expression of NKp30 on NK cells makes it an attractive target for quantitative imaging. Immunofluorescence staining on human RCC samples demonstrated the advantages of NKp30 targeting versus CD56 for detection of tumor infiltrating NK cells. This work advances PET imaging of NK cells and supports the translation of imaging agents for immunotherapy monitoring.

IL-6-induced CD39 expression on tumor-infiltrating NK cells predicts poor prognosis in esophageal squamous cell carcinoma.

Natural killer (NK) cells, a predominantinnatelymphocyte subset, mediates eradicating malignant cells. Purinergic signaling by ectonucleotidase CD39 can suppress T-cell response in caner. However, the role of CD39 in NK cells has not been fully elucidated. Here, we characterized CD39 expression on NK cells and its clinical relevance in esophageal squamous cell carcinoma (ESCC). Peripheral blood and tissue samples were collected from 36 ESCC patients. We observed that the proportion of NK cells significantly decreased but CD39 was obviously up-regulated on NK cells from cancerous tissues compared to paired peripheral blood in ESCC patients. Furthermore, tumor-infiltrating NK cells with high CD39 expression exhibited a phenotype of functional impairment. In vitro, conditioned media of ESCC cell lines could induce CD39 expression on peripheral NK cells from healthy donors. IL-6 was identified as the major cytokine produced by ESCC cell lines and also elevated in both tumor tissues and blood serum from ESCC patients. Recombinant IL-6 significantly induced surface CD39 expression in human NK cells, while IL-6-receptor antagonist tocilizumab prevented this effect. Finally, tumor-infiltrating CD39+ NK cells were correlated with poor prognosis in ESCC patients. Thus, tumor-derived IL-6 might impair NK cell functions through induction of CD39 expression. CD39+ NK cells may serve as a potential prognostic biomarker for ESCC patients.

Donor Lymphocyte-Derived Natural Killer Cells Control MHC Class I-Negative Melanoma.

Natural killer (NK) cells provide a natural defense against MHC-I-negative tumors, such as melanoma. Donor lymphocyte infusion (DLI) containing NK cells, a form of adoptive immunotherapy used after allogenic bone marrow transplantation (allo-BMT), promotes antitumor immune responses but is often associated with life-threatening complications such as graft-versus-host disease (GvHD). Here, we showed that without prior allo-BMT, DLI provoked melanoma control associated with the infiltration and persistence of the transferred NK cells. This allograft acceptance did not correlate with an increase of GvHD; instead it correlated with the expansion and activation of tumor-infiltrating NK cells that expressed the cytotoxic molecules (e.g., IFNγ and granzyme B) and maturation signatures (e.g., CD11bhiCD27lo and KLRGhi/CD43hi). The development of beneficial tumor-infiltrating NK cells of DLI origin required host CD4+ T-cell help in part by producing IL2, as well as by limiting regulatory CD4+ T cells (Treg). IL2 blockade impaired the NK-dependent melanoma control, which could not be rescued by IL2 administration beyond CD4+ T-cell help. Our findings linked NK allograft acceptance-CD4+ T-cell help crosstalk to melanoma development without the need of allo-BMT. We thereby helped define that tumor-infiltrating NK cells of DLI origin may serve as effective therapeutic targets for controlling melanoma.

Single-Cell RNA Sequencing of Tumor-Infiltrating NK Cells Reveals that Inhibition of Transcription Factor HIF-1α Unleashes NK Cell Activity

Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Hypoxia is a common feature of solid tumors, and cells adapt by upregulating the transcription factor HIF-1α. Here, we defined the transcriptional landscape of mouse tumor-infiltrating natural killer (NK) cells by using single-cell RNA sequencing. Conditional deletion of Hif1a in NK cells resulted in reduced tumor growth, elevated expression of activation markers, effector molecules, and an enriched NF-κB pathway in tumor-infiltrating NK cells. Interleukin-18 (IL-18) from myeloid cells was required for NF-κB activation and the enhanced anti-tumor activity of Hif1a-/- NK cells. Extended culture with an HIF-1α inhibitor increased human NK cell responses. Low HIF1A expression was associated with high expression of IFNG in human tumor-infiltrating NK cells, and an enriched NK-IL18-IFNG signature in solid tumors correlated with increased overall patient survival. Thus, inhibition of HIF-1α unleashes NK cell anti-tumor activity and could be exploited for cancer therapy.

Cell-based cancer immunotherapy using chimeric antigen receptor (CAR)-engineered natural killer (NK) cells surface-modified with drug-loaded nanoparticles targeting adenosine receptors

Abstract The immunosuppressive tumor microenvironment (TME) plays a key role in failed eradication of solid tumors and tumor recurrence upon cancer treatment, as resident immune cells lose their cancer surveillance abilities. One of the underlying mechanisms responsible for the loss of adoptively transferred immune cell function in the TME is the A2a adenosine receptor (A2aR) inhibitory pathway. Encapsulating SCH-58261 (SCH), a potent antagonists of A2aR, in crosslinked multilamellar liposomal vesicles (cMLVs) was shown to efficiently deliver the drug to adoptively transferred cells in tumors. We genetically modified human NK92 cells to express anti-mesothelin CAR (ameso.CAR.NK) to be used in this study. We hypothesized that the efficacy of NK cell adoptive transfer therapies could be enhanced if CAR.NK cells were used as active carriers to direct SCH-loaded cMLVs to the immunosuppressive TME to block the A2aR pathway. We first covalently attached cMLVs to NK cell surface. We next verified that cMLV conjugation did not have adverse effects on NK cell viability and migration. In addition, surface conjugation of cMLVs did not reduce IFN-γ secretion and cancer cell killing ability of NK cells, suggesting similar levels of cognate antigen-specific activation of ameso.CAR.NK cells by mesothelin-overexpressing ovarian cancer cells. In the future, we will test the in vitro function of irradiated CAR.NK cells, given the requirement of irradiating NK92 cell lines prior to clinical use. Next, we will test our platform in an ovarian cancer xenograft mouse model and examine the effects of our therapy on tumor growth. Finally, we will investigate the potential of this therapy to reverse immune suppression on the TME-resident tumor-infiltrating NK cells.

Abstract B05: Intestinal microbiota mediates pancreatic carcinogenesis through modulation of tumor infiltrating natural killer cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related death in the United States. Our group and others have demonstrated that the intestinal microbiota accelerates pancreatic carcinogenesis. The relationship of intestinal bacteria, immune response, and PDAC development is unclear. To that end, we investigated the role of intestinal bacterial soluble factors in modulating the immune environment of PDAC and its progression. Methods: Mice intestinal microbiota was depleted with wide-spectrum antibiotics. The human PDAC cell line, L3.6pl, was heterotopically implanted into Rag1-/- mice, while the syngeneic murine PDAC cell line, Pan02, was orthotopically implanted into the pancreas of C57Bl/6 mice. Tumor and pancreas infiltrated natural killer (NK) cells were quantitated by flow cytometry. In vivo NK cell depletion was attained by intraperitoneal injection of anti-Asialo-GM1 antibody twice weekly. Germ-free (GF) and conventionally housed Rag1-/- mice stool was cultured and bacteria-free supernatant extracted. The ability of these bacteria-free supernatants to regulate NK-92mi cell cytotoxicity and migration was tested in vitro by flow cytometry and a Transwell assay, respectively. Results: Compared to microbiota-intact Rag1-/- mice, microbiota depletion yielded 74% smaller tumors (p&amp;lt;0.05) with a 1.5-fold increase in PDAC infiltrating NK cells (p&amp;lt;0.001). Confirmation by immunohistochemistry demonstrated a 6.5-fold increase in NK cell tumor infiltration in microbiota-depleted mice (p&amp;lt;0.0001). Notably, a 3-fold increase of intrapancreatic NK cells in GF Rag1-/- mice versus conventionally housed mice was noted, suggesting that bacteria regulate immune cell trafficking. Antibody-mediated NK cell depletion with concomitant microbiota depletion increased PDAC tumor growth, negating the antitumor phenotype of microbiota depletion alone in both immunodeficient Rag1-/- (2.8-fold increase; p&amp;lt;0.05) and immunocompetent C57Bl/6 mice (2.4-fold increase; p&amp;lt;0.05). Quantitative PCR revealed 9-fold higher IFN-gamma gene expression in PDAC xenografts of microbiota-depleted mice versus microbiota-intact (p&amp;lt;0.05). Cell-free stool bacteria culture supernatant from conventionally housed Rag1-/- mice, but not from GF mice, inhibited NK-92mi cell migration by 26% (p&amp;lt;0.05) and cytotoxicity against L3.6pl cells by 51% (p&amp;lt;0.01). Compared to NK-92mi cells exposed to cultured stool supernatant from GF Rag1-/- mice, SPF cultured stool supernatant resulted in decreased gene expression associated with activation/recruitment of NK cells including FASLG, CCL18, IL-13, CXCR2, CCL4, and increased expression associated with inhibition of NK cell activity including CCR1 and IL-6. Conclusion: These findings suggest that intestinal bacteria modulate PDAC development through suppression of NK cell recruitment and activation, a phenomenon potentially mediated by yet to be identified small molecules. Citation Format: Qin Yu, Mark Beveridge, Ryan Thomas, Christian Jobin. Intestinal microbiota mediates pancreatic carcinogenesis through modulation of tumor infiltrating natural killer cells [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B05.