Extracellular vesicle-bound VEGF in oral squamous cell carcinoma and its role in resistance to Bevacizumab Therapy.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an important proangiogenic factor and has been considered as a key target of antiangiogenetic therapy in oral squamous cell carcinoma (OSCC). However, clinical application of bevacizumab, a specific VEGF antibody, didn't improve the survival rate of OSCC patients. One possible explanation is that VEGF gene expresses diverse isoforms, which associate with extracellular vesicles (EVs), and EVs potentially contribute to VEGF resistance to bevacizumab. However, clear solution is lacking in addressing this issue. METHODS: Expression of VEGF isoforms in OSCC cells was confirmed by reverse transcription and polymerase chain reaction (RT-PCR) and western blot. EVs isolated from OSCC cell's conditioned medium (CM) were characterized by western blot, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Flow cytometry, immunogold labeling and western blot were applied to study the VEGF on EVs. Tube formation assay and Matrigel plug angiogenesis assay were used for analyzing the angiogenesis capacity of EV-VEGF. RESULTS: The most popular isoforms expressed by VEGF gene are VEGF121, VEGF165 and VEGF189. In this study, we demonstrated that all three isoforms of mRNA could be detected at varying levels in OSCC cells, while only VEGF165 and VEGF189 proteins were found. CM derived from OSCC cells, both soluble and non-soluble forms of VEGF could be detected. We further confirmed the presence of VGEF189 bound to EVs as a non-soluble form. EV-bound VEGF189 presented angiogenic activity, which could not be neutralized by bevacizumab. It was found that VEGF189 bound to EVs by heparan sulfate proteoglycans (HSPG). In addition, the angiogenic effect of EV-VEGF could be reversed by surfen, a kind of HSPG antagonist both in vitro and in vivo. CONCLUSION: Antagonists targeting HSPG might potentially overcome the resistance of EV-VEGF to bevacizumab and serve as an alternative for anti-VEGF therapy in OSCC.

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.

Nanotherapy for bone repair: milk-derived small extracellular vesicles delivery of icariin.

Icariin (ICA) played an important role in the treatment of inflammatory bone defects. However, pharmacokinetic studies have shown that its poor bioavailability limited its application. In this context, we isolated bovine milk-derived sEV and prepared sEV-ICA to improve the osteogenic effect of ICA. In this study, we successfully constructed sEV-ICA. sEV-ICA was found to have significantly higher osteogenic efficiency than ICA in cell culture and cranial bone defect models. Mechanistically, bioinformatics analysis predicted that signal transducers and activators of transcription 5 (STAT5a) may bind to the GJA1 promoter, while luciferase activity assays and chromatin immunoprecipitation (ChIP) experiments confirmed that STAT5a directly binded to the GJA1 promoter to promote osteogenesis. We proved that compared with ICA, sEV-ICA showed a better effect of promoting bone repair in vivo and in vitro. In addition, sEV-ICA could promote osteogenesis by promoting the combination of STAT5a and GJA1 promoter. In summary, as a complex drug delivery system, sEV-ICA constituted a rapid and effective method for the treatment of bone defects and could improve the osteogenic activity of ICA.

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.

Research progress in membrane fusion-based hybrid exosomes for drug delivery systems.

Liposomes are the earliest and most widely used nanoparticles for targeted drug delivery. Exosomes are nanosized membrane-bound particles and important mediators of intercellular communication. Combining liposomes and exosomes using various membrane fusion methods gives rise to a novel potential drug delivery system called membrane fusion-based hybrid exosomes (MFHE). These novel MFHEs not only exhibit potential advantageous features, such as high drug loading rate and targeted cellular uptake via surface modification, but are also endowed with high biocompatibility and low immunogenicity. Here, we provide an overview of MFHEs' various preparation methods, characterization strategies, and their applications for disease treatment and scientific research.

Milk-derived small extracellular vesicles: nanomaterials to promote bone formation.

Small extracellular vesicles (sEVs) are an important component in the paracrine pathway. They can be used as a substitute for seed cells and have shown good application prospects in promoting bone regeneration. Cow's milk could be used as a source of sEVs with good biocompatibility and cost-effectiveness, with easy availability, low cost and low toxicity. This study focused on the role and mechanism of small extracellular vesicles derived from milk in bone repair. In order to explore the mechanism via which Milk-sEVs promote bone repair, we screened the differential gene GJA1 in Milk-sEV-treated osteoblasts through transcriptome chips, and verified the transcript AP3B1 of GJA1 through chromatin immunoprecipitation (CHIP). We have proved by in vivo and in vitro experiments that milk-derived sEVs (Milk-sEVs) increase the repair ability of bone tissue, and promote expression of the osteogenic gene GJA1 through the transcript AP3B1.

Spatial barcoding-enabled highly multiplexed immunoassay with digital microfluidics.

Digital microfluidics (DMF), facilitating independent manipulation of microliter samples, provides an ideal platform for immunoassay detection; however, suffering limited multiplexity. To address the need, herein we described a digital microfluidics (DMF) platform that realizes spatial barcoding on the Teflon-coated indium tin oxide (ITO) glass side to fulfill highly multiplexed immunoassay (10+) with low-volume samples (∼4 µL) in parallel, representing the highest multiplexing recorded to date for DMF-actuated immunoassay. Planar-based spatial immobilization of multiple capture antibodies was realized on a Teflon-coated ITO glass side, which was then used as the top plate of the DMF device. Droplets containing analytes, secondary antibodies, and fluorescent signaling reporters with low volume, which were electrically manipulated by our DMF control system, were shuttled sequentially along the working electrodes to complete the immuno-reaction. Evaluation of platform performance with recombinant proteins showed excellent sensitivity and reproducibility. To test the feasibility of our platform in analyzing multiplex biomarkers of the immune response, we used lipopolysaccharide-stimulated macrophages as a model system for protein secretion dynamics studies. As a result, temporal profiling of pro-inflammatory cytokine secretion dynamics was obtained. The spatial barcoding strategy presented here is easy-to-operate to enable a more comprehensive evaluation of protein abundance from biological samples, paving the way for new opportunities to realize multiplexity-associated applications with the DMF platform.

Angiogenesis and Functional Vessel Formation Induced by Interstitial Flow and Vascular Endothelial Growth Factor Using a Microfluidic Chip.

Angiogenesis occurs during both physiological and pathological processes. In this study, a microfluidic chip for the development of angiogenesis was utilized to assess angiogenic sprouting and functional vessel formation. We also found that vascular endothelial growth factor (VEGF) was a determinant of the initiation of vascular sprouts, while the direction of these sprouts was greatly influenced by interstitial flow. Isoforms of VEGF such as VEGF121, VEGF165, and VEGF189 displayed different angiogenic properties on the chip as assessed by sprout length and number, vessel perfusion, and connectivity. VEGF165 had the highest capacity to induce vascular sprouting among the three isoforms assessed and furthermore, also induced functional vessel formation. This chip could be used to analyze the effect of different angiogenic factors and drugs, as well as to explore the mechanism of angiogenesis induced by such factors.

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.

Enhanced Deep Utilization of Low-Organic Content Sludge by Processing Time-Extended Low-Temperature Thermal Pretreatment.

Anaerobic digestion is an important way for maintaining sewage sludge stability, reduction, and resource recovery. However, the low organic content generally limits methane production. Recently, thermal hydrolysis has been widely used for sludge pretreatment to improve the anaerobic digestion efficiency. Generally, an increased temperature is preferred to enhance the solubility of organic matters in the sludge. However, high energy requirement comes with increased temperature. Application of low-temperature thermal treatment could overcome this drawback. However, the appropriate low-temperature pretreatment time is still uncertain. In this study, an extended contact time with low thermal pretreatment (90 °C) was chosen to realize a more efficient and economical digestion process of low-organic content sludge. The results demonstrated that the solubilization of proteins and carbohydrates was significantly promoted by the contact time-extended thermal hydrolysis pretreatment. The following anaerobic digestion efficiency of low-organic content sludge was also dramatically improved with the prolonged contact time. The maximum methane production could reach around 294.73 mL/gVS after 36 h of 90 °C treatment, which was 5.56 times that of the untreated groups. Additionally, based on the energy balance calculation, extending the thermal hydrolysis time resulted in a more economically feasible anaerobic digestion than increasing the temperature. The dewatering properties and the stability of the heavy metals were also reinforced, implying the advanced deep utilization of the digested low-organic content sludge. In conclusion, sludge pretreated by low-temperature thermal hydrolysis with a prolonged contact time could be more effective for low-organic content sludge treatment and disposal.

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.

A biodegradable, mechanically tunable micro-arc oxidation AZ91D-based composite implant with calcium phosphate/chitosan coating promotes long-term bone tissue regeneration.

BACKGROUND: To reduce the biodegradable rate and develop the long-term osteogenic ability of magnesium (Mg) alloy, we prepared a new biodegradable micro arc oxidation AZ91D-based composite implant with calcium phosphate/chitosan coating (CaP-CS/MAO/AZ91D) and investigated its mechanical property and long-term bone tissue regeneration ability. MAIN METHODS AND MAJOR RESULTS: The results showed that the binding force and bioactivity of CaP-CS/MAO/AZ91D was better when the ratio of water to ethanol was 4:6 and MAO constant current was 0.1 A cm-2 . Compressive strengths of 4:6 sample were more than 1300 N when the soaking time was increased to 21 days. CaP-CS/MAO/AZ91D extracts promoted differentiation and proliferation of rat mesenchymal stem cells (RMSC), which achieved higher proliferation rates over 16 days of culture and exhibited early alkaline phosphatase activity and late bone sialoprotein markers. CONCLUSIONS AND IMPLICATIONS: CaP-CS/MAO/AZ91D was established to promote RMSC osteogenic differentiation within a proper range for at least 90 days through Wnt/ß-catenin pathway activation, which would allow sufficient time for bone healing. Collectively, our findings suggest that the CaP-CS/MAO/AZ91D coating could not only reduce the corrosion rate and lead to better long-term biocompatibility but also promote osteogenic mineralization.

CNTs-CaP/chitosan-coated AZ91D magnesium alloy extract promoted rat dorsal root ganglia neuron growth via activating ERK signalling pathway.

Increasing attention has been paid on the application of biodegradable materials such as magnesium and its alloys in neuron repair. AZ91D magnesium alloy coated with carbon nanotubes (CNTs) and/or calcium phosphate (CaP)/chitosan (CS) was fabricated in this study. To evaluate the bioactivity of these AZ91D-based composites, the extracts were prepared by immersing samples in modified simulated body fluid (m-SBF) for 0, 2, 8, 16, 24, 34, 44, 60, or 90 days. Immunofluorescence staining for neuronal class III ß-tubulin (TUJ1) revealed that both CNTs-CaP/CS-AZ91D and CaP/CS-AZ91D extracts promoted axon outgrowth of dorsal root ganglia (DRG) neurons, accompanied with increased expression of phosphorylated focal adhesion kinase (p-FAK) and growth associated protein-43 (GAP-43). Besides, the extracts increased the expression and the release of neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). ERK signalling was activated in DRG neurons after treating with either CNTs-CaP/CS-AZ91D or CaP/CS-AZ91D extracts, and its inhibition with U0126 counteracted the beneficial effects of these extracts on DRG neuron. Overall, the extracts from these AZ91D-based composites might promote DRG neuron growth via activating ERK signalling pathway. Notably, CNTs-CaP/CS-AZ91D extracts showed a better promoting effect on neuron growth than CaP/CS-AZ91D. Assessment of ion elements showed that the addition of CNTs coating enhanced magnesium corrosion resistance and reduced the deposition of calcium and phosphorus on the surface of CaP/CS-AZ91D alloy. These findings demonstrate that CNTs-CaP/CS-AZ91D likely provide a more suitable environment for neuron growth, which suggests a potential implantable biomaterial for the treatment of nerve injury. SIGNIFICANCE: AZ91D magnesium alloy coated with carbon nanotubes (CNTs) and/or calcium phosphate (CaP)/chitosan (CS) was fabricated and their immersion extracts were prepared using modified simulated body fluid in this study. Both extracts from CNTs-CaP/CS and CaP/CS-coated AZ91D magnesium alloy promotes rat dorsal root ganglia (DRG) neuron growth via activating ERK signalling pathway. Notably, the addition of CNTs improves the performance of CaP/CS-AZ91D. For the first time, our research demonstrates that CNTs-CaP/CS-AZ91D likely provide a suitable environment for neuron growth, suggesting these AZ91D-based composites as potential implantable biomaterials for the treatment of nerve injury.

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.

Rapid prototyping of PDMS microdevices viaµPLAT on nonplanar surfaces with flexible hollow-out mask.

A major goal of polydimethylsiloxane (PDMS) microfabrication is to develop a simple and inexpensive method for rapid fabrication. Despite the recent advancements in this field, facile PDMS microfabrication on non-planar surfaces remains elusive. Here we report a facile method for rapid prototyping of PDMS microdevices viaµPLAT (microscale plasma-activated templating) on non-planar surfaces through micropatterning of hydrophilic/hydrophobic (HL/HB) interface by flexible polyvinyl chloride (PVC) hollow-out mask. This mask can be easily prepared with flexible PVC film through a cutting crafter and applied as pattern definer during the plasma treatment for microscale HL/HB interface formation on different substrates. The whole process requires low inputs in terms of time as well as toxic chemicals. Inspired by liquid molding, we demonstrated its use for rapid prototyping of PDMS microstructures. Following the proof-of-concept study, we also demonstrated the use of the flexible hollow-out mask to facilitate cell patterning on curved substrates, which is difficult to realize with conventional methods. Collectively, our work utilizes flexible and foldable PVC film as mask materials for facile microscale HL non-planar surface modification to establish a useful tool for PDMS prototyping and cell patterning.