NK cell exhaustion in the tumor microenvironment

Natural killer (NK) cells kill mutant cells through death receptors and cytotoxic granules, playing an essential role in controlling cancer progression. However, in the tumor microenvironment (TME), NK cells frequently exhibit an exhausted status, which impairs their immunosurveillance function and contributes to tumor immune evasion. Emerging studies are ongoing to reveal the properties and mechanisms of NK cell exhaustion in the TME. In this review, we will briefly introduce the maturation, localization, homeostasis, and cytotoxicity of NK cells. We will then summarize the current understanding of the main mechanisms underlying NK cell exhaustion in the TME in four aspects: dysregulation of inhibitory and activating signaling, tumor cell-derived factors, immunosuppressive cells, and metabolism and exhaustion. We will also discuss the therapeutic approaches currently being developed to reverse NK cell exhaustion and enhance NK cell cytotoxicity in the TME

An amine-reactive tetraphenylethylene derivative for protein detection in SDS-PAGE

A new aggregation-induced emission (AIE) compound 1,2-bis[4-(isothiocyanatomethyl)phenyl]-1,2-diphenylethene (2) was synthesized for use in SDS-PAGE. The molecule is practically nonemissive in solution but becomes highly emissive after reacting with the amine groups of the proteins by either the prestaining or poststaining method. The sensitivity of 2 achieved in the prestaining method is the same as that of Coomassie brilliant blue (CBB), while that observed in the poststaining method is higher than that of CBB. Excellent linear responses with the amount of protein were obtained in both cases. The detection of a mixture of proteins with different molecular weights was successfully achieved

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Isolation and functional studies of D. discoideum cyclin B gene during growth and development

Cyclin B plays an essential role i n cell cycle regulation by complexing with Cdc2 protein kinase to direct cells into mitosis. A cyclin B gene (cycB) has been isolated from D. discoideum. The cyclin box region of the protein encoded by cycB has a high degree of sequence identity with B-type cyclins of other species. During celldivision synchronized growth, levels of cyclin B mRNA and protein were found to oscillate during the cell cycle with maximum accumulation occurring prior to and during cell division. Northern blot analysis showed that the cycB mRNA levels peaked twice during development, once during early aggregation (3 h) and again at the tipped aggregate stage (12 h). In contrast, the levels of cyclin B protein and histone H I kinase activity of the Cdc2-immunoprecipitate increased only during early development and then gradually decreased as development continued. The N-terminal region of the cyclin B protein has the conserved protein degradation motif, typical of cyclin B proteins. The 5' portion of the D. discoideum cycB gene was deleted and the truncated gene was cloned downstream from the regulatable discoidin promoter. Overexpression of this truncated cyclin B gene elevated the histone H I kinase activity of the Cdc2-immunoprecipitates, suggesting that the kinase activity was limited by the amount of the cyclin B i n the cells. The growth of these cyclin B overexpressing mutants was arrested during mitosis. These mitotically arrested cells formed very small fruiting bodies when allowed to undergo development. A protein that eluted together with the histidine tagged cyclin B fusion protein from a Ni-resin affinity column was identified as Cdc2, indicating a direct physical interaction between cyclin B and Cdc2. Indirect immunofluorescence staining using the cyclin B antibody revealed that D. discoideum cyclin B localized mainly in the nucleus. This is consistent with the finding that D. discoideum cyclin B is lacking the presumptive cytoplasmic signal sequence found in most of the B-type cyclins from multicellular organisms.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

High Shear Stresses under Exercise Condition Destroy Circulating Tumor Cells in a Microfluidic System

Circulating tumor cells (CTCs) are the primary targets of cancer treatment as they cause distal metastasis. However, how CTCs response to exercise-induced high shear stress is largely unknown. To study the effects of hemodynamic microenvironment on CTCs, we designed a microfluidic circulatory system that produces exercise relevant shear stresses. We explore the effects of shear stresses on breast cancer cells with different metastatic abilities, cancer cells of ovarian, lung and leukemic origin. Three major findings were obtained. 1) High shear stress of 60 dynes/cm2 achievable during intensive exercise killed more CTCs than low shear stress of 15 dynes/cm2 present in human arteries at the resting state. 2) High shear stress caused necrosis in over 90% of CTCs within the first 4 h of circulation. More importantly, the CTCs that survived the first 4 h-circulation, underwent apoptosis during 16–24 h of post-circulation incubation. 3) Prolonged high shear stress treatment effectively reduced the viability of highly metastatic and drug resistant breast cancer cells. As high shear stress had much less damaging effects on leukemic cells mimicking the white blood cells, we propose that intensive exercise may be a good strategy for generating high shear stress that can destroy CTCs and prevent cancer metastasis.MOE (Min. of Education, S’pore)Published versio

Shear Stress Drives the Cleavage Activation of Protease‐Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells

Abstract When circulating tumor cells (CTCs) travel in circulation, they can be killed by detachment‐induced anoikis and fluidic shear stress (SS)‐mediated apoptosis. Circulatory treatment, which can make CTCs detached but also generate SS, can increase metastasis of cancer cells. To identify SS‐specific mechanosensors without detachment impacts, a microfluidic circulatory system is used to generate arteriosus SS and compare transcriptome profiles of circulating lung cancer cells with suspended cells. Half of the cancer cells can survive SS damage and show higher invasion ability. Mesotrypsin (PRSS3), protease‐activated receptor 2 (PAR2), and the subunit of activating protein 1, Fos‐related antigen 1 (FOSL1), are upregulated by SS, and their high expression is responsible for promoting invasion and metastasis. SS triggers PRSS3 to cleave the N‐terminal inhibitory domain of PAR2 within 2 h. As a G protein‐coupled receptor, PAR2 further activates the Gαi protein to turn on the Src‐ERK/p38/JNK‐FRA1/cJUN axis to promote the expression of epithelial–mesenchymal transition markers, and also PRSS3, which facilitates metastasis. Enriched PRSS3, PAR2, and FOSL1 in human tumor samples and their correlations with worse outcomes reveal their clinical significance. PAR2 may serve as an SS‐specific mechanosensor cleavable by PRSS3 in circulation, which provides new insights for targeting metastasis‐initiating CTCs

A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z′ factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening

A prototypic microfluidic platform generating stepwise concentration gradients for real-time study of cell apoptosis

This work describes the development of a prototypic microfluidic platform for the generation of stepwise concentration gradients of drugs. A sensitive apoptotic analysis method is integrated into this microfluidic system for studying apoptosis of HeLa cells under the influence of anticancer drug, etoposide, with various concentrations in parallel; it measures the yellow fluorescent protein∕cyan fluorescent protein fluorescence resonance energy transfer (FRET) signal that responds to the activation of caspase-3, an indicator of cell apoptosis. Sets of microfluidic valves on the chip generate stepwise concentration gradient of etoposide in various cell-culture microchambers. The FRET signals from multiple chambers are simultaneously monitored under a fluorescent microscope for long-time observation and the on-chip results are compared with those from 96-well plate study and the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The microfluidic platform shows several advantages including high-throughput capacity, low drug consumption, and high sensitivity

Enhancement of the bioavailability of a novel anticancer compound (acetyltanshinone IIA) by encapsulation within mPEG-PLGA nanoparticles: A study of formulation optimization, toxicity, and pharmacokinetics

The Poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) (mPEG-PLGA) nanoparticles carrying acetyltanshinone IIA (ATA), a novel anti-breast cancer agent, were prepared by ultrasonic emulsion method to enhance the bioavailability and reduce the toxicity. Systematic optimization of encapsulation process was achieved using an orthogonal design. Drug efficacy analysis showed that ATA nanoparticles were as effective as free ATA against estrogen receptor positive breast cancer cells, but much less toxic towards human endothelial cells. Furthermore, in zebrafish, ATA nanoparticles displayed much lower toxicity than free ATA. More importantly, the blood concentration of ATA nanoparticles indicated by 24 hour-area under the curve (AUC0-24h) was 10 times higher than free ATA. These results indicated the potential of ATA-loaded mPEG-PLGA nanoparticles for the delivery of ATA in a clinical formulation, and their potential for use in tumor therapy in the future.Published versio