Consensus statement on extracellular vesicles in liquid biopsy for advancing laboratory medicine.

Extracellular vesicles (EVs) represent a diverse class of nanoscale membrane vesicles actively released by cells. These EVs can be further subdivided into categories like exosomes and microvesicles, based on their origins, sizes, and physical attributes. Significantly, disease-derived EVs have been detected in virtually all types of body fluids, providing a comprehensive molecular profile of their cellular origins. As a result, EVs are emerging as a valuable addition to liquid biopsy techniques. In this collective statement, the authors share their current perspectives on EV-related research and product development, with a shared commitment to translating this newfound knowledge into clinical applications for cancer and other diseases, particularly as disease biomarkers. The consensus within this document revolves around the overarching recognition of the merits, unresolved questions, and existing challenges surrounding EVs. This consensus manuscript is a collaborative effort led by the Committee of Exosomes, Society of Tumor Markers, Chinese anti-Cancer Association, aimed at expediting the cultivation of robust scientific and clinically applicable breakthroughs and propelling the field forward with greater swiftness and efficacy.

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.

Deciphering Cell-Cell Interactions with Integrative Single-Cell Secretion Profiling.

Cell-cell interactions are the fundamental behaviors to regulate cellular activities. A comprehensive evaluation of intercellular interactions requires direct profiling of various signaling behaviors simultaneously at the single-cell level, which remains lacking. Herein, an integrative single-cell secretion analysis platform is presented to profile different secreted factors (four proteins, three extracellular vesicles (EV) phenotypes), spatial distances, and migration information (distances and direction) simultaneously from high-throughput paired single cells using an antibody-barcode microchip. Applying the platform to analyze the tumor-stromal and tumor-immune interactions with the human oral squamous cell carcinoma (OSCC) cell lines and primary OSCC cells reveals that the initial distances between cells would determine their migratory distances and direction to approach stable organization. The cell-cell in close proximity enhances protein secretions while attenuating EV secretions. Migration has a more profound correlation with protein secretions than EV secretions, in which absolute migration distance affects protein secretions significantly but not the direction. These findings highlight the significance of spatial organization in regulating cell signaling behaviors and demonstrate that the integrative single-cell secretion profiling platform is well-suited for a comprehensive dissection of intercellular communication and interactions, providing new avenues for understanding cell-cell interaction biology and how different signaling behaviors coordinate within the tumor microenvironment.

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.

Deglycosylated EpCAM regulates proliferation by enhancing autophagy of breast cancer cells via PI3K/Akt/mTOR pathway.

Autophagy is an important regulator of cellular homeostasis and its dysregulation often results in cancer. Aberrant glycosylation induced by oncogenic transformation contributes to tumor invasion and metastasis. In a previous study, we have demonstrated that EpCAM, a glycosylation protein, is associated with cell growth and metastasis in breast cancer. But the effect of EpCAM glycosylation on autophagy is not clear. the precise mechanism of regulation remains largely unknown. In this study, breast cancer cells were transfected with N-glycosylation mutation EpCAM plasmid to express deglycosylated EpCAM. The result showed that deglycosylated EpCAM promoted autophagy in breast cancer cells. We further confirmed this conclusion with the activator (Rapamycin, RAP) and inhibitor (Wortmannin) of autophagy. We also found that deglycosylated EpCAM promoted apoptosis and inhibited proliferation through activating autophagy by suppressing Akt/mTOR signaling pathway in breast cancer cells. These findings represent a novel mechanism by which deglycosylated EpCAM inhibits proliferation by enhancing autophagy of breast cancer cells via PI3K/Akt/mTOR pathway. In conclusion, the combination of autophagy modulation and EpCAM targeted therapy is a promising therapeutic strategy in the treatment of breast cancer.

Pre-metastatic Niche Formation in Different Organs Induced by Tumor Extracellular Vesicles.

Primary tumors selectively modify the microenvironment of distant organs such as the lung, liver, brain, bone marrow, and lymph nodes to facilitate metastasis. This supportive metastatic microenvironment in distant organs was termed the pre-metastatic niche (PMN) that is characterized by increased vascular permeability, extracellular matrix remodeling, bone marrow-derived cells recruitment, angiogenesis, and immunosuppression. Extracellular vesicles (EVs) are a group of cell-derived membranous structures that carry various functional molecules. EVs play a critical role in PMN formation by delivering their cargos to recipient cells in target organs. We provide an overview of the characteristics of the PMN in different organs promoted by cancer EVs and the underlying mechanisms in this review.

High-Throughput Single-Cell Extracellular Vesicle Secretion Analysis on a Desktop Scanner without Cell Counting.

Single-cell EV (extracellular vesicle) secretion analysis is emerging for a better understanding of non-genetic cellular heterogeneity regulating human health and diseases through intercellular mediators. However, the requirements of expensive and bulky instrumentations hinder its widespread use. Herein, by combining gold nanoparticle-enhanced silver staining and the Poisson distribution, we reported the use of a home-use scanner to realize high-throughput single-cell EV secretion analysis without cell counting. We applied the platform to analyze the secretions of different EV phenotypes with the human oral squamous cell carcinoma cell line and primary cells from patients, which generated single-cell results comparable with those of the immunofluorescence approach. Notably, we also realized the quantification of the number of EVs secreted from every single cell using their respective titration curves obtained from population samples, making it possible to directly compare different EV phonotypes in regard to their secretion number, secretion rate, and so forth. The technology introduced here is simple, easy to operate, and of low cost, which make it a potential, easily accessible, and affordable tool for widespread use in both basic and clinical research.

LncRNA MALAT1 aggravates oxygen-glucose deprivation/reoxygenation-induced neuronal endoplasmic reticulum stress and apoptosis via the miR-195a-5p/HMGA1 axis.

BACKGROUND: This study aimed to investigate the potential role and molecular mechanism of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in cerebral ischemia/reperfusion injury. RESULTS: Using an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model, we determined that the expression of MALAT1 was significantly increased during OGD/R. MALAT1 knockdown reversed OGD/R-induced apoptosis and ER stress. Mechanistically, MALAT1 promoted OGD/R-induced neuronal injury through sponging miR-195a-5p to upregulating high mobility group AT-hook1 (HMGA1). CONCLUSIONS: Collectively, these data demonstrate the mechanism underlying the invovlvement of MALAT1 in cerebral ischemia/reperfusion injury, thus providing translational evidence that MALAT1 may serve as a novel biomarker and therapeutic target for ischemic stroke.

Single-Cell Secretion Analysis in the Engineered Tumor Microenvironment Reveals Differential Modulation of Macrophage Immune Responses.

It is increasingly recognized that the cellular microenvironment plays critical roles in regulating the fate and physiology of cells. Despite recent advancements in single-cell analysis technologies, engineering and integration of the microenvironment for single-cell analysis platforms remain limited. Here, we report a single-cell cytokine secretion analysis platform that integrated both the three-dimensional cell culture and the primary oral squamous cell carcinoma tumor cell co-culture to provide both physical and physiological cues for single cells to be analyzed. We apply the platform to investigate the immune responses of human macrophages stimulated with the ligand of toll-like receptor 4 lipopolysaccharide. Notably, we observe the differential modulation effect in cytokine secretions by the tumor microenvironment, in which antitumor cytokine TNF-a secretion was attenuated, and protumor cytokine IL-6 would increase. The differential modulation effect is conserved from cell line-derived macrophages to primary macrophages derived from healthy donors. Immunofluorescence staining further reveals that ∼50% of macrophage cells could be polarized from M1 to the M2 phenotype within 12 h in the engineered tumor microenvironment. This work demonstrates the significance of the cell microenvironment toward single-cell analysis, which could help to evaluate how immune cells will respond in the complex microenvironment more accurately.

LncRNA MALAT1 aggravates oxygen-glucose deprivation/reoxygenation-induced neuronal endoplasmic reticulum stress and apoptosis via the miR-195a-5p/HMGA1 axis

BACKGROUND: This study aimed to investigate the potential role and molecular mechanism of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in cerebral ischemia/reperfusion injury. RESULTS: Using an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model, we determined that the expression of MALAT1 was significantly increased during OGD/R. MALAT1 knockdown reversed OGD/R-induced apoptosis and ER stress. Mechanistically, MALAT1 promoted OGD/R-induced neuronal injury through sponging miR-195a-5p to upregulating high mobility group AT-hook1 (HMGA1). CONCLUSIONS: Collectively, these data demonstrate the mechanism underlying the invovlvement of MALAT1 in cerebral ischemia/reperfusion injury, thus providing translational evidence that MALAT1 may serve as a novel biomarker and therapeutic target for ischemic stroke.

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.

A Novel Tissue-Based Liver-Kidney-on-a-Chip Can Mimic Liver Tropism of Extracellular Vesicles Derived from Breast Cancer Cells.

Extracellular vesicles (EVs) from cancer cells remodel distant organs to promote metastasis in vivo. A biomimetic microsystem may compensate costly and time-consuming animal models to accelerate the study of EV organotropism. A tissue-based liver-kidney-on-a-chip is developed with precision-cut tissue slices (PTSs) cultured to represent individual organs. The organotropism of breast cancer EVs is modeled using the biomimetic microsystem. A traditional animal model of EV organotropism is used to investigate the physiological similarity of the microfluidic model to animal models. It is demonstrated that breast cancer EVs show strong liver tropism rather than kidney tropism on both the microfluidic and animal models. It is found that the metastatic inhibitor AMD3100 inhibits liver tropism effectively in both the microfluidic and animal models. Overall, the tropism of EVs to different organs is reconstituted on the microfluidic model. The liver-kidney-on-a-chip may expand the capabilities of traditional cell culture models and provide a faster alternative to animal models for EV studies.

Hypoxia modulates stem cell properties and induces EMT through N-glycosylation of EpCAM in breast cancer cells.

Epithelial cell adhesion molecule (EpCAM), which is a transmembrane glycoprotein, is related to tumor progression. We demonstrated that EpCAM plays important roles in proliferation, apoptosis, and metastasis during breast cancer (BC) progression. But the role of N-glycosylation in EpCAM in tumor aggressiveness is not clear. Here, we evaluated the role of N-glycosylation of EpCAM in stemness and epithelial-mesenchymal transition (EMT) characteristics. EpCAM overexpression increases the expression of stemness markers (NANOG，SOX2, and OCT4) and EMT markers (N-cadherin and vimentin) under the condition of hypoxia in BC. Knockdown of EpCAM and mutation of N-glycosylation of EpCAM maintained in severe hypoxia lead to a significant reduction of stemness/EMT markers. In addition, we found that N-glycosylation of EpCAM is a crucial factor during this process. This demonstrates that EpCAM has a novel regulatory role in stemness/EMT dependence of hypoxia-inducible factor 1-alpha via regulating nuclear factor kappa B in BC cells. Hence, our study reveals EpCAM glycosylation modification as a new regulator of stemness/EMT under hypoxic in BC and points out EpCAM as a potential therapeutic target.

MicroRNA­21 contributes to renal cell carcinoma cell invasiveness and angiogenesis via the PDCD4/c­Jun (AP­1) signalling pathway.

Accumulating evidence has demonstrated that microRNAs are associated with malignant biological behaviour, including tumorigenesis, cancer progression and metastasis via the regulation of target gene expression. Our previous study demonstrated that programmed cell death protein 4 (PDCD4), which is a tumour suppressor gene, is a target of microRNA­21 (miR­21), which affects the proliferation and transformation capabilities of renal cell carcinoma (RCC) cells. However, the role of miR­21 in the molecular mechanism underlying the migration, invasion and angiogenesis of RCC remains poorly understood. The effects of miR­21 on the invasion, migration and angiogenesis of RCC cells was determined through meta­analysis and regulation of miR­21 expression in vitro. After searching several databases, 6 articles including a total of 473 patients met the eligibility criteria for this analysis. The combined results of the meta­analysis revealed that increased miR­21 expression was significantly associated with adverse prognosis in patients with RCC, with a pooled hazard ratio estimate of 1.740. In in vitro experiments, we demonstrated that a miR­21 inhibitor decreased the number of migrating and invading A498 and 786­O RCC cells, along with a decrease in PDCD4, c­Jun, matrix metalloproteinase (MMP)2 and MMP9 expression. Additionally, inhibition of miR­21 was revealed to reduce tube formation and tube junctions in the endothelial cell line HMEC­1 by affecting the expression of angiotensin­1 and vascular endothelial growth factor A, whereas PDCD4 small interfering RNA exerted opposite effects on the same cells. Overall, these findings, along with evidence­based molecular biology, demonstrated that miR­21 expression promoted the migration, invasion and angiogenic abilities of RCC cells by directly targeting the PDCD4/c­Jun signalling pathway. The results may help elucidate the molecular mechanism underlying the development and progression of RCC and provide a promising target for microRNA­based therapy.

MicroRNA-29b-3p suppresses oral squamous cell carcinoma cell migration and invasion via IL32/AKT signalling pathway.

Oral squamous cell carcinoma (OSCC) is aggressive accompanied with poor prognosis. We previously isolated the most invasive cells resembling the invasive tumour front by microfluidic technology and explored their differentially expressed microRNAs (miRNAs) in our previous work. Here, we verified the miR-29b-3p as a guarder that suppressed migration and invasion of OSCC cells and was down-regulated in the most invasive cells. Besides that, the invasion suppression role of miR-29b-3p was achieved through the IL32/AKT pathway. Thus, miR-29b-3p and IL32 might serve as therapeutic targets for blocking the progression and improving the outcome of OSCC.

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.

Upregulation of miR-34a by Inhibition of IRE1α Has Protective Effect against Aß-Induced Injury in SH-SY5Y Cells by Targeting Caspase-2.

BACKGROUND: Neurotoxicity induced by the amyloid-ß (Aß) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Based on accumulating evidence in AD research, both endoplasmic reticulum stress (ER stress) and alterations in the microRNA (miRNA) network contribute to the pathogenesis of the disease, making them potential therapeutic targets for AD. The present study was performed to investigate whether miR-34a and the inositol-requiring enzyme 1 (IRE1) are involved in the regulation of Aß-induced cytotoxicity. METHODS: Human neuroblastoma SH-SY5Y cells were treated with Aß1-40. Cell viability was assessed by the MTT assay. The integrity of the plasma membrane was assessed by LDH release. The expression levels of XBP1s, IRE1α, p-IRE1α, and Caspase-2 were detected by Western blot analysis. Spliced-XBP1 mRNA and miR-34a were detected by reverse transcription- (RT-) PCR and quantitative real-time PCR, respectively. Caspase-2 activity was measured using the Caspase-2 cellular activity assay kit. The IRE1 inhibitor (STF-083010) was used to determine the role of IRE1α on miR-34a expression. SH-SY5Y cells were transfected with miR-34a mimics to assess the role of miR-34a on the activation of Caspase-2 and the viability of Aß-exposed SH-SY5Y cells. RESULTS: We showed that Aß caused concentration- and duration-dependent death of SH-SY5Y cells. The expression levels of XBP1s, p-IRE1α, and Caspase-2 were increased, along with a corresponding decrease in the miR-34a levels in Aß-exposed SH-SY5Y cells. The IRE1 inhibitor (STF-083010) upregulated the expression of miR-34a and suppressed the activation of Caspase-2, effectively alleviating the Aß-induced death of SH-SY5Y cells. Transfection studies show that miR-34a mimics inhibit the expression of Caspase-2 and restore the viability of Aß-exposed SH-SY5Y cells. CONCLUSION: Aß peptide induced downregulation of miR-34a through the activation of IRE1α, which may induce cytotoxicity by targeting Caspase-2. Upregulation of miR-34a by inhibition of IRE1α has protective effects against Aß-induced injury in SH-SY5Y cells.

Application of Microfluidic Chips in Separation and Analysis of Extracellular Vesicles in Liquid Biopsy for Cancer.

Extracellular vesicles (EVs) are becoming a promising biomarker in liquid biopsy of cancer. Separation EV from cell culture medium or biofluids with high purity and quality remains a technique challenge. EV manipulation techniques based on microfluidics have been developed in the last decade. Microfluidic-based EV separation techniques developed so far can be classified into two categories: surface biomarker-dependent and size-dependent approaches. Microfluidic techniques allow the integration of EV separation and analysis on a single chip. Integrated EV separation and on-chip analysis have shown great potential in cancer diagnosis and monitoring treatment of responses. In this review, we discuss the development of microfluidic chips for EV separation and analysis. We also detail the clinical application of these microfluidic chips in the liquid biopsy of various cancers.

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.