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.

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.

Extracellular Vesicles from Carcinoma-associated Fibroblasts Promote EMT of Salivary Adenoid Cystic Carcinoma Via IL-6.

BACKGROUND: Carcinoma-associated fibroblasts (CAFs) play a pivotal role in cancer progression. Salivary adenoid cystic carcinoma (SACC) has a high tendency to invade and metastasize. Understanding how CAFs interact with SACC cells is essential for developing new targeted therapies for SACC. Extracellular vesicles (EVs) play important roles in intercellular communication. However, the role of CAFs-derived EVs in SACC invasion remains poorly understood. AIM OF THE STUDY: To show that CAFs EVs are involved in the EMT of SACC and promote tumor invasion. METHODS: CAFs-derived EVs were characterized by western blot and transmission electron microscopy. Wound healing and transwell assay were performed for assessing biological foundation of CAFs-EVs for tumor cells. RNA interference transfection, western blot, wound healing and transwell assay were applied to study the effect of IL6 from CAFs-EVs on SACC cells and the mechanism. A subcutaneous xenograft model was used to evaluate the EMT of SACC induced by CAFs in vivo. RESULTS: In this study, we show that CAFs EVs promote the migration and invasion of SACC cells. The expression of biomarkers of epithelial-mesenchymal transition (EMT) was higher in SACC cells treated with CAFs EVs than in the negative controls, and high levels of IL6 were detected in CAFs and their EVs. Knockdown of IL6 in CAFs decreased tissue invasiveness and EMT biomarker expression in SACC cells induced by CAFs EVs. CAFs EV-associated IL6 promoted SACC EMT by activating the JAK2/STAT3 signaling pathway. CONCLUSION: CAFs-derived EVs carry IL6 to improve EMT of SACC by activating the JAK2/STAT3 signaling pathway.

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.

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.

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.