Natural Killer (NK) cells are innate lymphocytes that are key to intrinsic cancer immunosurveillance and an important target for cancer immunotherapy. Understanding fundamental human NK cell metabolism provides opportunities for optimizing NK cell therapies. Little is known about how glutamine, an important cell nutrient and carbon source, is utilized by human NK cells. To address this, we performed U13C-glutamine tracing experiments by Liquid Chromatography Mass Spectrometry and Gas Chromatography Mass Spectrometry analysis of human NK cells stimulated with IL-2 for 18 h to provide a global overview of glutamine usage by these cells. Our results show that glutamine is taken up by resting NK cells and that this increases further upon IL-2 stimulation. Metabolite labelling analysis identified that IL-2 stimulation results in greater conversion of glutamine to glutamate, allowing for anaplerotic flux into the TCA cycle. The fate of the glutamine-derived carbons diverged at oxaloacetate allowing both bioenergetic and biosynthetic outcomes - some carbons continued around the TCA cycle while others were exported, converted to aspartate and subsequently used for pyrimidine synthesis. Nucleotide synthesis by IL-2 activated NK cells was found to be essential for expression of the activation marker CD69. The data indicate that glutamine is a key nutrient taken up by human NK cells, and that IL-2 drives glutaminolysis. Subsequent glutamate is used to support the TCA cycle, generating energy and providing intermediates for de novo pyrimidine synthesis.

High-grade serous ovarian cancer (HGSOC) remains an urgent unmet clinical need, with more than 70% of patients presenting with metastatic disease. Many patients develop large volumes of ascites, which promotes metastasis and is associated with poor therapeutic response and survival. Immunotherapy trials have shown limited success, highlighting the need to better understand HGSOC immunology. Here, we analyzed cytotoxic lymphocytes [natural killer (NK), T, and innate T cells] from patients with HGSOC and observed widespread dysfunction across primary and metastatic sites. Although nutrient rich, ascites was immunosuppressive for all lymphocyte subsets. NK cell dysfunction was driven by uptake of polar lipids, with associated dysregulation in lipid storage. Phosphatidylcholine was a key immunosuppressive metabolite, disrupting NK cell membrane order and cytotoxicity. Blocking lipid uptake through SR-B1 protected NK cell antitumor functions in ascites. These findings offer insights into immune suppression in HGSOC and have important implications for the design of future immunotherapies.

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γδ T cells are important tissue-resident, innate T cells that are critical for tissue homeostasis. γδ cells are associated with positive prognosis in most tumors; however, little is known about their heterogeneity in human cancers. Here, we phenotyped innate and adaptive cells in human colorectal (CRC) and endometrial cancer. We found striking differences in γδ subsets and function in tumors compared to normal tissue, and in the γδ subsets present in tumor types. In CRC, an amphiregulin (AREG)-producing subset emerges, while endometrial cancer is infiltrated by cytotoxic cells. In humanized CRC models, tumors induced this AREG phenotype in Vδ1 cells after adoptive transfer. To exploit the beneficial roles of γδ cells for cell therapy, we developed an expansion method that enhanced cytotoxic function and boosted metabolic flexibility, while eliminating AREG production, achieving greater tumor infiltration and tumor clearance. This method has broad applications in cellular therapy as an ‘off-the-shelf’ treatment option.

Resistance to platinum‐based chemotherapy is the major cause of death from high‐grade serous ovarian cancer (HGSOC). We hypothesise that detection of specific DNA methylation changes may predict platinum resistance in HGSOC. Using a publicly available “discovery” dataset we examined epigenomic and transcriptomic alterations between primary platinum‐sensitive (n = 32) and recurrent acquired drug resistant HGSOC (n = 28) and identified several genes involved in immune and chemoresistance‐related pathways. Validation via high‐resolution melt analysis of these findings, in cell lines and HGSOC tumours, demonstrated the most consistent changes were observed in three of the genes: APOBEC3A, NKAPL and PDCD1 . Plasma samples from an independent HGSOC cohort (n = 17) were analysed using droplet digital PCR. Hypermethylation of NKAPL was detected in 46% and hypomethylation of APOBEC3A in 69% of plasma samples taken from women with relapsed HGSOC (n = 13), with no alterations identified in disease‐free patients (n = 4). Following these results, and using a CRISPR‐Cas9 approach, we were also able to demonstrate that in vitro NKAPL promoter demethylation increased platinum sensitivity by 15%. Overall, this study demonstrates the importance of aberrant methylation, especially of the NKAPL gene, in acquired platinum resistance in HGSOC.

Objectives Development of therapeutic resistance is a major cause of mortality in high-grade serous ovarian cancer (HGSOC), thus a better understanding of acquired resistance mechanisms is needed. This study aimed to investigate how epigenomic events might be associated with acquired-drug resistance in HGSOC patients. Methods Methylation and gene expression differences between primary platinum-sensitive (n=32) and recurrent acquired-resistant samples (n=28) was explored using a HGSOC dataset. High resolution melting was used to validate results using epithelial ovarian cancer cell lines and HGSOC tumours. A CRISPR-Cas9 approach was used to investigate the effects of DNA methylation editing in vitro. Plasma samples from HGSOC patients (n=17) and age-matched healthy controls (n=20) were used to investigate longitudinal methylation dynamics via droplet digital PCR. Results Comparison of methylation and gene expression analysis identified several genes, known to be involved in diverse immune and chemoresistance-related pathways, that significantly differentiated between paired platinum-sensitive and acquired-resistant HGSOC samples, with three genes displaying the most consistent methylation changes (PDCD1, NKAPL, APOBEC3A). A CRISPR-Cas9 approach was used to interrogate the effects of APOBEC3A and NKAPL promoter methylation editing on platinum sensitivity, with demethylation of NKAPL promoter being associated with increased platinum sensitivity. Hypermethylation of NKAPL and APOBEC3A were detected in 46% and 69%, respectively, of plasma samples from women with relapsed HGSOC. Conclusions Promoter methylation has been identified as potentially involved in HGSOC drug resistance. Further research is warranted to understand the future use of these methylation patterns as prognostic/predictive markers in the OC clinical setting.

High risk neuroblastoma is responsible for 15% of deaths in pediatric cancer patients. The introduction of anti-GD2 immunotherapy has significantly improved outcomes but there is still only approximately a 50% 5 year event-free-survival for these children and improvements in treatments are urgently required. Anti-GD2 immunotherapy uses the patients’ own immune system to kill cancer cells. In particular, Natural Killer (NK) cells kill antibody coated tumor cells by a process called antibody dependent cellular cytotoxicity (ADCC). However, our previous work has highlighted metabolic exhaustion of NK cells in circulating blood of adult cancer patients, identifying this as a potential therapeutic target. In this study, we investigated circulating NK cells in patients newly diagnosed with neuroblastoma. We found evidence of activation of NK cells in vivo by the cancer itself. While some evidence of NK cell dysfunction was observed in terms of IFNγ production, most results indicated that the NK cell compartment remained relatively intact. In fact, some aspects of metabolic and functional activities were actually increased in patients compared to controls. Glycolytic responses, which we show are crucial for ADCC, were actually enhanced in patients and CD16, the NK cell receptor that mediates ADCC, was also expressed at high levels in some patients. Overall, the data suggest that patient NK cells could be harvested at diagnosis for subsequent beneficial autologous use during immunotherapy. Enhancing glycolytic capacity of cell therapies could also be a strategic goal of future cell therapies for patients with neuroblastoma and indeed other cancers.

Introduction/Background* The tumour microenvironment (TME) in metastatic high grade serous ovarian cancer (HGSOC) is not well described. We present a multimodal characterisation of intraepitelial TILs (iTILS) and stromal TILs (sTILS) using flow cytometry (FACS) immunohistochemistry (IHC) in matched primary and metastatic HGSOC samples. Methodology FACs and IHC for CD4 and CD8 were performed on 26 samples from seven women with HGSOC. Tissue samples, labelled with fluorescent antibodies against CD3, CD4, CD8, checkpoints TIGIT, PD1 and cytokine IFN-γ also were analysed with a FACS Fortessa (BD Biosciences). IHC was performed on samples and images annotated to assess intra-epithelial and stromal CD4 and CD8 expression using ImageScope (Aperio), and analysed using the Aperio Nuclear Algorithm v9 (figure 1). Statistical analysis was performed using IBM SPSS 24 or Prism Graph Pad. Quantitative variables were assessed with one way ANOVA and Mann Whitney test. Result(s)* FACs demonstrated that, compared to primary samples, the frequency of CD8+ TILs(p= 0.017) , TIGIT (p=0.013) and PD1 (p=0.017) expression was reduced in matched metastatic sites. CD4+ TILs levels were unchanged between primary and metastatic samples. Consistent with a reduced level of cytotoxic activity, IFN-γ on CD8+ TILs was reduced in the metastatic TME (p=0.034). IHC demonstrated that the majority of primary samples (5/7 (71.4%) showed a higher proportion of CD8+ sTILs compared to iTILs (figure 3b). In the one BRCA mutated patient, the CD8 iTILs were higher than sTILs (figure 4a). In FACs, this sample also had the highest frequency of CD8+ TILs within the ovarian tumour (figure 4b). In IHC from 4/6 different metastatic sites (omentum, vagina, spleen and peritoneum) the density of CD8+ sTILs was higher than iTILs, demonstrating these tumours were immune excluded. Serosal liver and diaphragmatic metastases demonstrated increased CD8+ iTILs compared with primary tumours. Although not as marked, this pattern was replicated in CD4 sTILs (figure 5b, c). Conclusion* Using a multimodal approach, including IHC and FACs, we demonstrate that the metastatic TME in HGSOC is significantly different to the primary TME. These findings provide an initial explanation as to why immune checkpoint inhibitors have failed in HGSOC and warrant further investigation.

BackgroundNatural killer (NK) cells provide important immune protection from cancer and are a key requirement for particular immunotherapies. There is accumulating evidence that NK cells become dysfunctional during cancer. Overcoming NK cell exhaustion would be an important step to allow them to function optimally in a range of NK cell therapies, including those that depend on autologos circulating NK cells. We have previously demonstrated that NK cells undergo a normal metabolic reprogramming in response to cytokine activation and that this is required for optimal function. The objective of this work was to investigate if cellular metabolism of circulating NK cells is dysregulated in patients with metastatic breast cancer and if so, to gain insights into potential mechanisms underpinning this. Such discoveries would provide important insights into how to unleash the full activity of NK cells for maximum immunotherapy output.MethodsSingle-cell analysis, metabolic flux and confocal analysis of NK cells from patients with metastatic breast cancer and healthy controlsResultsIn addition to reduced interferon-γ production and cytotoxicity, peripheral blood NK cells from patients had clear metabolic deficits including reduced glycolysis and oxidative phosphorylation. There were also distinct morphologically alterations in the mitochondria with increased mitochondrial fragmentation observed. Transforminggrowth factor-β (TGFβ) was identified as a key driver of this phenotype as blocking its activity reversed many metabolic and functional readouts. Expression of glycoprotein-A repetitions predominant (GARP) and latency associated peptide (LAP), which are involved with a novel TGFβ processing pathway, was increased on NK cells from some patients. Blocking the GARP–TGFβ axis recapitulated the effects of TGFβ neutralization, highlighting GARP as a novel NK cell immunotherapy target for the first time.ConclusionsTGFβ contributes to metabolic dysfunction of circulating NK cells in patients with metastatic breast cancer. Blocking TGFβ and/or GARP can restore NK cell metabolism and function and is an important target for improving NK cell-based immunotherapies.

NK cells are innate lymphocytes which play an essential role in protection against cancer and viral infection. Their functions are dictated by many factors including the receptors they express, cytokines they respond to and changes in the external environment. These cell processes are regulated within NK cells at many levels including genetic, epigenetic and expression (RNA and protein) levels. The last decade has revealed cellular metabolism as another level of immune regulation. Specific immune cells adopt metabolic configurations that support their functions, and this is a dynamic process with cells undergoing metabolic reprogramming during the course of an immune response. Upon activation with pro-inflammatory cytokines, NK cells upregulate both glycolysis and oxphos metabolic pathways and this supports their anti-cancer functions. Perturbation of these pathways inhibits NK cell effector functions. Anti-inflammatory cytokines such as TGFβ can inhibit metabolic changes and reduce functional outputs. Although a lot remains to be learned, our knowledge of potential molecular mechanisms involved is growing quickly. This review will discuss our current knowledge on the role of TGFβ in regulating NK cell metabolism and will draw on a wider knowledge base regarding TGFβ regulation of cellular metabolic pathways, in order to highlight potential ways in which TGFβ might be targeted to contribute to the exciting progress that is being made in terms of adoptive NK cell therapies for cancer.

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric neoplasm, primarily diagnosed in those below the age of three. MRTs most commonly arise in the central nervous system and kidneys. A poor prognosis accompanies the MRT diagnosis, with a reported 2‑year survival rate of 30%. Thus, there is an urgent need for the development of new therapies for this malignancy. Members of the inhibitor of apoptosis protein (IAP) family have previously been reported to be overexpressed in various cancers. As such, small molecule inhibitors of these family members have entered clinical trials. However, the role of IAPs in MRT has not been examined yet. The present study is the first report of the expression of a range of IAPs, including X‑linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis protein 1 (cIAP1), cellular inhibitor of apoptosis protein 2 (cIAP2), livin and survivin in MRT cell lines. Furthermore, the results demonstrated the ability of the XIAP inhibitor, embelin, to sensitise MRT cell lines to TNF‑related apoptosis‑inducing ligand (TRAIL) treatment. The enhanced cell death detected upon cotreatment was dependent on caspase‑8 and co‑occurred with caspase‑8 and caspase‑3 cleavage, suggesting engagement of the extrinsic apoptotic pathway. Sensitisation to TRAIL was accompanied by livin cleavage, alongside downregulation of survivin and the caspase‑8 inhibitor FLIPL. In addition, knockdown of XIAP using siRNA enhanced TRAIL‑mediated cell death, suggesting that this process may in part mediate sensitisation. In conclusion, the present results suggested that IAP inhibition may present a novel avenue for the treatment of MRT.

Natural Killer (NK) cells provide important protection from cancer and are a key requirement for particular immunotherapies. In activated NK cells, a metabolic response towards increased glycolysis and oxidative phosphorylation is crucial for NK cell effector functions. However, there is accumulating evidence that NK cells become dysfunctional during chronic inflammatory diseases, such as human breast cancer. This dysfunction is apparent in peripheral blood NK cells and can impact on normal NK cell immune responses and their effective targeting during immunotherapy. Herein, we demonstrate that prolonged cytokine stimulation combined with metabolic restriction, through inhibition of mTORC1, is sufficient to induce persistent dysfunction in human NK cells. TGFβ, also restricted NK cell metabolism and promoted persistent NK cell dysfunction. NK cells from patients with metastatic breast cancer had profound metabolic defects in glycolysis and mitochondrial function, and clear structural differences in NK cell mitochondrial morphology. Importantly, blocking elevated TGFβ improved readouts of metabolism and restored IFNγ production in patient NK cells.