Spatial transcriptomics: Technologies, applications and experimental considerations.

The diverse cell types of an organ have a highly structured organization to enable their efficient and correct function. To fully appreciate gene functions in a given cell type, one needs to understand how much, when and where the gene is expressed. Classic bulk RNA sequencing and popular single cell sequencing destroy cell structural organization and fail to provide spatial information. However, the spatial location of gene expression or of the cell in a complex tissue provides key clues to comprehend how the neighboring genes or cells cross talk, transduce signals and work together as a team to complete the job. The functional requirement for the spatial content has been a driving force for rapid development of the spatial transcriptomics technologies in the past few years. Here, we present an overview of current spatial technologies with a special focus on the commercially available or currently being commercialized technologies, highlight their applications by category and discuss experimental considerations for a first spatial experiment.

Anti-EBV antibodies: Roles in diagnosis, pathogenesis, and antiviral therapy.

Epstein-Barr virus (EBV) infection is prevalent in global population and associated with multiple malignancies and autoimmune diseases. During the infection, EBV-harbored or infected cell-expressing antigen could elicit a variety of antibodies with significant role in viral host response and pathogenesis. These antibodies have been extensively evaluated and found to be valuable in predicting disease diagnosis and prognosis, exploring disease mechanisms, and developing antiviral agents. In this review, we discuss the versatile roles of EBV antibodies as important biomarkers for EBV-related diseases, potential driving factors of autoimmunity, and promising therapeutic agents for viral infection and pathogenesis.

Efficacy and safety of erythropoietin for traumatic brain injury.

BACKGROUND: Recent studies regarding the effects of erythropoietin (EPO) for treating traumatic brain injury (TBI) have been inconsistent. This study conducts a meta-analysis of randomized controlled trials (RCTs) to assess the safety and efficacy of EPO for TBI patients at various follow-up time points. METHODS: A literature search was performed using PubMed, Web of Science, MEDLINE, Embase, Google Scholar and the Cochrane Library for RCTs studying EPO in TBI patients published through March 2019. Non-English manuscripts and non-human studies were excluded. The assessed outcomes include mortality, neurological recovery and associated adverse effects. Dichotomous variables are presented as risk ratios (RR) with a 95% confidence interval (CI). RESULTS: A total of seven RCTs involving 1197 TBI patients (611 treated with EPO, 586 treated with placebo) were included in this study. Compared to the placebo arm, treatment with EPO did not improve acute hospital mortality or short-term mortality. However, there was a significant improvement in mid-term (6 months) follow-up survival rates. EPO administration was not associated with neurological function improvement. Regarding adverse effects, EPO treatment did not increase the incidence of thromboembolic events or other associated adverse events. CONCLUSIONS: This meta-analysis indicates a slight mortality benefit for TBI patients treated with EPO at mid-term follow-up. EPO does not improve in-hospital mortality, nor does it increase adverse events including thrombotic, cardiovascular and other associated complications. Our analysis did not demonstrate a significant beneficial effect of EPO intervention on the recovery of neurological function. Future RCTs are required to further characterize the use of EPO in TBI.

ATP6V1H Deficiency Impairs Bone Development through Activation of MMP9 and MMP13.

ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We identified two generations of individuals in which short stature and osteoporosis co-segregated with a mutation in ATP6V1H. Since V-ATPases are highly conserved between human and zebrafish, we generated loss-of-function mutants in atp6v1h in zebrafish through CRISPR/Cas9-mediated gene knockout. Homozygous mutant atp6v1h zebrafish exhibited a severe reduction in the number of mature calcified bone cells and a dramatic increase in the expression of mmp9 and mmp13. Heterozygous adults showed curved vertebra that lack calcified centrum structure and reduced bone mass and density. Treatment of mutant embryos with small molecule inhibitors of MMP9 and MMP13 significantly restored bone mass in the atp6v1h mutants. These studies have uncovered a new, ATP6V1H-mediated pathway that regulates bone formation, and defines a new mechanism of disease that leads to bone loss. We propose that MMP9/MMP13 could be therapeutic targets for patients with this rare genetic disease.

Correction: ATP6V1H Deficiency Impairs Bone Development through Activation of MMP9 and MMP13.

[This corrects the article DOI: 10.1371/journal.pgen.1006481.].

Wnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression.

AIMS: Wnt signaling plays a critical role in vascular calcification (VC). Wnt factors induce different physiological and pathological effects on cardiovascular functions. Wnt1, a ligand of Wnt/ß-catenin signaling, promotes pro-angiogenesis and reduces myocardial infarction. The role of Wnt1 on VC in chronic kidney disease (CKD) is not fully understood. METHODS AND RESULTS: We used human vascular smooth muscle cells (VSMCs) and a rat model of chronic renal failure (CRF), and observed a native protective mechanism by which VC is reduced via the activation of Wnt1 and its transcriptional target ANKH inorganic pyrophosphate transport regulator (ANKH) gene. ANKH is an essential calcification inhibitor that effluxes inorganic pyrophosphate (PPi) from VSMCs to play an inhibitory role in VC. Vascular ANKH and plasma PPi were significantly downregulated in the rat model of CRF. The knockdown or inhibition of ANKH reversed the effect of Wnt1 on VC in VSMCs. Clinical analysis revealed low plasma levels of Wnt1 and PPi were associated with CKD in patients. Applying a Wnt/ß-catenin signaling agonist can alleviate the progression of VC. CONCLUSION: This work reveals the ANKH regulation of Wnt1 in VSMCs is essential for blocking VC. Our findings may contribute to the development of medications that target Wnt signaling and/or ANKH to inhibit VC.

Spine surgeon specialty differences in single-level percutaneous kyphoplasty.

BACKGROUND: Percutaneous kyphoplasty (PKP) is a procedure performed by a spine surgeon who undergoes either orthopedic or neurosurgical training. The relationship between short-term adverse outcomes and spine specialty is presently unknown. To compare short-term adverse outcomes of single-level PKP when performed by neurosurgeons and orthopedic surgeons in order to develop more concretely preventive strategies for patients under consideration for single-level PKP. METHODS: We evaluated patients who underwent single-level PKP from 2012 to 2014 through the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP). We used univariate analysis and multivariate logistic regression to assess the association between spine surgeon specialty and short-term adverse events, including postoperative complication and unplanned readmission, and to identify different independent risk predictors between two specialties. RESULTS: Of 2248 patients who underwent single-level PKP procedure, 1229 patients (54.7%) had their operations completed by a neurosurgeon. There were no significant differences in the development of the majority of postoperative complications and the occurrence of unplanned readmission between the neurosurgical cohort (NC) and the orthopedic cohort (OC). A difference in the postoperative blood transfusion rate (0.7% NS vs. 1.7% OC, P = 0.039) was noted and may due to the differences in comorbidities between patients. Multivariate regression analysis revealed different independent predictors of postoperative adverse events for the two spine specialties. CONCLUSIONS: By comparing a large range of demographic feature, preoperative comorbidities, and intraoperative factors, we find that short-term adverse events in single-level PKP patients does not affect by spine surgeon specialty, except that the OC had higher postoperative blood transfusion rate. In addition, the different perioperative predictors of postoperative complications and unplanned readmissions were identified between the two specialties. These findings can lead to better evidence-based patient counseling and provide valuable information for medical evaluation and potentially devise methods to reduce patients' risk.

Thioredoxin attenuates oxidized low-density lipoprotein induced oxidative stress in human umbilical vein endothelial cells by reducing NADPH oxidase activity.

Oxidative stress is recognized as one of the most important contributing factors to the development of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) can induce vascular reactive oxygen species (ROS) production, trigger endothelial dysfunction and initiate the progression of atherosclerosis. Previous studies have demonstrated that thioredoxin-1 (Trx) is one of the key regulators of intracellular redox, which is pivotal in atherogenesis. However, the regulation mechanism is still unclear. In this study, we investigated the effects of Trx1 on NADPH oxidase in human umbilical vein endothelial cells (HUVECs), whose ROS level is mainly produced by NADPH oxidase, especially Nox4 isoform. Our data demonstrated that Trx decreased NADPH oxidase activity, ROS production and ICAM-1 expression in ox-LDL treated HUVECs. Genetic gain-of-function and loss-of-function studies showed that Trx1 suppressed ox-LDL-induced Nox4 and p22phox expression. A co-immunoprecipitation assay indicated that Trx1 decreased Nox4-p22phox complex level during ox-LDL stimulation. Transient transfection of Nox4 and p22phox significantly increased intracellular ROS generation, which could be blocked by Trx overexpression. In addition, Trx overexpression also prevented ox-LDL-induced Nox2 and Rac1 protein levels. These results suggest that Trx suppresses NADPH oxidase activity in vascular endothelia under pathological conditions and may prevent the initiation of atherosclerosis by attenuating exceeding ROS production.

Leucine supplementation via drinking water reduces atherosclerotic lesions in apoE null mice.

AIM: Recent evidence suggests that the essential amino acid leucine may be involved in systemic cholesterol metabolism. In this study, we investigated the effects of leucine supplementation on the development of atherosclerosis in apoE null mice. METHODS: ApoE null mice were fed with chow supplemented with leucine (1.5% w/v) in drinking water for 8 week. Aortic atherosclerotic lesions were examined using Oil Red O staining. Plasma lipoprotein-cholesterol levels were measured with fast protein liquid chromatography. Hepatic gene expression was detected using real-time PCR and Western blot analyses. RESULTS: Leucine supplementation resulted in 57.6% reduction of aortic atherosclerotic lesion area in apoE null mice, accompanied by 41.2% decrease of serum LDL-C levels and 40.2% increase of serum HDL-C levels. The body weight, food intake and blood glucose level were not affected by leucine supplementation. Furthermore, leucine supplementation increased the expression of Abcg5 and Abcg8 (that were involved in hepatic cholesterol efflux) by 1.28- and 0.86-fold, respectively, and significantly increased their protein levels. Leucine supplementation also increased the expression of Srebf1, Scd1 and Pgc1b (that were involved in hepatic triglyceride metabolism) by 3.73-, 1.35- and 1.71-fold, respectively. Consequently, leucine supplementation resulted in 51.77% reduction of liver cholesterol content and 2.2-fold increase of liver triglyceride content. Additionally, leucine supplementation did not affect the serum levels of IL-6, IFN-γ, TNF-α, IL-10 and IL-12, but markedly decreased the serum level of MCP-1. CONCLUSION: Leucine supplementation effectively attenuates atherosclerosis in apoE null mice by improving the plasma lipid profile and reducing systemic inflammation.

Formyl peptide enhances cancer immunotherapy by activating antitumoral neutrophils, and T cells.

Neutrophils are heterogeneous and plastic, with the ability to polarize from antitumour to protumour phenotype and modulate tumour microenvironment components. While some advances have been made, the neutrophil-targeting therapy remains underexplored. Activation of formyl peptide receptors (FPRs) by formylated peptides is needed for local control of infection through the recruitment of activated neutrophils while the potential contribution of antitumour activity remains underexplored. Here, we demonstrate that neutrophils can be harnessed to suppress tumour growth through the action of the formyl peptide (FP) on the formyl peptide receptor (FPR). Mechanistically, FP efficiently recruits neutrophils to produce reactive oxygen species production (ROS), resulting in the direct killing of tumours. Antitumour functions disappeared when neutrophils were depleted by anti-Ly6G antibodies. Interestingly, extensive T-cell activation was observed in mouse tumours treated with FP, showing the potential to alter the immune suppressed tumour microenvironment (TME) and further sensitize mice to anti-PD1 therapy. Transcriptomic and flow cytometry analyses revealed the mechanisms of FP-sensitized anti-PD1 therapy, mainly including stimulated neutrophils and an altered immune-suppressed tumour microenvironment. Collectively, these data establish FP as an effective combination partner for sensitizing anti-PD1 therapy by stimulating tumour-infiltrated neutrophils.

Potent human monoclonal antibodies targeting Epstein-Barr virus gp42 reveal vulnerable sites for virus infection.

Epstein-Barr virus (EBV) is linked to various malignancies and autoimmune diseases, posing a significant global health challenge due to the lack of specific treatments or vaccines. Despite its crucial role in EBV infection in B cells, the mechanisms of the glycoprotein gp42 remain elusive. In this study, we construct an antibody phage library from 100 EBV-positive individuals, leading to the identification of two human monoclonal antibodies, 2B7 and 2C1. These antibodies effectively neutralize EBV infection in vitro and in vivo while preserving gp42's interaction with the human leukocyte antigen class II (HLA-II) receptor. Structural analysis unveils their distinct binding epitopes on gp42, different from the HLA-II binding site. Furthermore, both 2B7 and 2C1 demonstrate potent neutralization of EBV infection in HLA-II-positive epithelial cells, expanding our understanding of gp42's role. Overall, this study introduces two human anti-gp42 antibodies with potential implications for developing EBV vaccines targeting gp42 epitopes, addressing a critical gap in EBV research.

T-box20 suppresses oxidized low-density lipoprotein-induced human vascular endothelial cell injury by upregulation of PPAR-γ.

BACKGROUND: Atherosclerosis is a chronic inflammation disease which is initiated by endothelial cell injury Oxidized low-density lipoprotein (ox-LDL) is directly associated with chronic vascular inflammation. Many transcription factors take part in the initiation and progression of atherosclerosis. As a transcription factor mainly expressed in cardiovascular system, T-box20 (Tbx20) plays an important role in embryonic cardiovascular system development and homeostasis. However, the role of Tbx20 in endothelial cell injury and atherosclerosis is still not clear. We showed that Tbx20 might affect ox-LDL-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: First, Tbx20 expression was down regulated in the C57BL/6 mice with high-fat diet-induced artery injury, which was accompanied by elevated reactive oxygen species (ROS) generation and cell adhesion molecule expression. Second, ox-LDL led to concurrent decreased Tbx20 expression and increased levels of ROS and adhesion molecules in the HUVECs. Third, over-expression of Tbx20 by adenovirus reduced ox-LDL-induced HUVEC injury via attenuation of ROS generation and cell adhesion molecule expression. Fourth, knock down of Tbx20 by siRNA significantly increased adhesion molecule expression and decreased cell viability. Moreover, Tbx20 could directly regulate PPAR-γ expression, as shown by Tbx20 knock down and PPAR-γ inhibition, which significantly reversed Tbx20's HUVEC protection effect. CONCLUSIONS: These results indicate that misregulation of Tbx20 could reduce HUVEC tolerance of ox-LDL-induced cell injury, suggesting that Tbx20 might be a crucial regulator and potential therapeutic target for atherosclerosis.

Chronic unpredictable mild stress promotes atherosclerosis via adipose tissue dysfunction in ApoE-/- mice.

Background: Chronic unpredictable mild stress (CUMS) has been shown to exacerbate atherosclerosis, but the underlying mechanism remains unknown. Adipose tissue is an energy storage organ and the largest endocrine organ in the human body, playing a key role in the development of cardiovascular disease. In this research, it was hypothesized that CUMS may exacerbate the development of atherosclerosis by inducing the hypertrophy and dysfunction of white adipocytes. Methods: The CUMS-induced atherosclerosis model was developed in Western diet-fed apolipoprotein E (ApoE)-/- mice. White adipose tissue (WAT), serum, aortic root, and the brachiocephalic trunk were collected and tested after 12 weeks of CUMS development. The mouse model of CUMS was evaluated for depression-like behavior using the open field test (OFT) and the elevated plus maze (EPM) test. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect serum noradrenaline and urine adrenaline protein levels. Serological assays were used to detect serum low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (TC), and free fatty acid (FFA) concentrations. Hematoxylin and eosin (H&E) staining and oil red O were used to detect atherosclerotic plaque area, lipid deposition, and adipocyte size. The mRNA levels of genes related to aberrant adipose tissue function were determined using real-time PCR. Immunofluorescence assay and western blotting were conducted to examine the expression of proteins in the adipose tissue samples. Results: CUMS aggravated vascular atherosclerotic lesions in ApoE-/- mice. It decreased body weight while increasing the percentage of WAT. The serological results indicated that the concentration of HDL decreased in CUMS mice. Notably, adipocyte hypertrophy increased, whereas the mRNA levels of Pparg and its target genes (Slc2a4 (encodes for GLUT4), Adipoq, and Plin1) decreased. Further investigation revealed that CUMS increased subcutaneous inguinal WAT (iWAT) lipid synthesis and adipocyte inflammation while decreasing lipid hydrolysis and the expression of HDL-associated protein ApoA-I. Moreover, CUMS aggravated insulin resistance in mice and inhibited the insulin pathway in iWAT. Conclusions: These findings indicated that CUMS induces adipose tissue dysfunction via a mechanism that leads to dyslipidemia, increased inflammation, and insulin resistance in the body, thereby exacerbating atherosclerosis. Notably, CUMS that is involved in decreasing the expression of HDL-associated proteins in adipose tissue may be a crucial link between adipose hypertrophy and advanced atherosclerosis. This study reveals a novel mechanism via which CUMS exacerbates atherosclerosis from the novel perspective of abnormal adipose function and identifies a novel potential therapeutic target for this disease.

mRNA-based Vaccines Targeting the T-cell Epitope-rich Domain of Epstein Barr Virus Latent Proteins Elicit Robust Anti-Tumor Immunity in Mice.

Epstein-Barr virus (EBV) is associated with various malignancies and infects >90% of the global population. EBV latent proteins are expressed in numerous EBV-associated cancers and contribute to carcinogenesis, making them critical therapeutic targets for these cancers. Thus, this study aims to develop mRNA-based therapeutic vaccines that express the T-cell-epitope-rich domain of truncated latent proteins of EBV, including truncatedlatent membrane protein 2A (Trunc-LMP2A), truncated EBV nuclear antigen 1 (Trunc-EBNA1), and Trunc-EBNA3A. The vaccines effectively activate both cellular and humoral immunity in mice and show promising results in suppressing tumor progression and improving survival time in tumor-bearing mice. Furthermore, it is observed that the truncated forms of the antigens, Trunc-LMP2A, Trunc-EBNA1, and Trunc-EBNA3A, are more effective than full-length antigens in activating antigen-specific immune responses. In summary, the findings demonstrate the effectiveness of mRNA-based therapeutic vaccines targeting the T-cell-epitope-rich domain of EBV latent proteins and providing new treatment options for EBV-associated cancers.

Activation of nuclear factor-kappa B accelerates vascular calcification by inhibiting ankylosis protein homolog expression.

Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

Ajuba Overexpression Promotes Breast Cancer Chemoresistance and Glucose Uptake through TAZ-GLUT3/Survivin Pathway.

The LIM protein Ajuba has been implicated in the development of human cancers. To date, its expression pattern and biological significance in breast cancers (BC) have not been fully investigated. In the current study, we examined Ajuba protein levels in 93 invasive ductal carcinoma specimens by immunohistochemistry. The Ajuba expression level was elevated in breast cancer tissue compared with normal tissue. Ajuba overexpression is correlated with advanced tumor-node-metastasis (TNM) stage, positive node status, and adverse patient outcomes. The Ajuba protein level was also higher in BC cell lines compared to normal breast epithelial cell line MCF-10A. Ectopically expressed Ajuba in MCF-7 cells stimulated in vitro and in vivo cell growth, invasion, cell cycle progression, and decreased paclitaxel-induced apoptosis. RNA-sequencing (RNA-seq) followed by gene set enrichment analysis (GSEA) analysis showed that Ajuba overexpression regulated the Hippo signaling pathway. Ajuba overexpression also increased glucose uptake and increased expression of TAZ, GLUT3, and Survivin. TAZ knockdown abolished the role of Ajuba on GLUT3 and Survivin induction. The ChIP assay showed that TEAD4, a major TAZ binding transcription factor, could bind to the GLUT3 and Survivin promoter regions. In conclusion, our data demonstrated that elevated Ajuba expression is correlated with poor BC prognosis and regulated malignant behavior through TAZ-GLUT3/Survivin signaling in BC cells.

Distinct patterns of responses in endothelial cells and smooth muscle cells following vascular injury.

Vascular smooth muscle cells (SMCs) are heterogeneous, and their differential responses to vascular injury are not well understood. To address this question, we performed single-cell analysis of vascular cells to a ligation injury in mouse carotid arteries after 3 days. While endothelial cells had a homogeneous activation of mesenchymal genes, less than 30% of SMCs responded to the injury and generated 2 distinct clusters - i.e., proinflammatory SMCs and stress-responsive SMCs. Proinflammatory SMCs were enriched with high levels of inflammatory markers such as vascular cell adhesion molecule-1 while stress-responsive SMCs overexpressed heat shock proteins. Trajectory analysis suggested that proinflammatory SMCs were potentially derived from a specific subpopulation of SMCs. Ligand-receptor pair analysis showed that the interaction between macrophages and proinflammatory SMCs was the major cell-cell communication among all cell types in the injured arteries. In vitro coculture demonstrated that VCAM1+ SMCs had a stronger chemotactic effect on macrophage recruitment than VCAM1- SMCs. Consistently, the number of VCAM1+ SMCs significantly increased in injured arteries and atherosclerotic lesions of ApoE-/- mice and human arteries. These findings provide insights at the single-cell level on the distinct patterns of endothelial cells and SMC responses to vascular injury.

Mitochondrial reactive oxygen species promote p65 nuclear translocation mediating high-phosphate-induced vascular calcification in vitro and in vivo.

Hyperphosphatemia is the major risk factor associated with vascular calcification (VC) in end-stage renal disease. As oxidative stress is increased in uremia, we studied the role of mitochondrial reactive oxygen species (ROS) and nuclear factor-κB signaling in phosphate-induced VC. In an in vitro calcification model (ß-glycerophosphate (BGP) induction) using bovine aortic smooth muscle cells, the production of intracellular and mitochondrial ROS, or superoxide anion, was stimulated by increased mitochondrial membrane potential. This effect was blocked by the superoxide dismutase (SOD) mimic MnTMPyP, a respiratory chain inhibitor rotenone, or a protonophore. Calcium deposition and the switch of smooth muscle cells from a contractile to an osteogenic phenotype were decreased when mitochondrial ROS generation was inhibited by the respiratory chain inhibitor, MnTMPyP, or the overexpression of SOD1 and SOD2 and uncoupling protein 2. The phosphorylation of IkKß, IκBα degradation, and p65 nuclear translocation were increased by BGP but reversed when mitochondrial ROS production was blocked by protonophore or MnTMPyP. Knockdown of endogenous p65 or overexpression of IκBα reduced calcium deposition in the cultured cells. Furthermore, in a rat model of dietary adenine-induced chronic renal failure, MnTMPyP reduced aortic ROS levels, p65 activation, and calcium deposition. Thus, mitochondrial ROS-mediated p65 nuclear translocation is involved in phosphate-induced VC.

Advances in pathogenesis and precision medicine for nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a squamous carcinoma with apparent geographical and racial distribution, mostly prevalent in East and Southeast Asia, particularly concentrated in southern China. The epidemiological trend over the past decades has suggested a substantial reduction in the incidence rate and mortality rate due to NPC. These results may reflect changes in lifestyle and environment, and more importantly, a deeper comprehension of the pathogenic mechanism of NPC, leading to much progress in the preventing, screening, and treating for this cancer. Herein, we present the recent advances on the key signal pathways involved in pathogenesis of NPC, the mechanism of Epstein-Barr virus (EBV) entry into the cell, and the progress of EBV vaccine and screening biomarkers. We will also discuss in depth the development of various therapeutic approaches including radiotherapy, chemotherapy, surgery, targeted therapy, and immunotherapy. These research advancements have led to a new era of precision medicine in NPC.

FDA-Approved and Emerging Next Generation Predictive Biomarkers for Immune Checkpoint Inhibitors in Cancer Patients.

A patient's response to immune checkpoint inhibitors (ICIs) is a complex quantitative trait, and determined by multiple intrinsic and extrinsic factors. Three currently FDA-approved predictive biomarkers (progra1mmed cell death ligand-1 (PD-L1); microsatellite instability (MSI); tumor mutational burden (TMB)) are routinely used for patient selection for ICI response in clinical practice. Although clinical utility of these biomarkers has been demonstrated in ample clinical trials, many variables involved in using these biomarkers have poised serious challenges in daily practice. Furthermore, the predicted responders by these three biomarkers only have a small percentage of overlap, suggesting that each biomarker captures different contributing factors to ICI response. Optimized use of currently FDA-approved biomarkers and development of a new generation of predictive biomarkers are urgently needed. In this review, we will first discuss three widely used FDA-approved predictive biomarkers and their optimal use. Secondly, we will review four novel gene signature biomarkers: T-cell inflamed gene expression profile (GEP), T-cell dysfunction and exclusion gene signature (TIDE), melanocytic plasticity signature (MPS) and B-cell focused gene signature. The GEP and TIDE have shown better predictive performance than PD-L1, and PD-L1 or TMB, respectively. The MPS is superior to PD-L1, TMB, and TIDE. The B-cell focused gene signature represents a previously unexplored predictive biomarker to ICI response. Thirdly, we will highlight two combined predictive biomarkers: TMB+GEP and MPS+TIDE. These integrated biomarkers showed improved predictive outcomes compared to a single predictor. Finally, we will present a potential nucleic acid biomarker signature, allowing DNA and RNA biomarkers to be analyzed in one assay. This comprehensive signature could represent a future direction of developing robust predictive biomarkers, particularly for the cold tumors, for ICI response.