Separate lifetime signatures of macaque S cones, M/L cones, and rods observed with adaptive optics fluorescence lifetime ophthalmoscopy.

In the retina, several molecules involved in metabolism, the visual cycle, and other roles exhibit intrinsic fluorescence. The overall properties of retinal fluorescence depend on changes to the composition of these molecules and their environmental interactions due to transient functional shifts, especially in disease. This behooves the understanding of the origins and deviations of these properties within the multilayered retina at high lateral and axial resolution. Of particular interest is the fluorescence lifetime, a potential biomarker of function and disease independent of fluorescence intensity that can be measured in the retina with adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO). This work demonstrates the utility of the phasor method of analysis, an alternate approach to traditional multiexponential fitting, to evaluate photoreceptor two-photon excited AOFLIO data and separate them based on functional differences. Phasor analysis on fluorescence lifetime decay data allowed the repeatable segregation of S from M/L cones, likely from differences in functional or metabolic demands. Furthermore, it is possible to track the lifetime changes in S cones after photodamage. Phasor analysis increases the sensitivity of AOFLIO to functional differences between cells and has the potential to improve our understanding of pathways involved in normal and diseased conditions at the cellular scale throughout the retina.

Effect of the Rehabilitation Program, ReStOre, on Serum Biomarkers in a Randomized Control Trial of Esophagogastric Cancer Survivors.

BACKGROUND: The Rehabilitation Strategies Following Esophagogastric cancer (ReStOre) randomized control trial demonstrated a significant improvement in cardiorespiratory fitness of esophagogastric cancer survivors. This follow-up, exploratory study analyzed the biological effect of exercise intervention on levels of 55 serum proteins, encompassing mediators of angiogenesis, inflammation, and vascular injury, from participants on the ReStOre trial. METHODS: Patients >6 months disease free from esophagogastric cancer were randomized to usual care or the 12-week ReStOre program (exercise training, dietary counselling, and multidisciplinary education). Serum was collected at baseline (T0), post-intervention (T1), and at 3-month follow up (T2). Serum biomarkers were quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirty-seven patients participated in this study; 17 in the control arm and 20 in the intervention arm. Exercise intervention resulted in significant alterations in the level of expression of serum IP-10 (mean difference (MD): 38.02 (95% CI: 0.69 to 75.35)), IL-27 (MD: 249.48 (95% CI: 22.43 to 476.53)), and the vascular injury biomarkers, ICAM-1 (MD: 1.05 (95% CI: 1.07 to 1.66)), and VCAM-1 (MD: 1.51 (95% CI: 1.04 to 2.14)) at T1. A significant increase in eotaxin-3 (MD: 2.59 (95% CI: 0.23 to 4.96)), IL-15 (MD: 0.27 (95% CI: 0 to 0.54)) and decrease in bFGF (MD: 1.62 (95% CI: -2.99 to 0.26)) expression was observed between control and intervention cohorts at T2 (p<0.05). CONCLUSIONS: Exercise intervention significantly altered the expression of a number of serum biomarkers in disease-free patients who had prior treatment for esophagogastric cancer. IMPACT: Exercise rehabilitation causes a significant biological effect on serum biomarkers in esophagogastric cancer survivors. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT03314311).

Tissue distribution of γδ T cell subsets in oesophageal adenocarcinoma.

The global obesity epidemic is contributing to increased prevalence of diseases fuelled by chronic inflammation, including cancer. Oesophageal adenocarcinoma (OAC) is an obesity-associated malignancy with increasing prevalence, dismal prognosis, and severely dysregulated immune processes. We previously reported that αß T cells migrate to omentum and liver in OAC and contribute to inflammation in these tissues. Here, we assessed the tissue distribution and phenotype of gamma/delta (γδ) T cells in the blood, omentum, liver and tumour of OAC patients. Our data show that the Vδ1 and Vδ3 subsets of γδ T cells are most prevalent in omentum and liver of OAC patients. Furthermore, γδ T cells are predominantly pro-inflammatory in these tissues, and co-express IFN-γ and IL-17. Moreover, γδ T cells exhibit cytotoxic capabilities in OAC omentum and liver. This study provides the first indication that γδ T cells contribute to obesity-associated inflammation in OAC and might be exploited therapeutically.

Linking Circulating Serum Proteins with Clinical Outcomes in Esophageal Adenocarcinoma-An Emerging Role for Chemokines.

Esophageal adenocarcinoma (EAC) is an aggressive cancer with poor prognosis and incidence is increasing rapidly in the Western world. Multi-modal treatment has improved survival outcomes but only for a minority of patients. Currently no markers have been identified to predict treatment response. This study investigated the association between clinical outcomes and pre-treatment levels of 54 serum proteins in n = 80 patients with EAC. Low tumor regression grade (TRG), corresponding to a favorable treatment response, was linked to prolonged overall survival (OS). CCL4 was higher in patients with a favorable treatment response, while Tie2 and CRP were higher in poor responders. Elevated CCL22 and CCL26 was associated with improved OS, while elevated IL-10 showed a negative association. CCL3, CCL4, IL-1α and IL-12/IL23p40 were highest in individuals with no adverse features of tumor biology, whereas levels of Tie2 and VEGF were lowest in this cohort. CCL4 was also elevated in patients with high tumor lymphocyte infiltration. Comparison of matched pre- and post-treatment serum (n = 28) showed a large reduction in VEGFC, and a concomitant increase in other cytokines, including CCL4. These data link several serum markers with clinical outcomes, highlighting an important role for immune cell trafficking in the EAC antitumor immune response.

Real-time metabolic profiling of oesophageal tumours reveals an altered metabolic phenotype to different oxygen tensions and to treatment with Pyrazinib.

Oesophageal cancer is the 6th most common cause of cancer related death worldwide. The current standard of care for oesophageal adenocarcinoma (OAC) focuses on neoadjuvant therapy with chemoradiation or chemotherapy, however the 5-year survival rates remain at < 20%. To improve treatment outcomes it is critical to further investigate OAC tumour biology, metabolic phenotype and their metabolic adaptation to different oxygen tensions. In this study, by using human ex-vivo explants we demonstrated using real-time metabolic profiling that OAC tumour biopsies have a significantly higher oxygen consumption rate (OCR), a measure of oxidative phosphorylation compared to extracellular acidification rate (ECAR), a measure of glycolysis (p = 0.0004). Previously, we identified a small molecule compound, pyrazinib which enhanced radiosensitivity in OAC. Pyrazinib significantly inhibited OCR in OAC treatment-naïve biopsies (p = 0.0139). Furthermore, OAC biopsies can significantly adapt their metabolic rate in real-time to their environment. Under hypoxic conditions pyrazinib produced a significant reduction in both OCR (p = 0.0313) and ECAR in OAC treatment-naïve biopsies. The inflammatory secretome profile from OAC treatment-naïve biopsies is heterogeneous. OCR was positively correlated with three secreted factors in the tumour conditioned media: vascular endothelial factor A (VEGF-A), IL-1RA and thymic stromal lymphopoietin (TSLP). Pyrazinib significantly inhibited IL-1ß secretion (p = 0.0377) and increased IL-3 (p = 0.0020) and IL-17B (p = 0.0181). Importantly, pyrazinib did not directly alter the expression of dendritic cell maturation markers or reduce T-cell viability or activation markers. We present a new method for profiling the metabolic rate of tumour biopsies in real-time and demonstrate the novel anti-metabolic and anti-inflammatory action of pyrazinib ex-vivo in OAC tumours, supporting previous findings in-vitro whereby pyrazinib significantly enhanced radiosensitivity in OAC.

CD1d expression and invariant natural killer T-cell numbers are reduced in patients with upper gastrointestinal cancers and are further impaired by commonly used chemotherapies.

Esophageal and gastric cancers collectively cause over 1.1 million deaths annually and only 20-30% of patients respond favorably to current therapies. Cellular therapies using invariant natural killer T (iNKT) cells are showing promise for patients with other cancers; therefore, we investigated if these cells are altered in esophageal and gastric cancer patients. Flow cytometric analysis of peripheral blood from 139 patients revealed that iNKT cells are depleted from patients with esophageal and gastric adenocarcinoma and esophageal squamous cell carcinoma, both before and after treatment. Interrogation of the KMPlot database of transcriptomic data from 876 gastric cancer patients revealed that low CD1d expression is associated with poor prognosis. These observations suggest that therapies that boost CD1d expression and iNKT cell responses may benefit these patients. However, we found that chemotherapies used for esophageal and gastric cancers have adverse effects on iNKT cells in vitro. Cisplatin caused a significant reduction of CD1d expression by esophageal tumor cell lines. Cisplatin, 5-fluorouracil and carboplatin induced dose-dependent apoptosis in primary lines of iNKT cells and inhibited CD1d-dependent interferon-γ production and cytolytic degranulation by viable iNKT cells. Interestingly, cisplatin increased granzyme B and perforin production and decreased the production of the granzyme B inhibitor PI9, which protects cytotoxic cells from self-damage by granzyme B. Thus, cisplatin-induced apoptosis of iNKT cells may be mediated in part by altering granzyme B and PI9 expression. Our data suggest that iNKT cell-based immunotherapies may benefit patients with gastrointestinal cancers, but may be negatively affected by chemotherapies used for these cancers.

Mucosal-Associated Invariant T Cells Display Diminished Effector Capacity in Oesophageal Adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is an aggressive malignancy with poor prognosis, and incidence is increasing rapidly in the Western world. Mucosal-associated invariant T (MAIT) cells recognize bacterial metabolites and kill infected cells, yet their role in OAC is unknown. We aimed to elucidate the role of MAIT cells during cancer development by characterizing the frequency, phenotype, and function of MAIT cells in human blood and tissues, from OAC and its pre-malignant inflammatory condition Barrett's oesophagus (BO). Blood and tissues were phenotyped by flow cytometry and conditioned media from explanted tissue was used to model the effects of the tumor microenvironment on MAIT cell function. Associations were assessed between MAIT cell frequency, circulating inflammatory markers, and clinical parameters to elucidate the role of MAIT cells in inflammation driven cancer. MAIT cells were decreased in BO and OAC blood compared to healthy controls, but were increased in oesophageal tissues, compared to BO-adjacent tissue, and remained detectable after neo-adjuvant treatment. MAIT cells in tumors expressed CD8, PD-1, and NKG2A but lower NKG2D than BO cohorts. MAIT cells produced less IFN-γ and TNF-α in the presence of tumor-conditioned media. OAC cell line viability was reduced upon exposure to expanded MAIT cells. Serum levels of chemokine IP-10 were inversely correlated with MAIT cell frequency in both tumors and blood. MAIT cells were higher in the tumors of node-negative patients, but were not significantly associated with other clinical parameters. This study demonstrates that OAC tumors are infiltrated by MAIT cells, a type of CD8 T cell featuring immune checkpoint expression and cytotoxic potential. These findings may have implications for immunotherapy and immune scoring approaches.

Leukaemia inhibitory factor is associated with treatment resistance in oesophageal adenocarcinoma.

Oesophageal cancer is an aggressive disease with a poor 5 year survival rate of <20% of diagnosed patients. Unfortunately, only 20-30% Oesophageal Adenocarinoma (OAC) patients show a beneficial response to neoadjuvant therapy (neoCT). Inflammation influences OAC given the increased risk of cancer development and poor outcome for obese patients where altered secretion of adipokines and cytokines from adipose tissue contributes a pro-tumourigenic environment. We carried out a large proteomics screen of 184 proteins to compare the inflammatory and oncogenic profiles of an isogenic radioresistant in-vitro model of OAC. We found that leukaemia inhibitory factor (LIF), an IL-6 type cytokine, was significantly elevated in radioresistant OAC cells (p=0.007). Furthermore, significantly higher circulating levels of LIF were present in the serum from treatment-naive OAC patients who had a subsequent poor pathological response to neo-adjuvant therapy, (p=0.037). Quantitative PCR analysis revealed expression of LIF receptor (LIFR) may function as a predictive indicator of response to neo-adjuvant chemoradiation therapy in OAC. LIF was demonstrated to be actively secreted from human OAC treatment-naïve biopsies and significantly correlated with the secretion of bFGF, VEGF-A and IL-8 (p<0.05, R=1), (p<0.05, R=0.9429), and (p<0.05, R=1) respectively. Importantly, LIF secretion negatively correlated with tumour infiltrating lymphocytes in pre-treatment OAC patient biopsies, (r=-0.8783, p=0.033). Elevated circulating LIF is a marker of poor response to neo-adjuvant treatment in OAC and secretion of this chemokine from the tumour is tightly linked with pro-tumourigenic mediators including bFGF, VEGF-A and IL-8. Targeting this pathway may be a novel mechanism enhance neoadjuvant treatment responses in OAC.

Effects of a multimodal rehabilitation programme on inflammation and oxidative stress in oesophageal cancer survivors: the ReStOre feasibility study.

PURPOSE: Physical, nutritional and quality-of-life compromises are known sequelae of oesophageal cancer (OC) treatment. Inflammation and oxidative stress may be relevant to adverse consequences. Multimodal rehabilitation involving exercise and diet prescription may attenuate some of the negative consequences and optimise survivorship, and this was assessed in this feasibility study in OC patients at least 1 year post-oesophagectomy. METHODS: The 12-week programme included supervised and home-based exercise, dietetic counselling to ensure energy balance and multidisciplinary education. Baseline and post-intervention assessments examined aerobic fitness, physical activity and body composition. Serum interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, IL-6 and IL-8 were measured via multiplex arrays. Lactate secretion, lipid peroxidation (4-HNE) and oxidative stress (8-iso-PGF2α) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Twelve patients (mean (SD) age 64(1.29) years) participated. IL-8 reduced significantly from pre- to post-intervention (percentage change -11.25 % (95 % CI -20.98 to -1.51 %), p = 0.03), and there was a non-significant trend towards lower expression patterns of other inflammatory mediators. At baseline, inflammatory status correlated inversely with sedentary behaviour (IL-6 rho = -0.74, IL-8 rho = -0.59, TNF-α rho = -0.69; p < 0.05). While energy metabolism did not change, post-intervention lactate concentration correlated strongly and inversely with aerobic fitness (rho = -0.68, p = 0.02). Body composition was maintained throughout the intervention. CONCLUSIONS: Results suggest that multimodal rehabilitation following OC treatment reduced inflammatory status without compromising body composition. Findings will be further examined in a larger randomised controlled trial.