Neogambogic acid enhances anti-PD-1 immunotherapy efficacy by attenuating suppressive function of MDSCs in pancreatic cancer.

BACKGROUND: Anti-PD-1-based immunotherapy has limited benefits in patients with pancreatic cancer. Accumulating data indicate that natural products exert antitumor activity by remodeling the tumor immune microenvironment. It has been reported that neogambogic acid (NGA), an active natural monomer extracted from Garcinia, has anti-inflammatory and antitumor effects. Nevertheless, there are few systematic studies on the antitumor efficacy and immunomodulatory effects of NGA in pancreatic cancer. METHODS: An orthotopic mouse model of pancreatic cancer was established and were treated with different doses of NGA. Tumor growth and ascites were observed. Flow cytometry and immunohistochemistry (IHC) were used to investigate the tumor immune microenvironment. CD11b+ MDSCs were infused back into mice with pancreatic cancer to observe tumor progression after NGA treatment. Bone marrow cells were induced to differentiate into MDSCs, and the effects of NGA on MDSCs were analyzed and the underlying mechanism was explored. The effects of NGA combined with an anti-PD-1 antibody on pancreatic cancer were further tested. RESULTS: NGA significantly inhibited the tumor growth and improve ascites character in pancreatic cancer model mice. Flow cytometry and IHC analysis revealed that NGA decreased the MDSCs proportion and infiltration in the tumor microenvironment. Moreover, adoptive MDSCs largely attenuated the inhibitory effect of NGA on the progression of pancreatic cancer. In addition, we showed that NGA significantly promoted apoptosis and inhibited the differentiation, migration and immunosuppressive function of MDSCs and decreased level of STAT3 and p-STAT3. Furthermore, we demonstrated that NGA synergistically enhanced the efficacy of anti-PD-1 antibodies against pancreatic cancer. CONCLUSION: NGA inhibited the progression of pancreatic cancer by inhibiting MDSCs in the tumor microenvironment, and enhanced the efficacy of anti-PD-1 therapy in the treatment of pancreatic cancer.

Hydroxygenkwanin suppresses peritoneal metastasis in colorectal cancer by modulating tumor-associated macrophages polarization.

Peritoneal metastasis is an important cause of high mortality and poor prognosis in colorectal cancer (CRC) patients. Therefore, the development of compounds with unique anti-CRC Peritoneal metastasis activities is urgently needed to improve the survival of CRC patients. Hydroxygenkwanin (HGK),a natural flavonoid compound, have been shown to display anti-inflammatory, antioxidant, antitumor, and immunoregulatory effects. Here, we employed CRC peritoneal metastasis mouse model with MC38 cells to examine the antitumor activity of HGK. The result showed that HGK not only inhibited peritoneal metastasis, but also significantly increased the proportion of M1-like macrophages while decreasing the proportion of M2-like macrophages within the tumor microenvironment (TME). Furthermore, we demonstrated that the inhibitory effect of HGK on peritoneal metastasis of CRC depended on macrophages in vitro and in vivo. Moreover, we revealed that HGK promoted the polarization of TAMs into M1-like macrophages and inhibited their polarization into M2-like macrophages in a LPS- or IL-4-induced bone marrow-derived macrophages (BMDMs) model and co-culture system. Finally, we also investigated the regulatory mechanism of HGK on TAMs polarization that HGK may active p-STAT5, p-NF-κB signaling in M1-like macrophages and inhibit p-STAT6, JMJD3, PPARγ expression in M2-like macrophages. Taken together, our findings suggest that HGK is a natural candidate for effective prevention of peritoneal metastasis in colorectal cancer, which provides a potential strategy for clinical treatment of colorectal cancer.

Saikosaponin a promotes neutrophil extracellular trap formation and bactericidal activity.

This study aimed to investigate the effects of SSa, one of the major triterpenoid saponins extracted from Radix bupleuri, on neutrophil extracellular trap (NET) formation and the mechanism associated with this process. Using Sytox green and immunofluorescence assays, we found SSa rapidly induced NET formation, which depended on NADPH oxidase (NOX)-independent ROS production and autophagy. Pharmacologic inhibitor studies indicated that ERK and PI3K/AKT signalling were also required for SSa-induced NET formation, whereas protein arginine deiminase 4 (PAD4) was not required. Furthermore, we found that SSa promoted neutrophil bactericidal activity mainly through NET formation. Based on flow cytometry and the Cell Counting Kit-8 (CCK-8) assays, the results demonstrated that SSa-induced NET formation occurred without neutrophil death. Taken together, these findings indicated that SSa could be a potential natural product to boost innate immune defense against pathogen attack via NET formation.

Pressure and Flame Propagation Characteristics of Suspended Coal Dust Explosions Induced by Gas Explosions.

In order to explore the pressure and flame propagation characteristics of gas-coal dust composite explosions, a semiclosed pipeline explosion test platform was built. The shock wave overpressure and explosion flame propagation law of different concentrations of suspended coal dust participating in gas explosions were studied in depth through experiments, and the coal dust motion law was simulated and analyzed based on Fluent software. The experimental results show that the peak pressure of gas-coal dust composite explosion is significantly higher than that of single-phase gas explosion, and the pressure peak increase ratio at the pipeline outlet is the highest; as the suspended coal dust concentration increases, the pressure rise rate at point 3 gradually decreases. Under the condition of 600 g/m3 coal dust participating in the explosion, the explosion pressure increase speed reduction ratio is 25.65%, the pressure wave secondary peak decreases, and the fluctuation frequency increases. When the explosion flame front passes through the suspended coal dust area, the flame shape changes from 'v' shape to 'finger' shape and propagates forward. The gas-coal dust composite explosion flame propagation speed shows a secondary acceleration phenomenon, after the flame front passes through the coal dust suspension area. As the coal dust concentration increases, the explosion core area moves away from the flame front. The coal dust cloud moves to the right, showing a concave rectangle; the larger the coal dust concentration, the smaller the moving speed. The experimental results and analysis provide an experimental basis for further exploring the mechanism and dynamic mechanism of gas-coal dust coupling explosion.

CD44 is a potential immunotherapeutic target and affects macrophage infiltration leading to poor prognosis.

CD44 plays a key role in the communication of CSCs with the microenvironment and the regulation of stem cell properties. UALCAN was used to analyze the expression of CD44 in bladder cancer (BLCA) and normal tissue. The UALCAN was utilized to analyze the prognostic value of CD44 in BLCA. The TIMER database was used to explore the relationship between CD44 and PD-L1; CD44 and tumor-infiltrating immune cells. The regulatory effect of CD44 on PD-L1 was verified by cell experiments in vitro. IHC confirmed the results of the bioinformatics analysis. GeneMania and Metascape were used to analyze protein-protein interaction (PPI) investigations and functional enrichment analysis. We found that BLCA patients with high CD44 expression had worse survival than those with low CD44 expression (P < 0.05). IHC and the TIMER database results showed that CD44 expression was positively correlated with PD-L1 expression (P < 0.05). At the cellular level, the expression of PD-L1 was significantly inhibited after CD44 expression was inhibited by siRNA. Immune infiltration analysis showed that CD44 expression levels in BLCA were significantly correlated with immune infiltration levels of different immune cells. IHC staining results further confirmed that the expression of CD44 in tumor cells was positively associated with the number of CD68+ macrophages and CD163+ macrophages (P < 0.05). Our results suggest that CD44 is a positive regulator of PD-L1 in BLCA and may be a key regulator of tumor macrophages infiltration and may be involved in M2 macrophage polarization. Our study provided new insights into the prognosis and immunotherapy of BLCA patients through macrophage infiltration and immune checkpoints.

Dioscin modulates macrophages polarization and MDSCs differentiation to inhibit tumorigenesis of colitis-associated colorectal cancer.

It has been reported that colitis is one of risk factors in colorectal cancer (CRC). Intervention of intestinal inflammation and in the early stage of tumorigenesis is of great significance to control the incidence and mortality of CRC. In recent years, natural active products of traditional Chinese medicine have been confirmed that they had made great progress in disease prevention. Here, we showed that Dioscin, a natural active product of Dioscorea nipponica Makino, inhibited initiation and tumorigenesis of AOM/DSS-induced colitis-associated colon cancer (CAC), including alleviating colonic inflammation, improving intestinal barrier function and decreasing tumor burden. In addition, we also explored the immunoregulatory effect of Dioscin on mice. The results showed that Dioscin modulated M1/M2 macrophages phenotype in spleen and decreased monocytic myeloid-derived suppressor cells (M-MDSCs) population in blood and spleen of mice. The in vitro assay demonstrated that Dioscin promoted M1 as well as inhibited M2 macrophages phenotype in LPS- or IL-4-induced bone marrow-derived macrophages (BMDMs) model. Based on the plasticity of MDSCs and its ability to differentiate into M1/M2 macrophages, we here found that Dioscin increased M1- and decreased M2-like phenotype during the process of MDSCs differentiation in vitro, suggesting Dioscin promoted MDSCs differentiate into M1 as well as inhibited its differentiation into M2 macrophages. Taken together, our study indicated that Dioscin had the inhibitory effect on the initial of tumorigenesis at early stage of CAC via the ant-inflammatory effect, which provided a natural active candidate for effective prevention of CAC.

Targeting PYCR2 inhibits intraperitoneal metastatic tumors of mouse colorectal cancer in a proline-independent approach.

Whether proline deficiency is a metabolic vulnerability in colorectal tumors is unknown. The aim of this study was to investigate the effects of proline metabolism-related genes and exogenous proline on the progression of colorectal cancer (CRC). We aimed to further clarify the role of pyrroline-5-carboxylate reductase (PYCR) 2, a key enzyme of proline synthesis, in the regulation of colorectal intraperitoneal metastatic tumors. This study was carried out based on The Cancer Genome Atlas (TCGA) data, database analysis, single-cell functional analysis, tissue microarray, cell experiments, and animal models. We found that, PYCR2 mRNA and protein levels were upregulated in CRC. The mRNA level of PYCR2 was closely related to the prognosis and tumor metastasis of CRC patients. The upregulated PYCR2 expression was at least partly due to low promoter methylation levels. The nomogram constructed based on PYCR2 expression and clinical characteristics of CRC showed good accuracy in predicting lymph node metastasis. Pycr2 knockdown inhibited epithelial-mesenchymal transition (EMT) of mouse CRC cells. Proline supplementation did not rescue the inhibition of mouse CRC cell proliferation and migration by Pycr2 knockdown. Proline supplementation also did not rescue the suppression of subcutaneous tumors and intraperitoneal metastatic tumors in mice by Pycr2 knockdown. PYCR2 co-expressed genes in TCGA-CRC were enriched in epigenetic modification-related biological processes and molecular functions. Four small molecules with the lowest binding energy to the PYCR2 protein were identified. Collectively, Pycr2 knockdown inhibited mouse CRC progression in a proline-independent approach. PYCR2 may be a promising tumor metastasis predictor and therapeutic target in CRC.

Imaging mass cytometry: High-dimensional and single-cell perspectives on the microenvironment of solid tumours.

Imaging mass cytometry (IMC) is a new technology integrating mass spectrometry, high-resolution laser ablation and immunohistochemistry/cytochemistry. A unique high-dimensional perspective comprehensively and accurately depicts the complex interaction of phenotype, signalling pathway and tumour microenvironment and is widely used in solid tumours. However, the application scenarios of IMC in basic medicine and clinical research in solid tumours lack systematic introduction and classification. This paper reviews the application of IMC in depicting the panorama of the tumour microenvironment, revealing tumour spatial heterogeneity, clarifying tumour pharmacological mechanisms, assisting in new drug development, and dynamically evaluating the efficacy of immunotherapy in solid tumours.

The antitumor activity of hPRDX5 against pancreatic cancer and the possible mechanisms.

Recombinant human peroxiredoxin-5 (hPRDX5), isolated from anti-cancer bioactive peptide (ACBPs), shows a homology of 89% with goat peroxiredoxin-5 (gPRDX5) and is reported to display anti-tumor activity in vivo. Herein, we explored the effect of hPRDX5 and the responsible mechanism in treating pancreatic cancer. Tumor-bearing mice were randomly divided into normal PBS group and treatment group (n=5; 10 mg/kg hPRDX5). Flow cytometry was employed to examine lymphocytes, myeloid-derived suppressor cell subsets, and the function proteins of natural killer (NK) cells in peripheral blood, spleen, and tumor tissues of mice. Western blot was used to measure the protein expressions of the key nodes in TLR4-MAPK-NF-κB signaling pathway. The rate of tumor suppression was 57.6% at a 10 mg/kg dose in orthotopic transplanted tumor mice. Moreover, the population of CD3+CD4+T cells, NK cells, and CD3+CD8+T cells was significantly increased in the tumor tissue of the hPRDX5 group, while the proportion of granulocytic-myeloid-derived suppressor cells decreased slightly. In addition, after treatment with hPRDX5, the percentage of NK cells in blood increased more than 4-fold. Our findings indicated that hPRDX5 effectively suppressed pancreatic cancer possibly via the TLR4-MAPK-NF-κB signaling cascade; hence hPRDX5 could be a prospective immunotherapy candidate for treating pancreatic cancer.

Comparison of diffusion kurtosis imaging and dynamic contrast enhanced MRI in prediction of prognostic factors and molecular subtypes in patients with breast cancer.

PURPOSE: To explore the clinical value of diffusional kurtosis imaging (DKI) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for predicting genotypes and prognostic factors of breast cancer. MATERIALS AND METHODS: A total of 130 female patients with pathologically-confirmed breast cancer and DKI and DCE-MRI data were reviewed retrospectively. Two radiologists independently evaluated mean diffusivity (MD) and mean kurtosis (MK) for the DKI model and volume transfer constant (Ktrans), reverse rate constant (Kep), and extracellular extravascular volume ratio (Ve) for the DCE-MRI model for post-processing analyses. Receiver operating characteristic (ROC) curves were used to analyse the diagnostic efficacies. RESULTS: MK, Ktrans, and Kep values were significantly higher in the high-grade Nottingham prognostic index (NPI) group (NPI ≥ 3.4) than in the low-grade NPI group (NPI < 3.4) (p < 0.01). The Ktrans had significantly greater area under ROC curve (AUC) than Kep and MK in predicting the NPI (p = 0.038 and 0.0217, respectively). Higher Ktrans, Kep, and MK values were observed in the high Ki-67 expression (≥14%) group than in the low Ki-67 expression (<14%) group (p < 0.05). Moreover, the MK value had better diagnostic performance than the Ktrans and Kep values in identifying Ki-67 expression status (p = 0.0097 and 0.0008, respectively). The combined model (MD + MK + Ktrans + Ve) had a significantly higher AUC than the single parameters for differentiating between luminal A/B and non-luminal subtypes (p = 0.003, < 0.001, 0.001, and < 0.001, respectively). The Human epidermal growth factor receptor 2-positive group had higher MD and Ve values than the other subtype groups (p < 0.05), and the Ve had a sensitivity of 100%. Moreover, the Ve AUC was significantly higher than that for MD in the identification of the triple-negative subtype (p = 0.048). CONCLUSION: Ktrans of DCE-MRI and MK of DKI demonstrated good diagnostic performance in predicting the prognostic factors of breast cancer. Additionally, the combination of the DCE-MRI and DKI models can improve the efficiency of predicting breast cancer genotypes.

[Effect of Xuanfu Daizhe decoction on stemness of esophageal cancer cells].

Objective: To investigate the effect of Xuanfu Daizhe decoction on the stemness of esophageal cancer cells. Methods: The BALB/c nude mice were randomly divided into the control group and experimental group, 5 mice in each group, which were continuously administered with normal saline and Xuanfu Daizhe decoction (9.89 g/kg) by gastrogavage, respectively. Human esophageal carcinoma cells ECA-109 (5×106) were subcutaneously injected into the mice on the 8th day. Tumor volume was measured twice a week. The mice were sacrificed 4 weeks after injection, and the tumor tissue and mouse serum were collected. The expressions of the major stemness-regulating transcription factors, i.e., NANOG, OCT4 and SOX2, were detected by RT-qPCR, Western Blot and immunohistochemistry. ECA-109 cells were treated with 10% fetal bovine serum and serum from the above two groups of mice for 48 hours respectively, and three replicate wells were set in each group, and the expressions of NANOG, OCT4, SOX2 and the levels of AKT and p-AKT were detected by RT-qPCR and Western Blot, respectively. ALDH activity in tumor cells was detected by flow cytometry; the number of spheroids of tumor cells was detected by the spheroidization experiment. Results: Compared with the control group, the growth and size of esophageal cancer tumors were significantly inhibited by Xuanfu Daizhe Decoction; the expressions of NANOG, OCT4, SOX2, the ALDH activity, the number of spheroids, and the levels of AKT and phosphorylated AKT (p-AKT) in esophageal cancer cells were significantly reduced by Xuanfu Daizhe Decoction both in vivo and in vitro. Conclusion: Xuanfu Daizhe Decoction inhibits the stemness of esophageal cancer cells, it may be a potentially effective drug for the treatment of esophageal cancer and provides a theoretical basis for the exploration of new effective drugs for the treatment of esophageal cancer.

3,4-dihydroxyphenylethyl alcohol glycoside reduces acetaminophen-induced acute liver failure in mice by inhibiting hepatocyte ferroptosis and pyroptosis.

APAP is one of the most commonly used antipyretic and pain medications, but excessive use can cause liver toxicity and damage. 3,4-dihydroxyphenylethyl alcohol glycoside (DAG) is a component isolated from Sargentodoxa cuneata known to have anti-apoptotic, anti-oxidation and anti-inflammatory effects. However, the effects of DAG on acute liver failure (ALF) are largely unknown. The purpose of this study is to study the protective effects and mechanism of DAG on APAP-induced ALF in mice. We established an ALF model in adult male pathogen-free C57BL/6 mice treated with APAP (300 mg/kg) by intraperitoneal injection and resolved by 24 h. Hematoxylin and eosin (HE) staining was used to evaluate the pathological changes in mouse liver tissue. The infiltration of neutrophils in liver tissue and reactive oxygen species (ROS) in AML12 cells were analyzed by flow cytometry. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione (GSH), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were analyzed using relevant kits. Our results show that DAG reduced APAP-induced serum ALT and AST levels, histopathological changes, liver neutrophil infiltration and proinflammatory cytokines production, also attenuated the accumulation of MDA and the exhaustion of GSH, CAT and SOD. In vitro experiment indicated that DAG dose-dependently inhibited APAP-induced the levels of pro-inflammatory factors (IL-1ß and IL18), and reactive oxygen species (ROS) and preventing GSH depletion in mouse AML12 hepatocytes. More interestingly, DAG inhibited the expression of ERK, HO-1, NLRP3, Caspase1 (p20) and Gasdermin-D and upregulated the expression of GPX4 in liver tissues and AML12hepatocytes. Therefore, our results indicate that DAG may act as a potential agent to treat ALF induced by APAP by inhibiting hepatocyte ferroptosis and pyroptosis.

Effects of different corticosteroid therapy on severe COVID-19 patients: a meta-analysis of randomized controlled trials.

BACKGROUND: To assess the efficacy and safety of corticosteroids in COVID-19 patients compared with standard care or placebo. METHODS: Electronic databases were searched to identify relevant studies. The mortality, adverse events, and other data from studies were pooled for statistical analysis. RESULTS: Ten randomized clinical trials were eligible for inclusion. Corticosteroid treatment in COVID-19 patients did not significantly reduce the risk of death (RR: 0.93; CI: 0.82, 1.05) and the need for mechanical ventilation (RR: 0.82; CI: 0.62, 1.08). No mortality reduction was also observed in the subgroup of patients requiring mechanical ventilation (RR: 0.90; CI: 0.79-1.03). The use of corticosteroids increased mortality in the subgroup of patients not requiring oxygen support (RR: 1.24; CI: 1.00-1.55). The survival benefit was observed in a low dosage of corticosteroids (RR: 0.90; CI: 0.84-0.97) and dexamethasone (RR: 0.90; 95% CI: 0.79-1.04). There was no difference in the rates of adverse events (RR: 1.13; CI: 0.58, 2.20) and secondary infections (RR: 0.87; CI: 0.66, 1.15). CONCLUSION: Corticosteroid treatment did not convincingly improve survival in severe COVID-19 patients. Low-dose dexamethasone could be considered as a drug for the treatment of COVID-19 patients. More high-quality trials are needed to further verify this conclusion.Expert Opinion: The effect of corticosteroids on patient survival highly depended on the selection of the right dosage and type and in a specific subgroup of patients. This meta-analysis, which included more RCTs, evaluated the safety and efficacy in severe COVID-19 patients and analyzed the effects of different types of corticosteroid treatments. Corticosteroid treatment did not convincingly improve survival in severe COVID-19 patients. But the low dose dexamethasone appear to have a role in the management of severe COVID-19 patients.

A novel nomogram for adult primary perihilar cholangiocarcinoma and considerations concerning lymph node dissection.

Objective: To construct a reliable nomogram available online to predict the postoperative survival of patients with perihilar cholangiocarcinoma. Methods: Data from 1808 patients diagnosed with perihilar cholangiocarcinoma between 2004 and 2015 were extracted from the National Cancer Institute Surveillance, Epidemiology, and End Results (SEER) database. They were randomly divided into training and validation sets. The nomogram was established by machine learning and Cox model. The discriminant ability and prediction accuracy of the nomogram were evaluated by concordance index (C-index), receiver operator characteristic (ROC) curve and calibration curve. Kaplan-Meier curves show the prognostic value of the associated risk factors and classification system. Results: Machine learning and multivariate Cox risk regression model showed that sex, age, tumor differentiation, primary tumor stage(T), lymph node metastasis(N), TNM stage, surgery, radiation, chemotherapy, lymph node dissection were associated with the prognosis of perihilar cholangiocarcinoma patients relevant factors (P < 0.05). A novel nomogram was established. The calibration plots, C-index and ROC curve for predictions of the 1-, 3-, and 5-year OS were in excellent agreement. In patients with stage T1 and N0 perihilar cholangiocarcinoma, the prognosis of ≥4 lymph nodes dissected was better than that of 1- 3 lymph nodes dissected (P < 0.01). Conclusion: The nomogram prognostic prediction model can provide a reference for evaluating the prognosis and survival rate of patients with perihilar cholangiocarcinoma. Patients with stage T1 and N0 perihilar cholangiocarcinoma have more benefits by increasing the number of lymph node dissection.

The antitumor activity of hPRDX5 against pancreatic cancer and the possible mechanisms

Recombinant human peroxiredoxin-5 (hPRDX5), isolated from anti-cancer bioactive peptide (ACBPs), shows a homology of 89% with goat peroxiredoxin-5 (gPRDX5) and is reported to display anti-tumor activity in vivo. Herein, we explored the effect of hPRDX5 and the responsible mechanism in treating pancreatic cancer. Tumor-bearing mice were randomly divided into normal PBS group and treatment group (n=5; 10 mg/kg hPRDX5). Flow cytometry was employed to examine lymphocytes, myeloid-derived suppressor cell subsets, and the function proteins of natural killer (NK) cells in peripheral blood, spleen, and tumor tissues of mice. Western blot was used to measure the protein expressions of the key nodes in TLR4-MAPK-NF-κB signaling pathway. The rate of tumor suppression was 57.6% at a 10 mg/kg dose in orthotopic transplanted tumor mice. Moreover, the population of CD3+CD4+T cells, NK cells, and CD3+CD8+T cells was significantly increased in the tumor tissue of the hPRDX5 group, while the proportion of granulocytic-myeloid-derived suppressor cells decreased slightly. In addition, after treatment with hPRDX5, the percentage of NK cells in blood increased more than 4-fold. Our findings indicated that hPRDX5 effectively suppressed pancreatic cancer possibly via the TLR4-MAPK-NF-κB signaling cascade; hence hPRDX5 could be a prospective immunotherapy candidate for treating pancreatic cancer.

Hydroxytyrosol Inhibits MDSCs and Promotes M1 Macrophages in Mice With Orthotopic Pancreatic Tumor.

The poor immunotherapy of pancreatic cancer is mainly due to its complex immunosuppressive microenvironment. The Mediterranean diet contributes to low cancer incidence. Hydroxytyrosol (HT) derived from olive oil has multiple health-promoting effects, but its therapeutic effect on pancreatic cancer remains controversial. Here, we evaluated the inhibitory effect of HT on mouse pancreatic cancer, and the effect of HT on the immune microenvironment. We found that HT can inhibit the proliferation of Panc 02 cells through signal transducer and activator of transcription (STAT) 3/Cyclin D1 signaling pathway. In the tumor-bearing mice treated with HT, the orthotopic pancreatic tumors were suppressed, accompanied by a decrease in the proportion of myeloid-derived suppressor cells (MDSCs) and an increase in the proportion of M1 macrophages. In addition, we found that HT inhibited the expression of immunosuppressive molecules in bone marrow (BM)-derived MDSCs, as well as down-regulated CCAAT/enhancer-binding protein beta (C/EBPß) and phosphorylation of STAT3. Moreover, HT enhanced the anti-tumor effect of anti-CD47 antibody in vivo. HT combined with plumbagin (PLB) induced more Panc 02 cells death than HT or PLB alone. This combination therapy not only inhibited the accumulation of MDSCs, but also promoted the infiltration of CD4+ and CD8+ T cells in the tumors. In summary, HT is a potential immunomodulatory drug for the treatment of pancreatic cancer.

miR-3065-3p promotes stemness and metastasis by targeting CRLF1 in colorectal cancer.

BACKGROUND: Colorectal cancer is one of the most common malignancy in the world. It has been reported that cancer stem cells (CSCs) serve as the primary drivers of tumorigenesis and tumor progression. There is an urgent need to explore novel molecules that regulate CSCs or their signatures. Increasing evidence has shown that miRNAs are involved in tumorigenesis and progression. Here, we aim to explore the regulatory effect and mechanism of miR-3065-3p on the stemness of colorectal cancer. METHODS: The expression of miR-3065-3p in colorectal cancer and the association of miR-3065-3p expression with prognosis of patients with colorectal cancer were analyzed using TCGA dataset or clinical cases. Gain or loss of function in different models, including colorectal cancer cell lines and orthotopic xenograft or liver metastatic mouse model, were used to investigate the effects of miR-3065-3p on colorectal cancer stemness and metastasis in vitro and in vivo. Cancer stemness was analyzed by detecting the ability of migration and invasion, NANOG, OCT4, and SOX2 expression, ALDH activity and sphere formation. In addition, the interaction of miR-3065-3p and cytokine receptor-like factor 1 (CRLF1) was analyzed theoretically and identified by the luciferase reporter assay. Moreover, the correlation between CRLF1 expression and miR-3065-3p was analyzed in colorectal cancer tissues. Finally, the effect of CRLF1 on the stemness and metastasis of colorectal cancer in vitro and in vivo was assessed. RESULTS: In this report, we found that miR-3065-3p was overexpressed in colorectal cancer and that its high expression was associated with poor prognosis of patients with colorectal cancer. miR-3065-3p promotes the stemness and metastasis of colorectal cancer. Furthermore, CRLF1 was the downstream target of miR-3065-3p and inhibited the stemness of colorectal cancer. In addition, CRLF1 expression was negatively correlated with miR-3065-3p in colorectal cancer tissues. And, CRLF1 mediated the effects of miR-3065-3p on promoting stemness of colorectal cancer cells. CONCLUSION: Our data suggest that miR-3065-3p promoted the stemness and metastasis of colorectal cancer by targeting CRLF1. miR-3065-3p might serve as a promising prognostic marker as well as a therapeutic target for colorectal cancer.

FTY720 induces neutrophil extracellular traps via a NADPH oxidase-independent pathway.

FTY720 is an immunosuppressive agent which has been approved to treat multiple sclerosis (MS). The main object of the present study is to investigate whether FTY720 has the potential to induce the formation of neutrophil extracellular traps (NETs) in vitro. Using Sytox Green assay and fluorescence microscopy, our results showed that FTY720 trigged the NET formation. In contrast to classic NET formation induced by Phorbol 12-myristate 13-acetate (PMA), FTY720-induced NETs were detected earlier and independent of NADPH oxidase (NOX) activity. Pharmacological inhibitor experiments indicated that autophagy was also required for the NET formation induced by FTY720. Moreover, p38 and AKT inhibitor significantly suppressed the NET formation by FTY720, whereas ERK inhibitor had no effect, suggesting that FTY720-induced NETs depended on the activation of p38 and AKT. We further found that citrullination of histone H3 and peptidylarginine deiminase 4 (PAD4) did not mediated FTY720-induced NET formation. Interestingly, necroptosis signaling activation was involved in the vital NET formation by FTY720, however, plasma membrane rupture resulting from necroptosis was not a major component of NET formation described here. Collectively, these findings indicated that FTY720 could be a potential antibacterial drug to protect host against pathogen infection.

Histone demethylase KDM6B inhibits breast cancer metastasis by regulating Wnt/ß-catenin signaling.

Tumor metastasis remains a major challenge for patients with breast cancer. Aberrant epigenetic factor lysine-specific demethylase 6B (KDM6B) has been associated with tumor progression. Here, we show that KDM6B is significantly down-regulated in human breast cancer tissues, and its low expression is associated with poor prognosis of patients with breast cancer. Furthermore, overexpression of KDM6B remarkably inhibited cell proliferation, invasion, migration and epithelial-mesenchymal transition markers of breast cancer cells in vitro and tumor growth and lung metastasis in vivo. Notably, the expression of KDM6B in breast cancer tissues was negatively correlated with that of ß-catenin, and overexpression of KDM6B decreased the expression of ß-catenin and its accumulation in the nucleus of breast cancer cells. Overall, our findings provide novel insights into suppression of metastasis of breast cancer cells by KDM6B via ß-catenin and suggest involvement of the KDM6B-Wnt/ß-catenin axis in breast cancer progression.

Cancer-derived exosomal miR-138-5p modulates polarization of tumor-associated macrophages through inhibition of KDM6B.

Rationale: Differential activation of macrophages correlates closely with tumor progression, and the epigenetic factor lysine demethylase 6B (KDM6B, previously named JMJD3) mediates the regulation of macrophage polarization through an unknown mechanism. Methods: We developed a suspension coculture system comprising breast cancer cells and macrophages and used RT-qPCR and western blotting to measure KDM6B expression. Bioinformatics and luciferase reporter assays were used to identify candidate microRNAs of cancer cells responsible for the downregulation of KDM6B. To determine if exosomes mediated the transfer of miR-138-5p between cancer cells to macrophages, we treated macrophages with exosomes collected from the conditioned medium of cancer cells. The effects of exosomal miR-138-5p on macrophage polarization were measured using RT-qPCR, flow cytometry, and chromatin immunoprecipitation assays. We employed a mouse model of breast cancer, metastatic to the lung, to evaluate the effects on tumor metastasis of macrophages treated with miR-138-5p-enriched exosomes. To develop a diagnostic evaluation index, the levels of exosomal miR-138-5p in samples from patients with breast cancer were compared to those of controls. Results: Coculture of breast cancer cells led to downregulation of KDM6B expression in macrophages. Cancer cell-derived exosomal miR-138-5p inhibited M1 polarization and promoted M2 polarization through inhibition of KDM6B expression in macrophages. Macrophages treated with exosomal miR-138-5p promoted lung metastasis, and the level of circulating exosomal miR-138-5p positively correlated with the progression of breast cancer. Conclusion: Our data suggest that miR-138-5p was delivered from breast cancer cells to tumor-associated macrophages via exosomes to downregulate KDM6B expression, inhibit M1 polarization, and stimulate M2 polarization. Therefore, exosomal miR-138-5p represents a promising prognostic marker and target for the treatment of breast cancer.