Vortexing-generated high throughput single-cell droplets for facile analysis of multiplexed microRNA dynamic secretion.

Discriminating secretory phenotypes provides a direct, intact, and dynamic way to evaluate the heterogeneity in cell states and activation, which is significant for dissecting non-genetic heterogeneity for human health studies and disease diagnostics. In particular, secreted microRNAs, soluble signaling molecules released by various cells, are increasingly recognized as a critical mediator for cell-cell communication and the circulating biomarkers for disease diagnosis. However, single-cell analysis of secreted miRNAs is still lacking due to the limited available tools. Herein, we realized three-plexed miRNA secretion analysis over four time points from single cells encapsulated in picoliter droplets with extreme simplicity, coupling vortexing-generated single-cell droplets with multiplexed molecular beacons. Notably, our platform only requires pipetting and vortexing steps to finish the assay setup within 5 min with minimal training, and customized software was developed for automatic data quantification. Applying the platform to human cancer cell lines and primary cells revealed previously undifferentiated heterogeneity and paracrine signaling underlying miRNA secretion. This platform can be used to dissect secretion heterogeneity and cell-cell interactions and has the potential to become a widely used tool in biomedical research.

Glycyrrhizin Protects Submandibular Gland Against Radiation Damage by Enhancing Antioxidant Defense and Preserving Mitochondrial Homeostasis.

Aims: Radiotherapy inevitably causes radiation damage to the salivary glands (SGs) in patients with head and neck cancers (HNCs). Excessive reactive oxygen species (ROS) levels and imbalanced mitochondrial homeostasis are serious consequences of ionizing radiation in SGs; however, there are few mitochondria-targeting therapeutic approaches. Glycyrrhizin is the main extract of licorice root and exhibits antioxidant activity to relieve mitochondrial damage in certain oxidative stress conditions. Herein, the effects of glycyrrhizin on irradiated submandibular glands (SMGs) and the related mechanisms were investigated. Results: Glycyrrhizin reduced radiation damage in rat SMGs at both the cell and tissue levels, and promoted saliva secretion in irradiated SMGs. Glycyrrhizin significantly downregulated high-mobility group box-1 protein (HMGB1) and toll-like receptor 5 (TLR5). Moreover, glycyrrhizin significantly suppressed the increases in malondialdehyde and glutathione disulfide (GSSG) levels; elevated the activity of some critical antioxidants, including superoxide dismutase, catalase, glutathione peroxidase, and glutathione (GSH); and increased the GSH/GSSG ratio in irradiated cells. Importantly, glycyrrhizin effectively enhanced thioredoxin-2 levels and scavenged mitochondrial ROS, inhibited the decline in mitochondrial membrane potential, improved adenosine triphosphate synthesis, preserved the mitochondrial ultrastructure, activated the proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)/nuclear respiratory factor 1/2 (NRF1/2)/mitochondrial transcription factor A (TFAM) signaling pathway, and inhibited mitochondria-related apoptosis in irradiated SMG cells and tissues. Innovation: Radiotherapy causes radiation sialadenitis in HNC patients. Our data suggest that glycyrrhizin could be a mitochondria-targeted antioxidant for the prevention of radiation damage in SGs. Conclusion: These findings demonstrate that glycyrrhizin protects SMGs from radiation damage by downregulating HMGB1/TLR5 signaling, maintaining intracellular redox balance, eliminating mitochondrial ROS, preserving mitochondrial homeostasis, and inhibiting apoptosis.

Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling.

Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2ß1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2ß1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.

The MocR family transcriptional regulator DnfR has multiple binding sites and regulates Dirammox gene transcription in Alcaligenes faecalis JQ135.

Microbial ammonia oxidation is vital to the nitrogen cycle. A biological process, called Dirammox (direct ammonia oxidation, NH3 →NH2 OH→N2 ), has been recently identified in Alcaligenes ammonioxydans and Alcaligenes faecalis. However, its transcriptional regulatory mechanism has not yet been fully elucidated. The present study characterized a new MocR-like transcription factor DnfR that is involved in the Dirammox process in A. faecalis strain JQ135. The entire dnf cluster was composed of 10 genes and transcribed as five transcriptional units, that is, dnfIH, dnfR, dnfG, dnfABCDE and dnfF. DnfR activates the transcription of dnfIH, dnfG and dnfABCDE genes, and represses its own transcription. The intact 1506-bp dnfR gene was required for activation of Dirammox. Electrophoretic mobility shift assays and DNase I footprinting analyses showed that DnfR has one binding site in the dnfH-dnfR intergenic region and two binding sites in the dnfG-dnfA intergenic region. Three binding sites of DnfR shared a 6-bp repeated conserved sequence 5'-GGTCTG-N17 -GGTCTG-3' which was essential for the transcription of downstream target genes. Cysteine and glutamate act as possible effectors of DnfR to activate the transcription of transcriptional units of dnfG and dnfABCDE, respectively. This study provided new insights in the transcriptional regulation mechanism of Dirammox by DnfR in A. faecalis JQ135.

Salidroside Ameliorates Radiation Damage by Reducing Mitochondrial Oxidative Stress in the Submandibular Gland.

Radiotherapy for patients with head and neck cancer inevitably causes radiation damage to salivary glands (SGs). Overproduction of reactive oxygen species (ROS) leads to mitochondrial damage and is critical in the pathophysiology of SG radiation damage. However, mitochondrial-targeted treatment is unavailable. Herein, both in vitro and in vivo models of radiation-damaged rat submandibular glands (SMGs) were used to investigate the potential role of salidroside in protecting irradiated SGs. Cell morphology was observed with an inverted phase-contrast microscope. Malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), mitochondrial ROS, mitochondrial membrane potential (MMP), and ATP were measured using relevant kits. The mitochondrial ultrastructure was observed under transmission electron microscopy. Cell apoptosis was determined by Western blot and TUNEL assays. Saliva was measured from Wharton's duct. We found that salidroside protected SMG cells and tissues against radiation and improved the secretion function. Moreover, salidroside enhanced the antioxidant defense by decreasing MDA, increasing SOD, CAT, and GSH, and scavenging mitochondrial ROS. Furthermore, salidroside rescued the mitochondrial ultrastructure, preserved MMP and ATP, suppressed cytosolic cytochrome c and cleaved caspase 3 expression, and inhibited cell apoptosis. Together, these findings first identify salidroside as a mitochondrial-targeted antioxidant for preventing SG radiation damage.

Biodegradation of Quinoline by a Newly Isolated Salt-Tolerating Bacterium Rhodococcus gordoniae Strain JH145.

Quinoline is a typical nitrogen-heterocyclic compound with high toxicity and carcinogenicity which exists ubiquitously in industrial wastewater. In this study, a new quinoline-degrading bacterial strain Rhodococcus sp. JH145 was isolated from oil-contaminated soil. Strain JH145 could grow with quinoline as the sole carbon source. The optimum growth temperature, pH, and salt concentration were 30 °C, 8.0, and 1%, respectively. 100 mg/L quinoline could be completely removed within 28 h. Particularly, strain JH145 showed excellent quinoline biodegradation ability under a high-salt concentration of 7.5%. Two different quinoline degradation pathways, a typical 8-hydroxycoumarin pathway, and a unique anthranilate pathway were proposed based on the intermediates identified by liquid chromatography-time of flight mass spectrometry. Our present results provided new candidates for industrial application in quinoline-contaminated wastewater treatment even under high-salt conditions.

Small extracellular vesicle-bound vascular endothelial growth factor secreted by carcinoma-associated fibroblasts promotes angiogenesis in a bevacizumab-resistant manner.

The blood vessel growth inhibitor bevacizumab targets vascular endothelial growth factor (VEGF), a crucial regulator of angiogenesis. Recently, small extracellular vesicles (sEVs) have been demonstrated to be important vehicles in the transport of growth factors to target cells. In this study, we isolated primary carcinoma-associated fibroblasts (CAFs) from four human oral squamous cell carcinoma (OSCC) specimens. Compared with other non-extracellular vesicle components, CAF-derived sEVs were found to be the main regulators of angiogenesis. The ability of CAF sEVs to activate VEGF receptor 2 (VEGFR2) signaling in human umbilical vein endothelial cells (HUVEC) was dependent on the association between sEVs and VEGF. In addition, sEV-bound VEGF secreted by CAFs further activated VEGFR2 signaling in HUVEC in a bevacizumab-resistant manner. VEGF was found to interact with heparan sulfate proteoglycans on the CAF sEV surface and could be released by heparinase I/III. The bioactivity of the dissociated VEGF was retained in vitro and in vivo and could be neutralized by bevacizumab. These findings suggest that the combined use of heparinase and bevacizumab might inhibit angiogenesis in patients with high levels of sEV-bound VEGF.

Extracellular vesicles of carcinoma-associated fibroblasts creates a pre-metastatic niche in the lung through activating fibroblasts.

OBJECTIVES: Carcinoma-associated fibroblasts (CAFs) have been known to promote cancer progression by modifying the primary tumor microenvironment. We aimed to elucidate the intercellular communication between CAFs and secondary organs in salivary adenoid cystic carcinoma (SACC) metastasis. METHODS: Pre-metastatic and metastatic animal models of SACC were established using extracellular vesicles (EVs) from CAFs and SACC cells. Lung fibroblasts (LFs) were treated with EVs and their transcriptomic alterations were identified by RNA sequencing. ITRAQ were performed to analyze EV cargos. TC I-15 was used to inhibit EV uptake by LFs and SACC lung metastasis in vivo. RESULTS: Here, we show that CAF EVs induced lung pre-metastatic niche formation in mice and consequently increased SACC lung metastasis. The pre-metastatic niche induced by CAF EVs was different from that induced by SACC EVs. CAF EVs presented a great ability for matrix remodeling and periostin is a potential biomarker characterizing the CAF EV-induced pre-metastatic niche. We found that lung fibroblast activation promoted by CAF EVs was a critical event at the pre-metastatic niche. Integrin α2ß1 mediated CAF EV uptake by lung fibroblasts, and its blockage by TC I-15 prevented lung pre-metastatic niche formation and subsequent metastasis. Plasma EV integrin ß1 was considerably upregulated in the mice bearing xenografts with high risk of lung metastasis. CONCLUSIONS: We demonstrated that CAF EVs participated in the pre-metastatic niche formation in the lung. Plasma EV integrin ß1 might be a promising biomarker to predict SACC metastasis at an early stage. An integrated strategy targeting both tumor and stromal cells is necessary to prevent SACC metastasis.

Multiplexed profiling of single-cell extracellular vesicles secretion.

Extracellular vesicles (EVs) are important intercellular mediators regulating health and diseases. Conventional methods for EV surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EV secretion. Herein, by using spatially patterned antibody barcodes, we realized multiplexed profiling of single-cell EV secretion from more than 1,000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to a deep understanding of previously undifferentiated single-cell heterogeneity underlying EV secretion. Notably, we observed that the decrement of certain EV phenotypes (e.g., CD63+EV) was associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EV secretion and cytokines secretion simultaneously from the same single cells to investigate the multidimensional spectrum of cellular communications, from which we resolved tiered functional subgroups with distinct secretion profiles by visualized clustering and principal component analysis. In particular, we found that different cell subgroups dominated EV secretion and cytokine secretion. The technology introduced here enables a comprehensive evaluation of EV secretion heterogeneity at single-cell level, which may become an indispensable tool to complement current single-cell analysis and EV research.

Nicotinamide Phosphoribosyltransferase Upregulation by Phenylephrine Reduces Radiation Injury in Submandibular Gland.

PURPOSE: Radiation therapy for head and neck cancer commonly leads to radiation sialadenitis. Emerging evidence has indicated that phenylephrine pretreatment reduces radiosensitivity in the salivary gland; however, the underlying cytoprotective mechanism remains unclear. Nicotinamide phosphoribosyltransferase (NAMPT) is not only a key enzyme for the nicotinamide adenine dinucleotide salvage pathway, but also a cytokine participating in cell survival, metabolism, and longevity, with a broad effect on cellular functions in physiology and pathology. However, the regulatory events of NAMPT in response to the irradiated salivary gland are unknown. METHODS AND MATERIALS: The cell viability of primary cultured submandibular gland cells was determined using the PrestoBlue assay. NAMPT expression was measured using reverse transcriptase polymerase chain reaction and Western blotting in vitro and in vivo. Silent information regulator 1 (SIRT1) and phosphorylated Akt protein levels were examined by Western blotting. The cellular locations of NAMPT and SIRT1 were detected by immunohistochemistry. NAMPT promoter activity was assessed using the luciferase reporter gene assay. RESULTS: NAMPT was mainly distributed in the cytoplasm of granular convoluted tubule cells and ductal cells in normal submandibular glands. mRNA and protein expression of NAMPT was downregulated after radiation but upregulated with phenylephrine pretreatment both in vivo and in vitro. Moreover, the protein expression of phosphorylated Akt and SIRT1 was decreased in irradiated glands, and phenylephrine pretreatment restored the expression of both. SIRT1 was mainly located in the cell nucleus and cytoplasm in the normal submandibular gland. Phenylephrine dramatically enhanced the expression of SIRT1, which was significantly reduced by radiation. Furthermore, phenylephrine induced a marked increase of NAMPT promoter activity. CONCLUSIONS: These findings reveal the regulatory mechanisms of NAMPT expression, which help to understand the mechanism of the cytoprotective role of phenylephrine on irradiated tissues.

Carcinoma-Associated Fibroblasts Lead the Invasion of Salivary Gland Adenoid Cystic Carcinoma Cells by Creating an Invasive Track.

Carcinoma-associated fibroblasts (CAFs) are critical in determining tumor invasion and metastasis. However the role of CAFs in the invasion of salivary gland adenoid cystic carcinoma (ACC) is poorly understood. In this study, we isolated primary CAFs from two ACC patients. ACC-derived CAFs expressed typical CAF biomarkers and showed increased migration and invasion activity. Conditioned medium collected from CAFs significantly promoted ACC cell migration and invasion. Co-culture of CAFs with ACC cells in a microfluidic device further revealed that CAFs localized at the invasion front and ACC cells followed the track behind the CAFs. Interfering of both matrix metalloproteinase and CXCL12/CXCR4 pathway inhibited ACC invasion promoted by CAFs. Overall, our study demonstrates that ACC-derived CAFs exhibit the most important defining feature of CAFs by promoting cancer invasion. In addition to secretion of soluble factors, CAFs also lead ACC invasion by creating an invasive track in the ECM.

Effect of Phenylephrine Pretreatment on the Expressions of Aquaporin 5 and c-Jun N-Terminal Kinase in Irradiated Submandibular Gland.

Radiotherapy for malignant tumors of the head and neck commonly leads to radiation-induced sialadenitis as a result of radiation-induced salivary gland dysfunction. We demonstrated previously that phenylephrine could protect the irradiated submandibular gland against apoptosis, although the mechanism is unclear. In this study, we investigated the influence of phenylephrine pretreatment on the expressions of aquaporin 5 (AQP5) and c-Jun N-terminal kinase (JNK) that were presumed to have a role in radiation-induced salivary gland dysfunction. Rats pretreated with phenylephrine (5 mg/kg) were locally irradiated (20 Gy) in the head and neck region. The submandibular glands were removed on day 7 after irradiation. The expression of AQP5 and activation of JNK were measured by immunohistochemistry and Western blot. The localization of AQP5 at the apical and lateral plasma membrane of acinar cells was significantly reduced by irradiation, but markedly enhanced with phenylephrine pretreatment. The protein expression of AQP5 was decreased by 84.91% in irradiated glands, whereas it was fully recovered to the control level in phenylephrine-pretreated glands. Moreover, many acinar, ductal and granular convoluted tubular cells in the irradiated glands exhibited intense immunoreactivity for p-JNK, while in the phenylephrine-pretreated irradiated glands, only a few acinar cells exhibited very faint immunoreactivity for p-JNK. The protein expression level of p-JNK was increased by 41.65% in the irradiated alone glands, but was significantly decreased in the phenylephrine-pretreated irradiated glands. These results suggest that the protective mechanism of phenylephrine might be related to the improved expression of AQP5 and decreased activation of JNK. Pretreatment with phenylephrine in patients undergoing radiotherapy may provide a helpful strategy for suppression of radiation-induced sialadenitis.

Myofibroblasts from salivary gland adenoid cystic carcinomas promote cancer invasion by expressing MMP2 and CXCL12.

OBJECTIVES: Salivary gland adenoid cystic carcinoma (ACC) is one of the most common malignant tumours in the oral and maxillofacial region, and has high aggressive potential. Tumour and stroma interactions are critical in determining the biological characteristics of malignancy. The aim of this study was to investigate the presence of myofibroblasts and their roles in the invasive characteristics of ACC. METHODS AND RESULTS: Immunohistochemistry was used to detect the expression of vimentin (VIM), α-smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP2) and CD34 in ACCs and normal salivary gland controls. A significant difference in α-SMA expression was found between normal controls and ACCs, suggesting the presence of myofibroblasts in ACCs. Immunohistochemical staining also demonstrated higher MMP2 expression in the stroma of ACCs than in the controls (P < 0.001). Primary culture of myofibroblasts from one ACC showed great invasive activity, with high expression of MMP2 and C-X-C motif chemokine 12 (CXCL12) by reverse transcription polymerase chain reaction (RT-PCR) analysis. CONCLUSIONS: This study demonstrated the presence of myofibroblasts in ACC. Myofibroblasts might be related to the aggressive growth behaviour of ACC, owing to their high levels of expression of MMP2 and CXCL12.

Underlying protective mechanism of α1-adrenoceptor activation against irradiation-induced damage in rat submandibular gland.

OBJECTIVES: Damage to salivary gland after radiotherapy for head and neck malignant tumours can lead to irreversible oral complaints, which severely impair quality of life. The protective effect of α1-adrenoceptor activation on the salivary glands after irradiation has previously been demonstrated. However, the exact mechanism remains unclear. In this study, we investigated the underlying cytoprotective mechanism of α1-adrenoceptor activation in rat submandibular glands after irradiation. STUDY DESIGN: Rats were locally irradiated using a linear accelerator in the head and neck region with a dose of 20Gy. After irradiation, phenylephrine (5mg/kg) was injected intraperitoneally for 7 successive days and the submandibular glands were then collected. The antiapoptotic effect of phenylephrine on the gland was examined by TUNEL, the proliferative cellular nuclei antigen (PCNA) was determined by immunohistochemistry, and the activation of c-Jun N-terminal kinase (JNK) was detected by Western blot. RESULTS: The irradiation only group showed severe atrophy, increased apoptosis, enhanced cell proliferation, and the phosphorylation of JNK was markedly increased by 26.89% (P<0.05), compared to the control. The phenylephrine-treated group, however, showed remarkably alleviated atrophy, decreased apoptosis, and further increased cell proliferation, and the phosphorylation of JNK was markedly decreased by 36.00% (P<0.05), compared to the irradiation only group. CONCLUSIONS: The data showed that the underlying protective mechanism of α1-adrenoceptor activation in irradiated gland might be related to improved cell proliferation, inhibited cell apoptosis, and depressed activation of JNK. It could be helpful in protecting salivary glands against irradiation damage.

Mechanism of the protective effect of phenylephrine pretreatment against irradiation-induced damage in the submandibular gland.

Irradiation is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of irradiation treatment is the irreversible damage to salivary glands in the radiation field. Although the protective effect of phenylephrine pretreatment on salivary glands following irradiation has previously been demonstrated, the exact mechanism remains unclear. In this study, we investigated the cytoprotective mechanisms of phenylephrine pretreatment in rat submandibular glands following irradiation. Rats were locally irradiated using a linear accelerator in the head and neck region with a single dose of 20 Gy. Phenylephrine (5 mg/kg) was injected intraperitoneally 30 min prior to irradiation and the submandibular glands were collected on day 7 after irradiation. In comparison with the control group, the irradiation-only group demonstrated severe atrophy, enhanced cell proliferation and increased apoptosis. The phenylephrine-pretreated group, however, demonstrated markedly alleviated atrophy, further increased cell proliferation and decreased apoptosis compared with the irradiation-only group. The data indicated that the cytoprotective mechanisms of phenylephrine pretreatment in the submandibular gland following irradiation may be related to improved cell proliferation and inhibition of cell apoptosis.