Cellular crosstalk of macrophages and therapeutic implications in non-small cell lung cancer revealed by integrative inference of single-cell transcriptomics.

Introduction: Non-small cell lung cancer (NSCLC) exhibits heterogeneity with diverse immune cell infiltration patterns that can influence tumor cell behavior and immunotherapy. A comprehensive characterization of the tumor microenvironment can guide precision medicine. Methods: Here, we generated a single-cell atlas of 398170 cells from 52 NSCLC patients, and investigated the imprinted genes and cellular crosstalk for macrophages. Subsequently, we evaluated the effect of tumor cells on macrophages and verified the expression of marker genes using co-culture experiments, flow cytometry and RT-qPCR assays. Results: Remarkable macrophage adaptability to NSCLC environment was observed, which contributed to generating tumor-associated macrophages (TAMs). We identified 5 distinct functional TAM subtypes, of which the majority were SELENOP-positive macrophages, with high levels of SLC40A1 and CCL13. The TAMs were also involved in mediating CD8+ T cell activity and form intercellular interaction with cancer cells, as indicated by receptor-ligand binding. Indirect coculture of tumor cells SPC-A1 and THP-1 monocytes, produced M2-like TAMs that highly expressed several markers of SELENOP-positive macrophages. The abundance of this type TAMs seemed to be associated with poorer overall survival rates [hazard ratio (HR) = 1.34, 95% confidence interval (CI) = 0.98-1.83, p = 0.068] based on deconvolution of TCGA-LUAD dataset. Discussion: In summary, we provided a high-resolution molecular resource of TAMs, and displayed the acquired properties in the tumor microenvironment. Dynamic crosstalk between TAMs and tumor cells via multiple ligand-receptor pairs were revealed, emphasizing its role in sustaining the pro-tumoral microenvironment and its implications for cancer therapy.

Novel insights into IL-37: an anti-inflammatory cytokine with emerging roles in anti-cancer process.

Interleukin-37 (IL-37) is a newly discovered member of IL-1 family. The cytokine was proved to have extensive protective effects in infectious diseases, allergic diseases, metabolic diseases, autoimmune diseases and tumors since its discovery. IL-37 was mainly produced by immune and some non-immune cells in response to inflammatory stimulus. The IL-37 precursors can convert into the mature forms after caspase-1 cleavage and activation intracellularly, and then bind to Smad-3 and transfer to the nucleus to inhibit the production and functions of proinflammatory cytokines; extracellularly, IL-37 binds to cell surface receptors to form IL-37/IL-18Rα/IL-1R8 complex to exert immunosuppressive function via inhibiting/activating multiple signal pathways. In addition, IL-37 can attenuate the pro-inflammatory effect of IL-18 through directly or forming an IL-37/IL-18BP/IL-18Rß complex. Therefore, IL-37 has the ability to suppress innate and acquired immunity of the host, and effectively control inflammatory stimulation, which was considered as a new hallmark of cancer. Specifically, it is concluded that IL-37 can inhibit the growth and migration of tumor cells, prohibit angiogenesis and mediate the immunoregulation in tumor microenvironment, so as to exert effective anti-tumor effects. Importantly, latest studies also showed that IL-37 may be a novel therapeutic target for cancer monitoring. In this review, we summarize the immunoregulation roles and mechanisms of IL-37 in anti-tumor process, and discuss its progress so far and potential as tumor immunotherapy.

Beyond liver cancer, more application scenarios for alpha-fetoprotein in clinical practice.

Alpha-fetoprotein (AFP) is a commonly used clinical biomarker. Before 1970, the two-way agar diffusion method was mainly used, and the specificity of AFP in the diagnosis of primary liver cancer was satisfactory. However, its positivity rate was not very high. The diagnostic value of AFP is changing with the evolution of detection methods. Here, we performed a literature search to identify English-language publications. The search was performed from January 2015 to April 2023 using the PubMed database and the following terms in [Titles/Abstracts]: alpha-fetoprotein, clinical practice, detection, etc. The references of retrieved articles were also screened to broaden the search. Studies referring to liver cancer and AFP detection methods were excluded. In this review, several clinical application scenarios for AFP were systematically reviewed, and its potential detection value in the future was discussed.

A study on inhibition of the Aß1-42-induced inflammatory response by the Huatuo Zaizao pill through the NF-κB signaling pathway.

Introduction: The pathology of Alzheimer's disease (AD) includes ß-amyloid (Aß) (plaques) and neurofibrillary tangles (NFTs). This study aimed to explore the efficacy of Huatuo Zaizao pill (HTZP) in an AD mouse model induced by injecting Aß1-42, and the neuroprotective mechanism of HTZP in AD. Material and methods: C57BL/6 (B6) mice were randomly divided into 4 groups (n = 10, per group): control group, AD model group, and 2 different doses of HTZP treated groups. The Morris water maze test was carried out on AD mice to assess the learning ability after treatment with HTZP for 15 day. The levels of inflammatory factors and the nuclear factor-κB (NF-κB) pathway were examined by western blot and real-time polymerase chain reaction (PCR). The content of microglia was investigated by immunofluorescence. Results: This study revealed that a cognitive disorder could be mitigated when the AD mice were treated with HTZP, which might be associated with the decreased level of pro-inflammatory factors, and the inhibitory activities of microglia. Additionally, phosphorylation of IκB and NF-κB p65 could be reduced by prohibiting the neuroinflammation of NF-κB activation in the hippocampus of AD mice. Conclusions: These results showed that HTZP could mitigate a cognitive disorder, diminish the activation of microglia, and inhibit the content of inflammatory factors through the NF-κB pathway in Aß1-42-induced AD mice. HTZP may be an appropriate agent for AD treatment in the future.

Noncanonical contribution of microglial transcription factor NR4A1 to post-stroke recovery through TNF mRNA destabilization.

Microglia-mediated neuroinflammation is involved in various neurological diseases, including ischemic stroke, but the endogenous mechanisms preventing unstrained inflammation is still unclear. The anti-inflammatory role of transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1) in macrophages and microglia has previously been identified. However, the endogenous mechanisms that how NR4A1 restricts unstrained inflammation remain elusive. Here, we observed that NR4A1 is up-regulated in the cytoplasm of activated microglia and localizes to processing bodies (P-bodies). In addition, we found that cytoplasmic NR4A1 functions as an RNA-binding protein (RBP) that directly binds and destabilizes Tnf mRNA in an N6-methyladenosine (m6A)-dependent manner. Remarkably, conditional microglial deletion of Nr4a1 elevates Tnf expression and worsens outcomes in a mouse model of ischemic stroke, in which case NR4A1 expression is significantly induced in the cytoplasm of microglia. Thus, our study illustrates a novel mechanism that NR4A1 posttranscriptionally regulates Tnf expression in microglia and determines stroke outcomes.

SP70 is a novel biomarker of hepatocellular carcinoma.

Background: Tumor-specific protein 70 (SP70) was identified as a new biomarker associated with the proliferation and invasion of cancer cells. This study aimed to investigate the expression of SP70 in hepatocellular carcinoma (HCC) and assess its clinical value in the diagnosis and prediction of early HCC recurrence. Methods: A total of 1049 subjects from the First Affiliated Hospital of Nanjing Medical University were recruited in this study. Serum SP70, alpha-fetoprotein (AFP) and prothrombin induced by vitamin K absence II (PIVKA-II) were measured. The diagnostic performance for HCC was obtained using the receiver operating characteristic (ROC) curve, and recurrence-free survival (RFS) was calculated using the Kaplan-Meier method. Univariate and multivariate analyses were performed to identify predictive factors of RFS. Results: SP70 was highly expressed in HCC cells and HCC tissue. Serum SP70 levels in the HCC group were significantly higher than in the benign liver diseases group and healthy control group (P<0.001). SP70 combined with AFP showed the best diagnostic performance (AUC=0.909, 95%CI [confidence interval]=0.890-0.929). Kaplan-Meier analysis revealed that patients with high SP70 levels had shorter median RFS than those with low SP70 levels (P=0.003). In addition, high SP70 levels were significantly associated with shorter RFS (P=0.037) in the AFP-negative subgroup. Univariate and multivariate analyses confirmed that preoperative serum SP70 level, serum AFP, tumor diameter and microvascular invasion were independent prognostic factors of RFS. Conclusion: SP70 is a promising biomarker in diagnosing HCC. High preoperative serum SP70 level is associated with an increased risk of early relapse and could be used as a valuable marker to predict early recurrence of HCC after resection.

Follow-up value of serum AFP and aminotransferases in chronic hepatitis B progression.

Introduction: Chronic viral hepatitis (CH) is a stage prior to cirrhosis and primary cancer. Standard protocols for CH assessment during the long follow-up period are of great importance for precise treatment and living quality improvement. In this study, we aimed to analyze multiple serum indexes in chronic hepatitis B (CHB)-infected patients and to discuss their combined values in clinical applications. Methods: Total 503 lines of laboratory data from 2012 to 2021 were extracted from103 CHB patients who were followed-up in our hospital. They were divided into the remission group and the progression group according to their complete clinical information and laboratory data. A series of models of serum indexes were analyzed to illustrate the fluctuation trend of @ach index in a time-dependent manner. Results: The models revealed that abundant serum alpha-fetoprotein (AFP) in the remission group was characteristically associated with hepatocyte destruction markers aspartate aminotransferase (AST) and alanine aminotransferase and favored a much longer progression-free period (P 0.0001). A model-derived equation consisting of serum AFP and AST values showed a good performance (83% reliability) to distinguish the two groups. Discussion: This study clearly demonstrates the intrinsic quantitative relationship between serum AFP and liver aminotransferases involving antivirus treatment response. The model-based equation compensates for serum hepatitis B virus DNA detection during outpatient follow-up and it may serve as a useful laboratory tool for CHB progression assessment.

Momordica charantia polysaccharide ameliorates D-galactose-induced aging through the Nrf2/ß-Catenin signaling pathway.

Aging is widely thought to be associated with oxidative stress. Momordica charantia (MC) is a classic vegetable and traditional herbal medicine widely consumed in Asia, and M. charantia polysaccharide (MCP) is the main bioactive ingredient of MC. We previously reported an antioxidative and neuroprotective effect of MCP in models of cerebral ischemia/reperfusion and hemorrhage injury. However, the role played by MCP in neurodegenerative diseases, especially during aging, remains unknown. In this study, we investigated the protective effect of MCP against oxidative stress and brain damage in a D-galactose-induced aging model (DGAM). The Morris water maze test was performed to evaluate the spatial memory function of model rats. The levels of malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were measured and telomerase activity was determined. The results showed that MCP treatment attenuated spatial memory dysfunction induced by D-galactose. In addition, MCP increased antioxidant capacity by decreasing MDA and increasing SOD and GSH levels. MCP treatment also improved telomerase activity in aging rats. Mechanistically, MCP promoted the entry of both Nrf2 and ß-Catenin into the nucleus, which is the hallmark of antioxidation signaling pathway activation. This study highlights a role played by MCP in ameliorating aging-induced oxidative stress injury and reversing the decline in learning and memory capacity. Our work provides evidence that MCP administration might be a potential antiaging strategy.

microRNA, a Subtle Indicator of Human Cytomegalovirus against Host Immune Cells.

Human cytomegalovirus (HCMV) is a double-stranded DNA virus that belongs to the ß-herpesvirus family and infects 40-90% of the adult population worldwide. HCMV infection is usually asymptomatic in healthy individuals but causes serious problems in immunocompromised people. We restricted this narrative review (PubMed, January 2022) to demonstrate the interaction and molecular mechanisms between the virus and host immune cells with a focus on HCMV-encoded miRNAs. We found a series of HCMV-encoded miRNAs (e.g., miR-UL112 and miR-UL148D) are explicitly involved in the regulation of viral DNA replication, immune evasion, as well as host cell fate. MiRNA-targeted therapies have been explored for the treatment of atherosclerosis, cardiovascular disease, cancer, diabetes, and hepatitis C virus infection. It is feasible to develop an alternative vaccine to restart peripheral immunity or to inhibit HCMV activity, which may contribute to the antiviral intervention for serious HCMV-related diseases.

Serum miR-27a is a biomarker for the prognosis of non-small cell lung cancer patients receiving chemotherapy.

BACKGROUND: Lung cancer has a high incidence and a 5-year survival rate of less than 15%. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases. Chemotherapy and immunotherapy are the most frequently used alternative treatments for patients with advanced-stage NSCLC in whom surgery failed. Previous studies have suggested that miR-27a is involved in cancer development and progression. The purpose of this study was to investigate the clinical value of miR-27a in the prognosis of NSCLC patients after chemotherapy. METHODS: Flow cytometry was used to detect the apoptosis rate of SPC-A1 cells treated with optical cisplatin at different times. Simultaneously, the expression of miR-27a in supernatants and cells was detected. Fifty-two newly diagnosed NSCLC patients were recruited. All patients received gemcitabine and cisplatin as first-line chemotherapy and docetaxel as second-line chemotherapy. At the end of every chemotherapy cycle, a therapeutic evaluation was performed according to the RECIST criteria. The expression of serum miR-27a was detected in each cycle. RESULTS: After treatment with 2.5 µg/mL cisplatin, the apoptosis rates of SPC-A1 cells were significantly greater than those of the paired untreated control groups at 12, 24, 48 and 72 h. The expression of miR-27a in supernatants and cells was also consistent with the apoptosis rate and changed a time-dependent manner. The chi-square test showed that an increase in miR-27a after chemotherapy was more common in patients who achieved partial response (PR) than in those who achieved no response (NR) (61.5% vs. 30.8%, P=0.026). Kaplan-Meier survival analysis indicated that patients with decreased miR-27a levels had poorer outcomes than those with increased miR-27a levels (P<0.05). Furthermore, dynamic changes in serum miR-27a with a gradual increasing trend during chemotherapy predicted a good prognosis. CONCLUSIONS: Collectively, our results suggest that miR-27a is involved in the apoptosis of lung cancer cells and that serum miR-27a levels are related to the prognosis of NSCLC patients. The expression levels of miR-27a in the serum may be an independent predictor for the prognosis of NSCLC.

Development and validation of a prediction model for microvascular invasion in hepatocellular carcinoma.

BACKGROUND: Microvascular invasion (MVI) is an important prognostic factor affecting early recurrence and overall survival in hepatocellular carcinoma (HCC) patients after hepatectomy and liver transplantation, but it can be determined only in surgical specimens. Accurate preoperative prediction of MVI is conducive to clinical decisions. AIM: To develop and validate a preoperative prediction model for MVI in patients with HCC. METHODS: Data from 454 patients with HCC who underwent hepatectomy at the First Affiliated Hospital of Nanjing Medical University between May 2016 and October 2019 were retrospectively collected. Then, the patients were nonrandomly split into a training cohort and a validation cohort. Logistic regression analysis was used to identify variables significantly associated with MVI that were then included in the nomogram. We evaluated the discrimination and calibration ability of the nomogram by using R software. RESULTS: MVI was confirmed in 209 (46.0%) patients by a pathological examination. Multivariate logistic regression analysis identified four risk factors independently associated with MVI: Tumor size [odds ratio (OR) = 1.195; 95% confidence interval (CI): 1.107-1.290; P < 0.001], number of tumors (OR = 4.441; 95%CI: 2.112-9.341; P < 0.001), neutrophils (OR = 1.714; 95%CI: 1.036-2.836; P = 0.036), and serum α-fetoprotein (20-400 ng/mL, OR = 1.955; 95%CI: 1.055-3.624; P = 0.033; >400 ng/mL, OR = 3.476; 95%CI: 1.950-6.195; P < 0.001). The concordance index was 0.79 (95%CI: 0.74-0.84) and 0.81 (95%CI: 0.74-0.89) in the training and validation cohorts, respectively. The calibration curves showed good agreement between the predicted risk by the nomogram and real outcomes. CONCLUSION: We have developed and validated a preoperative prediction model for MVI in patients with HCC. The model could aid physicians in clinical treatment decision making.

Relationship between White Matter Hyperintensities and Hematoma Volume in Patients with Intracerebral Hematoma.

The relationship of white-matter hyperintensity (WMH) to intracerebral hemorrhage (ICH) remains unclear. In this retrospective study, we investigated whether the severity and progression of WMH could be related to the hematoma volume and absorption in ICH. 2338 WMH patients with ICH aged≥40 years receiving brain computed tomography (CT) imaging within 12 hours of ICH symptom onset were screened, and 227 patients were included in the final study. The severity and progression of WMH were assessed using the software programs MRICRON and ITK-SNAP on brain magnetic resonance imaging (MRI) and the hematoma volumes and absorption with ITK-SNAP software on CT. We assessed the association of WMH severity with ICH volume in 227 patients at baseline. Totally 183 of 227 patients underwent repeated CT within 14 days of ICH onset. The relationship of WMH severity to ICH absorption was analyzed in 183 patients. Additionally, among all 227 patients, 37 subjected to another MRI before ICH onset were divided into two groups according to WMH progression: non-progression and progression groups. The link between WMH progression and hematoma volume was examined. The ICH volume was significantly larger in patients with the highest WMH scores than in those with the lowest WMH scores. Larger WMH volume was independently associated with larger ICH volume (odds ratio 1.00; 95% CI, 1.00 to 1.00; P = 0.049). There was a trend towards WMH progression being related to ICH volume (P =0.049). Contrastingly, the WMH volume was not linked with hematoma absorption (P = 0.79). In conclusion, we found that greater severity and progression of WMH were associated with larger ICH volume. Our findings suggest that WMH might provide important prognostic information about patients with ICH and may have implications for treatment stratification.

4-((5-(Tert-butyl)-3-chloro-2-hydroxybenzyl) amino)-2-hydroxybenzoic acid protects against oxygen-glucose deprivation/reperfusion injury.

AIMS: Oxidative stress is one of the most important pathological mechanisms which could aggravate ischemic stroke injury. In order to seek for better treatment therapies to alleviate stroke injury, novel chemicals have been synthetized. In the present study, a new compound 4-((5-(tert-butyl)-3-chloro-2-hydroxybenzyl) amino)-2- hydroxybenzoic acid, named LX009, was used to determine whether it could reduce the oxidative stress caused by oxygen-glucose deprivation (OGD)/reperfusion (RP) and exert neuroprotective effect both in mouse Neuro 2A (N2A) neuroblastoma cells and mouse primary cortical neurons. MAIN METHODS: OGD/RP was performed as an in vitro model to mimic the pathologic process of ischemic stroke. We explored the anti-apoptosis effect of LX009 through CCK8 assay, calcein acetoxymethylester/propidium iodide (calcein-AM/PI) staining, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit, caspase-3 activity assay. Besides, the anti-oxidative stress effect of the drug was determined by intracellular reactive oxygen species (ROS) detection, nitrite analysis, measurement of mitochondrial membrane potential (MMP), intracellular catalase (CAT) and Mn-superoxide dismutase (Mn-SOD) activity. KEY FINDINGS: Our results indicated that LX009 could alleviate OGD/RP-induced cell apoptosis. Furthermore, OGD/RP induced oxidative stress could be reserved by LX009, including measurements of intracellular ROS production, MMP, CAT and Mn-SOD activity. Mechanistically, the phosphorylation level of Akt, as well as the expression of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were elevated after LX009 treatment. SIGNIFICANCE: Our present study indicated that LX009 might have the potential to be an anti-oxidative stress agent in the future.

Lentivirus-Mediated HDAC3 Inhibition Attenuates Oxidative Stress in APPswe/PS1dE9 Mice.

Amyloid-ß (Aß) induces a burst of oxidative stress and plays a critical role in the pathogenesis of Alzheimer's disease (AD). Our previous results have shown that histone deacetylase 3 (HDAC3) inhibition ameliorates spatial memory deficits and decreases the Aß burden in the brains of 9-month-old APPswe/PS1dE9 (APP/PS1) mice. In this study, we investigated the role of HDAC3 inhibition in oxidative stress in vivo and in vitro models of AD. HDAC3 was detected mainly in the neurons, and HDAC3 inhibition significantly decreased reactive oxygen species generation and improved primary cortical neuron viability. In addition, HDAC3 inhibition attenuated spatial memory dysfunction in 6-month-old APP/PS1 mice, and decreased the apoptotic rate in the hippocampi as demonstrated by TUNEL staining. HDAC3 inhibition also reduced markers of lipid peroxidation, protein oxidation, and DNA/RNA oxidation in the hippocampi of APP/PS1 mice. Moreover, HDAC3 inhibition inactivated the c-Abl/MST1/YAP signaling pathway in the hippocampi of APP/PS1 mice. In conclusion, our data show that HDAC3 inhibition can attenuate spatial memory deficits and inhibit oxidative stress in APP/PS1 mice; these results indicate a potential strategy for AD treatment.

The Anti-inflammatory Effects of 4-((5-Bromo-3-chloro-2-hydroxybenzyl) amino)-2-hydroxybenzoic Acid in Lipopolysaccharide-Activated Primary Microglial Cells.

Over-activated microglial cells are known to be implicated in various neurological diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis. Our previous reports have shown that ZL006, a compound with a hydrophobic ring A and a hydrophilic ring B with a carboxyl group, exhibited stronger neuroprotective activity in vitro and in vivo. However, the directly anti-inflammatory effects of these compounds in the central nervous system (CNS) have not been elucidated. In the present study, as a part of our ongoing screening experiment to evaluate the anti-inflammatory effects of new compounds, a newly synthesized 4-((5-bromo-3-chloro-2-hydroxybenzyl) amino)-2-hydroxybenzoic acid (LX007) was used to examine whether it could reduce the inflammatory responses of activated microglia. Our results indicated that LX007 inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and prostaglandin E2 (PGE2) expression, as well as their regulatory gene-inducible NO syntheses (iNOS) and cyclooxygenase-2 (COX-2) in LPS-treated primary microglia. LPS-induced production from microglia of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF-α) was also significantly attenuated by LX007. Mechanistically, LX007 potently suppressed phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) p65 nuclear translocation in LPS-induced microglia. We therefore conclude that LX007 exhibits anti-inflammatory effects in LPS-stimulated microglial cells by inhibiting pro-inflammatory mediators corresponding to the downregulating of MAPKs and NF-κB activation. Taken together, the present study indicated that LX007 may have potential to be developed into an anti-inflammatory agent in the future.

Proteomic analysis of the effects of Nur77 on lipopolysaccharide-induced microglial activation.

Microglia are critical components of the immune response in the central nervous system. Our study aims to explore potential role of Nur77 in lipopolysaccharide (LPS)-induced microglial activation. Primary wild-type and Nur77-/- microglia were stimulated with LPS and protein extracts were detected via mass spectrometry. Q-PCR and western blotting were performed to validate candidate proteins. A total of 2004 proteins were identified, with 749 and 677 significantly differentially expressed proteins in wild-type and Nur77-/- microglia in resting and activated states, respectively. Signaling pathway analysis showed that significantly differentially expressed proteins in LPS-treated Nur77-/- microglia were present in important signaling pathways of microglial activation, including the Toll-like receptor signaling pathway, the MAPK signaling pathway, FcγR-mediated phagocytosis, and chemokine signaling pathways. Furthermore, we found that Nur77 could be the upstream protein of the vav1 and ERK1/2 signaling pathway. This study provided new insights into the understanding the mechanisms of the effects of Nur77 on LPS-activated microglia.