Human Macrophages Activate Bystander Neutrophils' Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis.

Neutrophils are dynamic cells, playing a critical role in pathogen clearance; however, neutrophil infiltration into the tissue can act as a double-edged sword. They are one of the primary sources of excessive inflammation during infection, which has been observed in many infectious diseases including pneumonia and active tuberculosis (TB). Neutrophil function is influenced by interactions with other immune cells within the inflammatory lung milieu; however, how these interactions affect neutrophil function is unclear. Our study examined the macrophage-neutrophil axis by assessing the effects of conditioned medium (MΦ-CM) from primary human monocyte-derived macrophages (hMDMs) stimulated with LPS or a whole bacterium (Mycobacterium tuberculosis) on neutrophil function. Stimulated hMDM-derived MΦ-CM boosts neutrophil activation, heightening oxidative and glycolytic metabolism, but diminishes migratory potential. These neutrophils exhibit increased ROS production, elevated NET formation, and heightened CXCL8, IL-13, and IL-6 compared to untreated or unstimulated hMDM-treated neutrophils. Collectively, these data show that MΦ-CM from stimulated hMDMs activates neutrophils, bolsters their energetic profile, increase effector and inflammatory functions, and sequester them at sites of infection by decreasing their migratory capacity. These data may aid in the design of novel immunotherapies for severe pneumonia, active tuberculosis and other diseases driven by pathological inflammation mediated by the macrophage-neutrophil axis.

Acidosis significantly alters immune checkpoint expression profiles of T cells from oesophageal adenocarcinoma patients.

Tumour acidosis contributes to cancer progression by inhibiting anti-tumour immunity. However, the effect of acidosis on anti-tumour T cell phenotypes in oesophageal adenocarcinoma (OAC) is unknown. Therefore, this study investigated the effect of acidosis on anti-tumour T cell profiles and if immune checkpoint blockade (ICB) could enhance anti-tumour T cell immunity under acidosis. Acidic conditions substantially altered immune checkpoint expression profiles of OAC patient-derived T cells, upregulating TIM-3, LAG-3 and CTLA-4. Severe acidosis (pH 5.5) significantly decreased the percentage of central memory CD4+ T cells, an effect that was attenuated by ICB treatment. ICB increased T cell production of IFN-γ under moderate acidosis (pH 6.6) but not severe acidosis (pH 5.5) and decreased IL-10 production by T cells under severe acidic conditions only. A link between lactate and metastasis was also depicted; patients with nodal metastasis had higher serum lactate levels (p = 0.07) which also positively correlated with circulating levels of pro-angiogenic factor Tie-2. Our findings establish that acidosis-induced upregulation of immune checkpoints on T cells may potentially contribute to immune evasion and disease progression in OAC. However, acidic conditions curtailed ICB efficacy, supporting a rationale for utilizing systemic oral buffers to neutralize tumour acidity to improve ICB efficacy. Study schematic-PBMCs were isolated from OAC patients (A) and expanded ex vivo for 7 days using anti-CD3/28 +IL-2 T cell activation protocol (B) and further cultured for 48 h under increasing acidic conditions in the absence or presence of immune checkpoint blockade (nivolumab, ipilimumab or dual nivolumab + ipilimumab) (C). Immunophenotyping was then carried out to assess immune checkpoint expression profiles and anti-tumour T cell phenotypes (D). Serum lactate was assessed in OAC patients (E-F) and levels were correlated with patient demographics (G) and the levels of circulating immune/pro-angiogenic cytokines that were determined by multiplex ELISA (H). Key Findings-severe acidic conditions upregulated multiple immune checkpoints on T cells (I). Efficacy of ICB was curtailed under severe acidic conditions (J). Circulating lactate levels positively correlated with circulating levels of pro-angiogenic factor tie-2 and higher serum lactate levels were found in patients who had nodal metastasis (K).

The Iron Chelator Desferrioxamine Increases the Efficacy of Bedaquiline in Primary Human Macrophages Infected with BCG.

For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1ß in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.

The Effect of Tuberculosis Antimicrobials on the Immunometabolic Profiles of Primary Human Macrophages Stimulated with Mycobacterium tuberculosis.

Tuberculosis (TB) remains a global health challenge. Patients with drug-sensitive and drug-resistant TB undergo long, arduous, and complex treatment regimens, often involving multiple antimicrobials. While these drugs were initially implemented based on their bactericidal effects, some studies show that TB antimicrobials can also directly affect cells of the immune system, altering their immune function. As use of these antimicrobials has been the mainstay of TB therapy for over fifty years now, it is more important than ever to understand how these antimicrobials affect key pathways of the immune system. One such central pathway, which underpins the immune response to a variety of infections, is immunometabolism, namely glycolysis and oxidative phosphorylation (OXPHOS). We hypothesise that in addition to their direct bactericidal effect on Mycobacterium tuberculosis (Mtb), current TB antimicrobials can modulate immunometabolic profiles and alter mitochondrial function in primary human macrophages. Human monocyte-derived macrophages (hMDMs) were differentiated from PBMCs isolated from healthy blood donors, and treated with four first-line and six second-line TB antimicrobials three hours post stimulation with either iH37Rv-Mtb or lipopolysaccharide (LPS). 24 h post stimulation, baseline metabolism and mitochondrial function were determined using the Seahorse Extracellular Flux Analyser. The effect of these antimicrobials on cytokine and chemokine production was also assayed using Meso Scale Discovery Multi-Array technology. We show that some of the TB antimicrobials tested can significantly alter OXPHOS and glycolysis in uninfected, iH37Rv-Mtb, and LPS-stimulated hMDMs. We also demonstrate how these antimicrobial-induced immunometabolic effects are linked with alterations in mitochondrial function. Our results show that TB antimicrobials, specifically clofazimine, can modify host immunometabolism and mitochondrial function. Moreover, clofazimine significantly increased the production of IL-6 in human macrophages that were stimulated with iH37Rv-Mtb. This provides further insight into the use of some of these TB antimicrobials as potential host-directed therapies in patients with early and active disease, which could help to inform TB treatment strategies in the future.

Combining 1,4-dihydroxy quininib with Bevacizumab/FOLFOX alters angiogenic and inflammatory secretions in ex vivo colorectal tumors.

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer-related mortality worldwide with one in every five patients diagnosed with metastatic CRC (mCRC). In mCRC cases, the 5-year survival rate remains at approximately 14%, reflecting the lack of effectiveness of currently available treatments such as the anti-VEGF targeting antibody Bevacizumab combined with the chemotherapy folinic acid, fluorouracil and oxaliplatin (FOLFOX). Approximately 60% of patients do not respond to this combined treatment. Furthermore, Bevacizumab inhibits dendritic cell (DC) maturation in poor responders, a key process for tumor eradication. METHOD: Following drug treatment, secreted expression levels of angiogenic and inflammatory markers in tumor conditioned media generated from human ex vivo colorectal tumors were measured by ELISA. Dendritic cell phenotypic and maturation markers were assessed by flow cytometry. RESULTS: Our novel compound, 1,4-dihydroxy quininib, acts in an alternative pathway compared to the approved therapy Bevacizumab. 1,4-dihydroxy quininib alone, and in combination with Bevacizumab or FOLFOX significantly reduced TIE-2 expression which is involved in the promotion of tumor vascularization. Combination treatment with 1,4-dihydroxy quininib significantly increased the expression level of DC phenotypic and maturation markers. CONCLUSION: Our results indicate the anti-angiogenic small molecule 1,4-dihydroxy quininib could be an alternative novel treatment in combination therapy for CRC patients.

Estimating influenza incidence using search query deceptiveness and generalized ridge regression.

Seasonal influenza is a sometimes surprisingly impactful disease, causing thousands of deaths per year along with much additional morbidity. Timely knowledge of the outbreak state is valuable for managing an effective response. The current state of the art is to gather this knowledge using in-person patient contact. While accurate, this is time-consuming and expensive. This has motivated inquiry into new approaches using internet activity traces, based on the theory that lay observations of health status lead to informative features in internet data. These approaches risk being deceived by activity traces having a coincidental, rather than informative, relationship to disease incidence; to our knowledge, this risk has not yet been quantitatively explored. We evaluated both simulated and real activity traces of varying deceptiveness for influenza incidence estimation using linear regression. We found that deceptiveness knowledge does reduce error in such estimates, that it may help automatically-selected features perform as well or better than features that require human curation, and that a semantic distance measure derived from the Wikipedia article category tree serves as a useful proxy for deceptiveness. This suggests that disease incidence estimation models should incorporate not only data about how internet features map to incidence but also additional data to estimate feature deceptiveness. By doing so, we may gain one more step along the path to accurate, reliable disease incidence estimation using internet data. This capability would improve public health by decreasing the cost and increasing the timeliness of such estimates.

Clinical and molecular consequences of disease-associated de novo mutations in SATB2.

PURPOSE: To characterize features associated with de novo mutations affecting SATB2 function in individuals ascertained on the basis of intellectual disability. METHODS: Twenty previously unreported individuals with 19 different SATB2 mutations (11 loss-of-function and 8 missense variants) were studied. Fibroblasts were used to measure mutant protein production. Subcellular localization and mobility of wild-type and mutant SATB2 were assessed using fluorescently tagged protein. RESULTS: Recurrent clinical features included neurodevelopmental impairment (19/19), absent/near absent speech (16/19), normal somatic growth (17/19), cleft palate (9/19), drooling (12/19), and dental anomalies (8/19). Six of eight missense variants clustered in the first CUT domain. Sibling recurrence due to gonadal mosaicism was seen in one family. A nonsense mutation in the last exon resulted in production of a truncated protein retaining all three DNA-binding domains. SATB2 nuclear mobility was mutation-dependent; p.Arg389Cys in CUT1 increased mobility and both p.Gly515Ser in CUT2 and p.Gln566Lys between CUT2 and HOX reduced mobility. The clinical features in individuals with missense variants were indistinguishable from those with loss of function. CONCLUSION: SATB2 haploinsufficiency is a common cause of syndromic intellectual disability. When mutant SATB2 protein is produced, the protein appears functionally inactive with a disrupted pattern of chromatin or matrix association.Genet Med advance online publication 02 February 2017.

Mutations in KIF11 cause autosomal-dominant microcephaly variably associated with congenital lymphedema and chorioretinopathy.

We have identified KIF11 mutations in individuals with syndromic autosomal-dominant microcephaly associated with lymphedema and/or chorioretinopathy. Initial whole-exome sequencing revealed heterozygous KIF11 mutations in three individuals with a combination of microcephaly and lymphedema from a microcephaly-lymphedema-chorioretinal-dysplasia cohort. Subsequent Sanger sequencing of KIF11 in a further 15 unrelated microcephalic probands with lymphedema and/or chorioretinopathy identified additional heterozygous mutations in 12 of them. KIF11 encodes EG5, a homotetramer kinesin motor. The variety of mutations we have found (two nonsense, two splice site, four missense, and six indels causing frameshifts) are all predicted to have an impact on protein function. EG5 has previously been shown to play a role in spindle assembly and function, and these findings highlight the critical role of proteins necessary for spindle formation in CNS development. Moreover, identification of KIF11 mutations in patients with chorioretinopathy and lymphedema suggests that EG5 is involved in the development and maintenance of retinal and lymphatic structures.

Mutation in vascular endothelial growth factor-C, a ligand for vascular endothelial growth factor receptor-3, is associated with autosomal dominant milroy-like primary lymphedema.

RATIONALE: Mutations in vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3 or FLT4) cause Milroy disease, an autosomal dominant condition that presents with congenital lymphedema. Mutations in VEGFR3 are identified in only 70% of patients with classic Milroy disease, suggesting genetic heterogeneity. OBJECTIVE: To investigate the underlying cause in patients with clinical signs resembling Milroy disease in whom sequencing of the coding region of VEGFR3 did not reveal any pathogenic variation. METHODS AND RESULTS: Exome sequencing of 5 such patients was performed, and a novel frameshift variant, c.571_572insTT in VEGFC, a ligand for VEGFR3, was identified in 1 proband. The variant cosegregated with the affected status in the family. An assay to assess the biological function of VEGFC activity in vivo, by expressing human VEGFC in the zebrafish floorplate was established. Forced expression of wild-type human VEGFC in the floorplate of zebrafish embryos leads to excessive sprouting in neighboring vessels. However, when overexpressing the human c.571_572insTT variant in the floorplate, no sprouting of vessels was observed, indicating that the base changes have a marked effect on the activity of VEGFC. CONCLUSIONS: We propose that the mutation in VEGFC is causative for the Milroy disease-like phenotype seen in this family. This is the first time a mutation in one of the ligands of VEGFR3 has been reported to cause primary lymphedema.

Obesity, Dietary Fats, and Gastrointestinal Cancer Risk-Potential Mechanisms Relating to Lipid Metabolism and Inflammation.

Obesity is a major driving factor in the incidence, progression, and poor treatment response in gastrointestinal cancers. Herein, we conducted a comprehensive analysis of the impact of obesity and its resulting metabolic perturbations across four gastrointestinal cancer types, namely, oesophageal, gastric, liver, and colorectal cancer. Importantly, not all obese phenotypes are equal. Obese adipose tissue heterogeneity depends on the location, structure, cellular profile (including resident immune cell populations), and dietary fatty acid intake. We discuss whether adipose heterogeneity impacts the tumorigenic environment. Dietary fat quality, in particular saturated fatty acids, promotes a hypertrophic, pro-inflammatory adipose profile, in contrast to monounsaturated fatty acids, resulting in a hyperplastic, less inflammatory adipose phenotype. The purpose of this review is to examine the impact of obesity, including dietary fat quality, on adipose tissue biology and oncogenesis, specifically focusing on lipid metabolism and inflammatory mechanisms. This is achieved with a particular focus on gastrointestinal cancers as exemplar models of obesity-associated cancers.

Real-time ex vivo monitoring of NK cell migration toward obesity-associated oesophageal adenocarcinoma following modulation of CX3CR1.

Oesophagogastric adenocarcinomas (OAC) are poor prognosis, obesity-associated cancers which may benefit from natural killer (NK) cell-based immunotherapies. Cellular immunotherapies encounter two key challenges to their success in OAC, namely recruitment to extratumoural tissues such as the omentum at the expense of the tumour and an immunosuppressive tumour microenvironment (TME) which can hamper NK cell function. Herein, we examined approaches to overcome the detrimental impact of obesity on NK cells and NK cell-based immunotherapies. We have demonstrated that NK cells migrate preferentially to the chemotactic signals of OAC patient-derived omentum over tumour in an ex vivo model of immune cell migration. We have identified CX3CR1 modulation and/or tumour chemokine profile remodelling as approaches to skew NK cell migration towards tumour. We also report targetable immunosuppressive facets of the obese OAC TME which dampen NK cell function, in particular cytotoxic capabilities. These data provide insights into approaches to therapeutically overcome key challenges presented by obesity and will inform superior design of NK cell-based immunotherapies for OAC.

FLT4/VEGFR3 and Milroy disease: novel mutations, a review of published variants and database update.

Milroy disease (MD) is an autosomal dominantly inherited primary lymphedema. In 1998, the gene locus for MD was mapped to 5q35.3 and variants in the VEGFR3 (FLT4) gene, encoding vascular endothelial growth factor receptor 3 (VEGFR3), were identified as being responsible for the majority of MD cases. Several reports have since been published detailing pathogenic FLT4 mutations. To date, a total of 58 different variants in FLT4, 20 of which are unpublished, have been observed in 95 families with MD. A review of published mutations is presented in this update. Furthermore, the unpublished variants are presented including clinical data. Comparison of clinical features in patients and their families with the same mutations reveals incomplete penetrance and variable expression, making genotype-phenotype correlations difficult. Most mutations are missense, but a few deletions and one splicing variant have also been reported. Several animal models have confirmed the role of VEGFR3 in lymphangiogenesis and studies show mutant VEGFR3 receptors are not phosphorylated. Here, an MD patient with the same p.Ile1053Phe change as seen in the Chy mouse is presented for the first time. This finding confirms that this mouse lineage is an excellent model for MD. All the data reviewed here has been submitted to a database based on the Leiden Open (source) Variation Database (LOVD) and is accessible online at www.lovd.nl/flt4.

Visceral adipose tissue secretome from early and late-stage oesophageal cancer patients differentially affects effector and regulatory T cells.

AIM: Visceral obesity is a key risk factor in the development of oesophagogastric junctional adenocarcinoma (OGJ), predominantly via generation of systemic low grade inflammation. Obesity-induced inflammation promotes resistance to current standards of care, enhancing tumour cell growth and survival. This study investigates the effect of the visceral adipose tissue secretome from OGJ patients with early versus advanced tumours on T-cell immunity and the role of immune checkpoint blockade in enhancing anti-tumour immunity. METHODS AND RESULTS: Visceral adipose conditioned media (ACM) from both early and late-stage OGJ patients significantly altered T cell activation status, upregulating co-stimulatory marker CD27 on T cells. ACM from both early and late-stage OGJ patients significantly altered immune checkpoint expression profiles downregulating immune checkpoints (ICs) on the surface of dual Th1/17-like and Th17-like cells and upregulating ICs on the surface of Th1-like cells and Treg cells. ACM derived from early-stage OGJ patients but not late-stage OGJ patients increased IFN-γ production by T cells. The addition of immune checkpoint blockers (ICBs) did not increase IFN-γ production by T cells in the presence of late-stage ACM, collectively highlighting the dichotomous immunostimulatory effect of early-stage ACM and immune-inhibitory effect of late-stage ACM. Interestingly, ACM from early-stage OGJ patients was more pro-inflammatory than ACM from late-stage patients, reflected by decreased levels of IL-17A/F, TNF-α, IL-1RA and IL-5. CONCLUSION: The ACM-induced upregulation of ICs on T cells highlights a therapeutic vulnerability that could be exploited by ICBs to harness anti-cancer immunity and improve clinical outcomes for OGJ patients. Schematic workflow - (A) visceral adipose tissue was taken from OAC patients at time of surgery and cultured for 72 h in media. (B) The harvested ACM was co-cultured with healthy donor PBMCs that were concurrently activated with anti-CD3/28 for 48 h and T cell immunophenotyping was carried out by flow cytometry. Key findings - (A) Early and late stage ACM enhanced a Th1-like phenotype and upregulated CTLA-4 on Th1-like cells. A Th17-like phenotype was also enhanced in addition with a Treg-like phenotype. CTLA-4 and PD-L1 were upregulated on the surface of Treg-like cells. (B) ICB-attenuated IL-17 production by T cells. However, ACM attenuated ICB-mediated reduction in IL-10 production by T cells. Higher levels of pro-inflammatory factors were found in early stage ACM compared with late stage ACM.

FLOT and CROSS chemotherapy regimens alter the frequency of CD27+ and CD69+ T cells in oesophagogastric adenocarcinomas: implications for combination with immunotherapy.

Combining immunostimulatory chemotherapies with immunotherapy is an attractive strategy to enhance treatment responses in oesophagogastric junctional adenocarcinoma (OGJ). This study investigates the immunostimulatory properties of FLOT, CROSS and MAGIC chemotherapy regimens in the context of OGJ using in vitro and ex vivo models of the treatment-naïve and post-chemotherapy treated tumour microenvironment. FLOT and CROSS chemotherapy regimens increased surrogate markers of immunogenic cell death (HMGB1 and HLA-DR), whereas the MAGIC treatment regimen decreased HMGB1 and HLA-DR on OGJ cells (markedly for epirubicin). Tumour-infiltrating and circulating T cells had significantly lower CD27 expression and significantly higher CD69 expression post-FLOT and post-CROSS treatment. Similarly, the supernatant from FLOT- and CROSS-treated OGJ cell lines and from FLOT- and CROSS-treated OGJ biopsies cultured ex vivo also decreased CD27 and increased CD69 expression on T cells. Following 48 h treatment with post-FLOT and post-CROSS tumour conditioned media the frequency of CD69+ T cells in culture negatively correlated with the levels of soluble immunosuppressive pro-angiogenic factors in the conditioned media from ex vivo explants. Supernatant from FLOT- and CROSS-treated OGJ cell lines also increased the cytotoxic potential of healthy donor T cells ex vivo and enhanced OGJ patient-derived lymphocyte mediated-killing of OE33 cells ex vivo. Collectively, this data demonstrate that FLOT and CROSS chemotherapy regimens possess immunostimulatory properties, identifying these chemotherapy regimens as rational synergistic partners to test in combination with immunotherapy and determine if this combinatorial approach could boost anti-tumour immunity in OGJ patients and improve clinical outcomes.

PD-1 blockade attenuates surgery-mediated immunosuppression and boosts Th1 immunity perioperatively in oesophagogastric junctional adenocarcinoma.

Introduction: This timely study assesses the immunosuppressive effects of surgery on cytotoxic Th1-like immunity and investigates if immune checkpoint blockade (ICB) can boost Th1-like immunity in the perioperative window in upper gastrointestinal cancer (UGI) patients. Methods: PBMCs were isolated from 11 UGI patients undergoing tumour resection on post-operative days (POD) 0, 1, 7 and 42 and expanded ex vivo using anti-CD3/28 and IL-2 for 5 days in the absence/presence of nivolumab or ipilimumab. T cells were subsequently immunophenotyped via flow cytometry to determine the frequency of T helper (Th)1-like, Th1/17-like, Th17-like and regulatory T cell (Tregs) subsets and their immune checkpoint expression profile. Lymphocyte secretions were also assessed via multiplex ELISA (IFN-γ, granzyme B, IL-17 and IL-10). The 48h cytotoxic ability of vehicle-, nivolumab- and ipilimumab-expanded PBMCs isolated on POD 0, 1, 7 and 42 against radiosensitive and radioresistant oesophageal adenocarcinoma tumour cells (OE33 P and OE33 R) was also examined using a cell counting kit-8 (CCK-8) assay to determine if surgery affected the killing ability of lymphocytes and whether the use of ICB could enhance cytotoxicity. Results: Th1-like immunity was suppressed in expanded PBMCs in the immediate post-operative setting. The frequency of expanded circulating Th1-like cells was significantly decreased post-operatively accompanied by a decrease in IFN-γ production and a concomitant increase in the frequency of expanded regulatory T cells with an increase in circulating levels of IL-10. Interestingly, PD-L1 and CTLA-4 immune checkpoint proteins were also upregulated on expanded Th1-like cells post-operatively. Additionally, the cytotoxic ability of expanded lymphocytes against oesophageal adenocarcinoma tumour cells was abrogated post-surgery. Of note, the addition of nivolumab or ipilimumab attenuated the surgery-mediated suppression of lymphocyte cytotoxicity, demonstrated by a significant increase in tumour cell killing and an increase in the frequency of Th1-like cells and Th1 cytokine production. Conclusion: These findings support the hypothesis of a surgery-mediated suppression in Th1-like cytotoxic immunity and highlights a rationale for the use of ICB within the perioperative setting to abrogate tumour-promoting effects of surgery and ameliorate the risk of recurrence.

Nutrient deprivation and hypoxia alter T cell immune checkpoint expression: potential impact for immunotherapy.

AIM: Use of immune checkpoint blockade to enhance T cell-mediated immunity within the hostile tumour microenvironment (TME) is an attractive approach in oesophageal adenocarcinoma (OAC). This study explored the effects of the hostile TME, including nutrient deprivation and hypoxia, on immune checkpoint (IC) expression and T cell phenotypes, and the potential use of nivolumab to enhance T cell function under such conditions. METHODS AND RESULTS: ICs were upregulated on stromal immune cells within the tumour including PD-L2, CTLA-4 and TIGIT. OAC patient-derived PBMCs co-cultured with OE33 OAC cells upregulated LAG-3 and downregulated the co-stimulatory marker CD27 on T cells, highlighting the direct immunosuppressive effects of tumour cells on T cells. Hypoxia and nutrient deprivation altered the secretome of OAC patient-derived PBMCs, which induced upregulation of PD-L1 and PD-L2 on OE33 OAC cells thus enhancing an immune-resistant phenotype. Importantly, culturing OAC patient-derived PBMCs under dual hypoxia and glucose deprivation, reflective of the conditions within the hostile TME, upregulated an array of ICs on the surface of T cells including PD-1, CTLA-4, A2aR, PD-L1 and PD-L2 and decreased expression of IFN-γ by T cells. Addition of nivolumab under these hostile conditions decreased the production of pro-tumorigenic cytokine IL-10. CONCLUSION: Collectively, these findings highlight the immunosuppressive crosstalk between tumour cells and T cells within the OAC TME. The ability of nivolumab to suppress pro-tumorigenic T cell phenotypes within the hostile TME supports a rationale for the use of immune checkpoint blockade to promote anti-tumour immunity in OAC. Study schematic: (A) IC expression profiles were assessed on CD45+ cells in peripheral whole blood and infiltrating tumour tissue from OAC patients in the treatment-naïve setting. (B) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then co-cultured for 48 h with OE33 cells. T cell phenotypes were then assessed by flow cytometry. (C) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then further cultured under conditions of nutrient deprivation or hypoxia for 48 h and T cell phenotypes were then assessed by flow cytometry. KEY FINDINGS: (A) TIGIT, CTLA-4 and PD-L2 were upregulated on CD45+ immune cells and CTLA-4 expression on CD45+ cells correlated with a subsequent decreased response to neoadjuvant regimen. (B) Following a 48 h co-culture with OE33 cells, T cells upregulated LAG-3 and decreased CD27 co-stimulatory marker. (C) Nutrient deprivation and hypoxia upregulated a range of ICs on T cells and decreased IFN-γ production by T cells. Nivolumab decreased IL-10 production by T cells under nutrient deprivation-hypoxic conditions.

Energy Metabolism, Metabolite, and Inflammatory Profiles in Human Ex Vivo Adipose Tissue Are Influenced by Obesity Status, Metabolic Dysfunction, and Treatment Regimes in Patients with Oesophageal Adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is a poor prognosis cancer with limited response rates to current treatment modalities and has a strong link to obesity. To better elucidate the role of visceral adiposity in this disease state, a full metabolic profile combined with analysis of secreted pro-inflammatory cytokines, metabolites, and lipid profiles were assessed in human ex vivo adipose tissue explants from obese and non-obese OAC patients. These data were then related to extensive clinical data including obesity status, metabolic dysfunction, previous treatment exposure, and tumour regression grades. Real-time energy metabolism profiles were assessed using the seahorse technology. Adipose explant conditioned media was screened using multiplex ELISA to assess secreted levels of 54 pro-inflammatory mediators. Targeted secreted metabolite and lipid profiles were analysed using Ultra-High-Performance Liquid Chromatography coupled with Mass Spectrometry. Adipose tissue explants and matched clinical data were collected from OAC patients (n = 32). Compared to visceral fat from non-obese patients (n = 16), visceral fat explants from obese OAC patients (n = 16) had significantly elevated oxidative phosphorylation metabolism profiles and an increase in Eotaxin-3, IL-17A, IL-17D, IL-3, MCP-1, and MDC and altered secretions of glutamine associated metabolites. Adipose explants from patients with metabolic dysfunction correlated with increased oxidative phosphorylation metabolism, and increases in IL-5, IL-7, SAA, VEGF-C, triacylglycerides, and metabolites compared with metabolically healthy patients. Adipose explants generated from patients who had previously received neo-adjuvant chemotherapy (n = 14) showed elevated secretions of pro-inflammatory mediators, IL-12p40, IL-1α, IL-22, and TNF-ß and a decreased expression of triacylglycerides. Furthermore, decreased secreted levels of triacylglycerides were also observed in the adipose secretome of patients who received the chemotherapy-only regimen FLOT compared with patients who received no neo-adjuvant treatment or chemo-radiotherapy regimen CROSS. For those patients who showed the poorest response to currently available treatments, their adipose tissue was associated with higher glycolytic metabolism compared to patients who had good treatment responses. This study demonstrates that the adipose secretome in OAC patients is enriched with mediators that could prime the tumour microenvironment to aid tumour progression and attenuate responses to conventional cancer treatments, an effect which appears to be augmented by obesity and metabolic dysfunction and exposure to different treatment regimes.

Ergolide mediates anti-cancer effects on metastatic uveal melanoma cells and modulates their cellular and extracellular vesicle proteomes.

Background: Uveal melanoma is a poor prognosis cancer. Ergolide, a sesquiterpene lactone isolated from Inula Brittanica, exerts anti-cancer properties. The objective of this study was to 1) evaluate whether ergolide reduced metastatic uveal melanoma (MUM) cell survival/viability in vitro and in vivo; and 2) to understand the molecular mechanism of ergolide action. Methods: Ergolide bioactivity was screened via long-term proliferation assay in UM/MUM cells and in zebrafish MUM xenograft models. Mass spectrometry profiled proteins modulated by ergolide within whole cell or extracellular vesicle (EVs) lysates of the OMM2.5 MUM cell line. Protein expression was analyzed by immunoblots and correlation analyses to UM patient survival used The Cancer Genome Atlas (TCGA) data. Results: Ergolide treatment resulted in significant, dose-dependent reductions (48.5 to 99.9%; p<0.0001) in OMM2.5 cell survival in vitro and of normalized primary zebrafish xenograft fluorescence (56%; p<0.0001) in vivo, compared to vehicle controls. Proteome-profiling of ergolide-treated OMM2.5 cells, identified 5023 proteins, with 52 and 55 proteins significantly altered at 4 and 24 hours, respectively ( p<0.05; fold-change >1.2). Immunoblotting of heme oxygenase 1 (HMOX1) and growth/differentiation factor 15 (GDF15) corroborated the proteomic data. Additional proteomics of EVs isolated from OMM2.5 cells treated with ergolide, detected 2931 proteins. There was a large overlap with EV proteins annotated within the Vesiclepedia compendium. Within the differentially expressed proteins, the proteasomal pathway was primarily altered. Interestingly, BRCA2 and CDKN1A Interacting Protein (BCCIP) and Chitinase Domain Containing 1 (CHID1), were the only proteins significantly differentially expressed by ergolide in both the OMM2.5 cellular and EV isolates and they displayed inverse differential expression in the cells versus the EVs. Conclusions: Ergolide is a novel, promising anti-proliferative agent for UM/MUM. Proteomic profiling of OMM2.5 cellular/EV lysates identified candidate pathways elucidating the action of ergolide and putative biomarkers of UM, that require further examination.

The most common form of adult eye cancer is uveal melanoma (UM). Once UM cancer cells spread to organs in the rest of the body, metastatic UM (MUM), there is a poor prognosis for patients with only one approved drug treatment. Hence, it is vital to better understand the cellular and extracellular proteins that regulate UM pathology in order to uncover biomarkers of disease and therapeutic targets. In this original study, we demonstrate a compound called ergolide is capable of severely reducing the metabolic activity and growth of UM cancer cells, grown as isolated monolayers. Ergolide was also able to reduce the growth of human MUM cells growing as tumors in transplanted zebrafish larvae. We identify that ergolide alters specific proteins found in the human UM cells. These proteins once analyzed in detail offer opportunities to understand how new treatment strategies can be developed for UM.

Potential of damage associated molecular patterns in synergising radiation and the immune response in oesophageal cancer.

BACKGROUND: There is an intimate crosstalk between cancer formation, dissemination, treatment response and the host immune system, with inducing tumour cell death the ultimate therapeutic goal for most anti-cancer treatments. However, inducing a purposeful synergistic response between conventional therapies and the immune system remains evasive. The release of damage associated molecular patterns (DAMPs) is indicative of immunogenic cell death and propagation of established immune responses. However, there is a gap in the literature regarding the importance of DAMP expression in oesophageal adenocarcinoma (OAC) or by immune cells themselves. AIM: To investigate the effects of conventional therapies on DAMP expression and to determine whether OAC is an immunogenic cancer. METHODS: We investigated the levels of immunogenic cell death-associated DAMPs, calreticulin (CRT) and HMGB1 using an OAC isogenic model of radioresistance. DAMP expression was also assessed directly using ex vivo cancer patient T cells (n = 10) and within tumour biopsies (n = 9) both pre and post-treatment with clinically relevant chemo(radio)therapeutics. RESULTS: Hypoxia in combination with nutrient deprivation significantly reduces DAMP expression by OAC cells in vitro. Significantly increased frequencies of T cell DAMP expression in OAC patients were observed following chemo(radio)therapy, which was significantly higher in tumour tissue compared with peripheral blood. Patients with high expression of HMGB1 had a significantly better tumour regression grade (TRG 1-2) compared to low expressors. CONCLUSION: In conclusion, OAC expresses an immunogenic phenotype with two distinct subgroups of high and low DAMP expressors, which correlated with tumour regression grade and lymphatic invasion. It also identifies DAMPs namely CRT and HMGB1 as potential promising biomarkers in predicting good pathological responses to conventional chemo(radio)therapies currently used in the multimodal management of locally advanced disease.

Metformin is a metabolic modulator and radiosensitiser in rectal cancer.

Resistance to neoadjuvant chemoradiation therapy, is a major challenge in the management of rectal cancer. Increasing evidence supports a role for altered energy metabolism in the resistance of tumours to anti-cancer therapy, suggesting that targeting tumour metabolism may have potential as a novel therapeutic strategy to boost treatment response. In this study, the impact of metformin on the radiosensitivity of colorectal cancer cells, and the potential mechanisms of action of metformin-mediated radiosensitisation were investigated. Metformin treatment was demonstrated to significantly radiosensitise both radiosensitive and radioresistant colorectal cancer cells in vitro. Transcriptomic and functional analysis demonstrated metformin-mediated alterations to energy metabolism, mitochondrial function, cell cycle distribution and progression, cell death and antioxidant levels in colorectal cancer cells. Using ex vivo models, metformin treatment significantly inhibited oxidative phosphorylation and glycolysis in treatment naïve rectal cancer biopsies, without affecting the real-time metabolic profile of non-cancer rectal tissue. Importantly, metformin treatment differentially altered the protein secretome of rectal cancer tissue when compared to non-cancer rectal tissue. Together these data highlight the potential utility of metformin as an anti-metabolic radiosensitiser in rectal cancer.