Increased monocyte-platelet aggregates and monocyte-endothelial adhesion in healthy individuals with vitamin D deficiency

Vitamin D deficiency is a major public health problem worldwide, linked to several chronic diseases including cardiovascular diseases. While immunomodulatory effects of vitamin D on monocytes have been reported in cardiovascular and metabolic diseases, there is limited understanding on monocyte phenotype in healthy individuals with suboptimal vitamin D levels and without any clinical diseases. In this work, we performed label-free, microfluidic isolation of monocytes, and characterized their functional phenotype using flow cytometry and in vitro vascular models in healthy subjects with (n = 7) and without vitamin D deficiency (n = 16). Vitamin D deficient (VitD-Def) subjects (25(OH)D3 level < 26 ng/mL) expressed significant downregulation of vitamin D receptor (VDR) on monocytes as compared to controls (P < .0001), and VDR expression was well-associated with serum 25(OH)D3 levels. Increased monocyte-platelet aggregates (MPA), a marker for platelet activation, were also observed in VitD-Def subjects (P < .05) which suggests a pro-inflammatory monocyte phenotype. Monocyte adhesion to endothelial cells, an early-stage atherosclerosis event, was also higher in VitD-Def individuals, and inversely correlated to serum 25(OH)D3 level (P < .05). Taken together, these results indicate the pro-inflammatory state and atherogenic potential of monocytes in VitD-Def healthy subjects, and propound the use of vitamin D supplementation as a prospective immunomodulatory and anti-inflammatory therapy in atherosclerosis.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Ministry of Health (MOH)National Medical Research Council (NMRC)This research is supported by the National Research Foundation Singapore under its CBRG-NIG (NMRC/BNIG/2038/2015) and administered by the Singapore Ministry of Health's, National Medical Research Council and Biomedical Research Council (BMRC), ASTAR. HWH would like to acknowledge support from an NTU start-up grant and the Singapore Ministry of Education (MOE) Academic Research Fund Tier 1 (RG53/18)

Label-free leukocyte sorting and impedance-based profiling for diabetes testing

Circulating leukocytes comprise of approximately 1% of all blood cells and efficient enrichment of these cells from whole blood is critical for understanding cellular heterogeneity and biological significance in health and diseases. In this work, we report a novel microfluidic strategy for rapid (< 1 h) label-free leukocyte sorting and impedance-based profiling to determine cell activation in type 2 diabetes mellitus (T2DM) using whole blood. Leukocytes were first size-fractionated into different subtypes (neutrophils, monocytes, lymphocytes) using an inertial spiral sorter prior to single-cell impedance measurement in a microfluidic device with coplanar electrode design. Significant changes in membrane dielectric properties (size and opacity) were detected between healthy and activated leukocytes (TNF-α/LPS stimulated), during monocyte differentiation and among different monocyte subsets (classical, intermediate, non-classical). As proof-of-concept for diabetes testing, neutrophil/monocyte dielectric properties in T2DM subjects (n = 8) were quantified which were associated with cardiovascular risk factors including lipid levels, C-reactive protein (CRP) and vascular functions (LnRHI) (P < 0.05) were observed. Overall, these results clearly showed that T2DM subjects have pro-inflammatory leukocyte phenotypes and suggest leukocyte impedance signature as a novel surrogate biomarker for inflammation.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore)Accepted versio

Engineering a 3D microfluidic culture platform for tumor-treating field application

The limitations of current cancer therapies highlight the urgent need for a more effective therapeutic strategy. One promising approach uses an alternating electric field; however, the mechanisms involved in the disruption of the cancer cell cycle as well as the potential adverse effects on non-cancerous cells must be clarified. In this study, we present a novel microfluidic device with embedded electrodes that enables the application of an alternating electric field therapy to cancer cells in a 3D extracellular matrix. To demonstrate the potential of our system to aid in designing and testing new therapeutic approaches, cancer cells and cancer cell aggregates were cultured individually or co-cultured with endothelial cells. The metastatic potential of the cancer cells was reduced after electric field treatment. Moreover, the proliferation rate of the treated cancer cells was lower compared with that of the untreated cells, whereas the morphologies and proliferative capacities of the endothelial cells were not significantly affected. These results demonstrate that our novel system can be used to rapidly screen the effect of an alternating electric field on cancer and normal cells within an in vivo-like microenvironment with the potential to optimize treatment protocols and evaluate synergies between tumor-treating field treatment and chemotherapy

The pro-inflammatory phenotype of the human non-classical monocyte subset is attributed to senescence

Abstract Human primary monocytes comprise a heterogeneous population that can be classified into three subsets based on CD14 and CD16 expression: classical (CD14high/CD16−), intermediate (CD14high/CD16+), and non-classical (CD14low/CD16+). The non-classical monocytes are the most pro-inflammatory in response to TLR stimulation in vitro, yet they express a remarkably high basal level of miR-146a, a microRNA known to negatively regulate the TLR pathway. This concurrence of a pro-inflammatory status and a high miR-146a level has been associated with cellular senescence in other cell types. Hence, we assessed the three monocyte subsets for evidence of senescence, including proliferative status, telomere length, cellular ROS levels, and mitochondrial membrane potential. Indeed, the non-classical subset exhibited the clearest hallmarks of senescence, followed by the intermediate and then the classical subset. In addition, the non-classical subset secreted pro-inflammatory cytokines basally in vitro. The highly pro-inflammatory nature of the non-classical monocytes could be a manifestation of the senescence-associated secretory phenotype (SASP), likely induced by a high basal NF-κB activity and IL-1α production. Finally, we observed an accumulation of the non-classical monocytes, in conjunction with higher levels of plasma TNF-α and IL-8, in the elderly. These factors may contribute to inflamm-aging and age-related inflammatory conditions, such as atherosclerosis and osteoarthritis. With our new understanding that the non-classical monocyte subset is a senescent population, we can now re-examine the role of this subset in disease conditions where this subset expands

Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention

Background: Emergence of drug-resistant cancer phenotypes is a challenge for anti-cancer therapy. Cancer stem cells are identified as one of the ways by which chemoresistance develops. Method: We investigated the anti-inflammatory combinatorial treatment (DA) of doxorubicin and aspirin using a preclinical microfluidic model on cancer cell lines and patient-derived circulating tumour cell clusters. The model had been previously demonstrated to predict patient overall prognosis. Results: We demonstrated that low-dose aspirin with a sub-optimal dose of doxorubicin for 72 h could generate higher killing efficacy and enhanced apoptosis. Seven days of DA treatment significantly reduced the proportion of cancer stem cells and colony-forming ability. DA treatment delayed the inhibition of interleukin-6 secretion, which is mediated by both COX-dependent and independent pathways. The response of patients varied due to clinical heterogeneity, with 62.5% and 64.7% of samples demonstrating higher killing efficacy or reduction in cancer stem cell (CSC) proportions after DA treatment, respectively. These results highlight the importance of using patient-derived models for drug discovery. Conclusions: This preclinical proof of concept seeks to reduce the onset of CSCs generated post treatment by stressful stimuli. Our study will promote a better understanding of anti-inflammatory treatments for cancer and reduce the risk of relapse in patients.Singapore. National Medical Research Council (Grant NMRC

Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention

10.1038/s41416-018-0301-9BRITISH JOURNAL OF CANCER1204407-42

Prognostic value of CD8 + PD-1+ immune infiltrates and PDCD1 gene expression in triple negative breast cancer

Abstract The role of programmed cell death protein-1 (PD-1)/programmed cell death ligand 1 (PD-L1) in triple negative breast cancer (TNBC) remains to be fully understood. In this study, we investigated the role of PD-1 as a prognostic marker for TNBC in an Asian cohort (n = 269). Samples from patients with TNBC were labeled with antibodies against PD-L1 and PD-1, and subjected to NanoString assays to measure the expression of immune-related genes. Associations between disease-free survival (DFS), overall survival (OS) and biomarker expression were investigated. Multivariate analysis showed that tumors with high PD-1+ immune infiltrates harbored significantly increased DFS, and this increase was significant even after controlling for clinicopathological parameters (HR 0.95; P = 0.030). In addition, the density of cells expressing both CD8 and PD-1, but not the density of CD8−PD-1+ immune infiltrates, was associated with improved DFS. Notably, this prognostic significance was independent of clinicopathological parameters and the densities of total CD8+ cell (HR 0.43, P = 0.011). At the transcriptional level, high expression of PDCD1 within the tumor was significantly associated with improved DFS (HR 0.38; P = 0.027). In line with these findings, high expression of IFNG (HR 0.38; P = 0.001) and IFN signaling genes (HR 0.46; p = 0.027) was also associated with favorable DFS. Inclusion of PD-1 immune infiltrates and PDCD1 gene expression added significant prognostic value for DFS (ΔLRχ2 = 6.35; P = 0.041) and OS (ΔLRχ2 = 9.53; P = 0.008), beyond that provided by classical clinicopathological variables. Thus, PD-1 mRNA and protein expression status represent a promising, independent indicator of prognosis in TNBC

Targeting glycolysis in macrophages confers protection against pancreatic ductal adenocarcinoma

Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage- specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1β inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes