Mapping secretome-mediated interaction between paired neuron-macrophage single cells.

Neuron-immune interaction through secreted factors contributes significantly to the complex microenvironment in the central nervous system that could alter cell functionalities and fates in both physiological and pathological conditions, which remains poorly characterized at the single-cell level. Herein, using a spatially patterned antibody barcode microchip, we realized the mapping of 12 different secretomes, covering cytokines, neurotrophic factors (NFs), and neuron-derived exosomes (NDEs) from high-throughput, paired single cells (≥ 600) simultaneously under normal conditions and an Alzheimer's disease (AD) model induced with amyloid beta protein 1-42 (Aß1-42). We applied the platform to analyze the secretion profiles from paired neuron-macrophage and neuron-microglia single cells with human cell lines. We found that pairwise neuron-macrophage interaction would trigger immune responses and attenuate neuron cells' secretion, while neuron-microglia interaction generally results in opposite outcomes in secretion. When neuron cells are induced with Aß1-42 protein into the AD model, both neuron-macrophage and neuron-microglia interactions lead to increased cytokines and NDEs and decreased NFs. Further analysis of AD patients' serum showed that NDEs were significantly higher in patients' samples than in the control group, validating our observation from the interaction assay. Furthermore, we resolved previously undifferentiated heterogeneity underlying the secretions from single-neuron cells. We found that the NDE and NF secretion was less dependent on the paracrine signaling between one another and that secretions from neuron cells would attenuate after differentiation with Aß1-42. This study demonstrates the mapping of the different secretomes from paired neuron-immune single cells, providing avenues for understanding how neurons and immune cells interact through the complex secretome network.

Identification of Stutzerimonas stutzeri volatile organic compounds that enhance the colonization and promote tomato seedling growth.

AIMS: Volatile organic compounds (VOCs) have an important function in plant growth-promoting rhizobacteria (PGPR) development and plant growth. This study aimed to identify VOCs of the PGPR strain, Stutzerimonas stutzeri NRCB010, and investigate their effects on NRCB010 biofilm formation, swarming motility, colonization, and tomato seedling growth. METHODS AND RESULTS: Solid-phase microextraction and gas chromatography-mass spectrometry were performed to identify the VOCs produced during NRCB010 fermentation. A total of 28 VOCs were identified. Among them, seven (e.g. γ-valerolactone, 3-octanone, mandelic acid, 2-heptanone, methyl palmitate, S-methyl thioacetate, and 2,3-heptanedione), which smell well, are beneficial for plant, or as food additives, and without serious toxicities were selected to evaluate their effects on NRCB010 and tomato seedling growth. It was found that most of these VOCs positively influenced NRCB010 swarming motility, biofilm formation, and colonization, and the tomato seedling growth. Notably, γ-valerolactone and S-methyl thioacetate exhibited the most positive performances. CONCLUSION: The seven NRCB010 VOCs, essential for PGPR and crop growth, are potential bioactive ingredients within microbial fertilizer formulations. Nevertheless, the long-term sustainability and replicability of the positive effects of these compounds across different soil and crop types, particularly under field conditions, require further investigation.

Cell-in-cell promotes lung cancer malignancy by enhancing glucose metabolism through mitochondria transfer.

Heterotypic cell-in-cell structure (CICs) is the definition of the entry of one type of living cells into another type of cell. CICs between immune cells and tumor cells have been found to correlate with malignancy in many cancers. Since tumor immune microenvironment promotes non-small cell lung cancer (NSCLC) progression and drug resistance, we wondered the potential significance of heterotypic CICs in NSCLC. Heterotypic CICs was analyzed by histochemistry in an expanded spectrum of clinical lung cancer tissue specimens. In vitro study was performed using the mouse lung cancer cell line LLC and splenocytes. Our results revealed that CICs formed by lung cancer cells and infiltrated lymphocytes were correlated with malignancy of NSCLC. In addition, we found CICs mediated the transfer of lymphocyte mitochondria to tumor cells, and promoted cancer cell proliferation and anti-cytotoxicity by activating MAPK pathway and up-regulating PD-L1 expression. Furthermore, CICs induces glucose metabolism reprogramming of lung cancer cells by upregulating glucose intake and glycolytic enzyme. Our findings suggest that CICs formed by lung cancer cell and lymphocyte contribute to NSCLC progression and reprogramming of glucose metabolism, and might represent a previously undescribed pathway for drug resistance of NSCLC.

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.

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.

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.

PDMS Microwell Stencil Based Multiplexed Single-Cell Secretion Analysis.

Multiplexed single-cell protein secretion analysis provides an in-depth understanding of cellular heterogeneity in intercellular communications mediated by secreted proteins in both fundamental and clinical research. However, it has been challenging to increase the proteomic parameters co-profiled from every single cell in a facile way. Herein, a simple method to improve the multiplexed proteomic parameters of PDMS microwell based single-cell secretion analysis platform by sandwiching PDMS stencil in between two antibody-coated glass slides is introduced. Two different antibody panels can be immobilized easily by static coating, without using sophisticated fluid handling or bulky equipment. 5-plexed, 3-fluorescence color single-cell secretion assay is demonstrated with this platform to investigate human monocytic U937 cells in response to lipopolysaccharide and phorbol myristate acetate stimulation, which identified the existence of functional subsets dictated by different cytokine profiles. The technology introduced here is simple, easy to operate, which holds great potential to become a powerful tool for profiling multiplexed single-cell cytokine secretion at high throughput to dissect cellular heterogeneity in secretome signatures.

Heterotypic cell-in-cell structures in colon cancer can be regulated by IL-6 and lead to tumor immune escape.

Heterotypic CICs (cell-in-cell structures) have been found between tumor cells and various immune cells in a variety of cancer tissues. The frequency of CICs has been found to correlate with tumor malignancy in some studies but not in others. Herein, we examined in depth the CICs observed in colon cancer to determine their potential significance in disease progression. Heterotypic CICs were observed by histochemistry between epithelial cells and lymphocytes in an expanded spectrum of colon tissue from colitis to cancer and in vitro studies were performed using the colonic tumor cell line HCT8 and human peripheral blood lymphocytes. Our data revealed that the CICs formed by colonic epithelial cells and infiltrated lymphocytes not only positively correlated with tumor malignancy but also were upregulated by the inflammatory cytokine IL-6. In addition, we observed that colon cancer cells could initiate autophagy for survival after cytotoxic lymphocyte internalization and that IL-6 could also be involved in this process to promote the death of lymphocytes in CIC structures. Furthermore, certain changes were observed in tumor cells after experiencing CICs. Our findings suggest that CICs formed by colon cancer cells and lymphocytes contribute to tumor escape from immune surveillance, which could be facilitated by IL-6, and might represent a previously undescribed pathway for tumor cells to adapt and evade host immune defense.

Electrolyte Solvation Manipulation Enables Unprecedented Room-Temperature Calcium-Metal Batteries.

Calcium-metal batteries (CMBs) provide a promising option for high-energy and cost-effective energy-storage technology beyond the current state-of-the-art lithium-ion batteries. Nevertheless, the development of room-temperature CMBs is significantly impeded by the poor reversibility and short lifespan of the calcium-metal anode. A solvation manipulation strategy is reported to improve the plating/stripping reversibility of calcium-metal anodes by enhancing the desolvation kinetics of calcium ions in the electrolyte. The introduction of lithium salt changes the electrolyte structure considerably by reducing coordination number of calcium ions in the first solvation shell. As a result, an unprecedented Coulombic efficiency of up to 99.1 % is achieved for galvanostatic plating/stripping of the calcium-metal anode, accompanied by a very stable long-term cycling performance over 200 cycles at room temperature. This work may open up new opportunities for development of practical CMBs.

Paper-Based 3D Scaffold for Multiplexed Single Cell Secretomic Analysis.

Despite rapid progresses in single-cell analysis technologies, efforts to control the three-dimensional microenvironment for single cell measurements have been lacking. Here, we report a simple method to incorporate three-dimensional scaffolds, including polyvinylidene fluoride (PVDF) membranes and PVDF membrane replicated analog polydimethylsiloxane, into multiplexed single cell secretomic analysis platforms (including a microwell array and a single cell barcode microchip) to mimic the extracellular physical matrix and mechanical support for single cells. Applying this platform to brain tumor cell line U87 to investigate single cell protein secretion behavior on different substrates, we revealed that single cell protein secretions were regulated differently in three-dimensional (3D) microenvironments. This finding was further verified with intracellular cytokine staining, highlighting the significance of 3D single cell microenvironments. This new single cell biomimetic platform can be easily adaptable to other three-dimensional cell culture scaffolds or other single cell assays and may become a broadly applicable three-dimensional single cell analysis system to study the effect of microenvironment conditions on cellular functional heterogeneity in vitro.

Inoculation with Stutzerimonas stutzeri strains decreases N2O emissions from vegetable soil by altering microbial community composition and diversity.

Inoculation with plant growth-promoting rhizobacteria (PGPR) strains has promoted plant growth and decreased nitrous oxide (N2O) emissions from agricultural soils simultaneously. However, limited PGPR strains can mitigate N2O emissions from agricultural soils, and the microbial ecological mechanisms underlying N2O mitigation after inoculation are poorly understood. In greenhouse pot experiments, the effects of inoculation with Stutzerimonas stutzeri NRCB010 and NRCB025 on tomato growth and N2O emissions were investigated in two vegetable agricultural soils with contrasting textures. Inoculation with NRCB010 and NRCB025 significantly promoted tomato growth in both soils. Moreover, inoculation with NRCB010 decreased the N2O emissions from the fine- and coarse-textured soils by 38.7% and 52.2%, respectively, and inoculation with NRCB025 decreased the N2O emissions from the coarse-textured soil by 76.6%. Inoculation with NRCB010 and NRCB025 decreased N2O emissions mainly by altering soil microbial community composition and the abundance of nitrogen-cycle functional genes. The N2O-mitigating effect might be partially explained by a decrease in the (amoA + amoB)/(nosZI + nosZII) and (nirS + nirK)/(nosZI + nosZII) ratios, respectively. Soil pH and organic matter were key variables that explain the variation in abundance of N-cycle functional genes and subsequent N2O emission. Moreover, the N2O-mitigating effect varied depending on soil textures and individual strain after inoculation. This study provides insights into developing biofertilizers with plant growth-promoting and N2O-mitigating effects. IMPORTANCE: Plant growth-promoting rhizobacteria (PGPR) have been applied to mitigate nitrous oxide (N2O) emissions from agricultural soils, but the microbial ecological mechanisms underlying N2O mitigation are poorly understood. That is why only limited PGPR strains can mitigate N2O emissions from agricultural soils. Therefore, it is of substantial significance to reveal soil ecological mechanisms of PGPR strains to achieve efficient and reliable N2O-mitigating effect after inoculation. Inoculation with Stutzerimonas stutzeri strains decreased N2O emissions from two soils with contrasting textures probably by altering soil microbial community composition and gene abundance involved in nitrification and denitrification. Our findings provide detailed insight into soil ecological mechanisms of PGPR strains to mitigate N2O emissions from vegetable agricultural soils.

Monolithically integrated micro-supercapacitors with high areal number density produced by surface adhesive-directed electrolyte assembly.

Accurately placing very small amounts of electrolyte on tiny micro-supercapacitors (MSCs) arrays in close proximity is a major challenge. This difficulty hinders the development of densely-compact monolithically integrated MSCs (MIMSCs). To overcome this grand challenge, we demonstrate a controllable electrolyte directed assembly strategy for precise isolation of densely-packed MSCs at micron scale, achieving scalable production of MIMSCs with ultrahigh areal number density and output voltage. We fabricate a patterned adhesive surface across MIMSCs, that induce electrolyte directed assembly on 10,000 highly adhesive MSC regions, achieving a 100 µm-scale spatial separation between each electrolyte droplet within seconds. The resultant MIMSCs achieve an areal number density of 210 cells cm-2 and a high areal voltage of 555 V cm-2. Further, cycling the MIMSCs at 190 V over 9000 times manifests no performance degradation. A seamlessly integrated system of ultracompact wirelessly-chargeable MIMSCs is also demonstrated to show its practicality and versatile applicability.

Pseudomonas aeruginosa exotoxin A as a novel allergen induced Non-TH2 inflammation in a murine model of steroid-insensitive asthma.

Background: Despite the immediate in vivo occurrence of anaphylactic and allergic reactions following treatment with Pseudomonas aeruginosa exotoxin A (PEA)-based immunotoxins, the immunological role of PEA in asthma pathogenesis remains unclear. Objective: This study investigated the allergenic potential of PEA and the specific type of asthma induced. Methods: Recombinant PEA (rPEA) lacking domain Ia (to eliminate non-specific cytotoxicity) was expressed, purified, and employed to detect serum PEA-specific IgE levels in asthmatic patients. Competitive ELISA assays were used to assess rPEA's IgE binding capacity and allergenicity. Additionally, rPEA-challenged C57BL/6 mice were subjected to inflammatory endotyping and therapeutic assays to characterize the allergic nature of PEA. Results: PEA-specific IgE was identified in 17 (14.2 %) of 120 asthma patients. The rPEA-sensitized and challenged mice had increased PEA-specific immunoglobulins (such as IgE, IgG1 and IgG2a) and developed asthma-like phenotypes with airway hyperresponsiveness, severe airway inflammation, and airway remodeling. Lungs from these mice displayed significant increases in neutrophils and IL-17A+ cells. Innate lymphoid cells (ILCs) produced type 2 cytokines (IL-4, IL-5, and IL-13), whereas Th cells did not. Nonetheless, airway inflammation, rather than hyperresponsiveness, was elicited in non-sensitized mice upon challenge with rPEA. Importantly, rPEA-induced asthmatic mice were unresponsive to dexamethasone treatment. Conclusion: PEA is a novel allergen that sensitizes asthmatic patients. Furthermore, mice developed steroid-resistant asthma, characterized by an atypical cytokine profile associated with non-TH2 inflammation, only after being sensitized and challenged with rPEA. These findings suggest a potentially significant role for PEA in asthma development, warranting consideration in clinical diagnosis and treatment strategies.

Ferroptotic alveolar epithelial type II cells drive TH2 and TH17 mixed asthma triggered by birch pollen allergen Bet v 1.

Asthma is a common allergic disease characterized by airway hypersensitivity and airway remodeling. Ferroptosis is a regulated death marked by iron accumulation and lipid peroxidation. Several environmental pollutants and allergens have been shown to cause ferroptosis in epithelial cells, but the relationship between birch pollinosis and ferroptosis in asthma is poorly defined. Here, for the first time, we have identified ferroptosis of type II alveolar epithelial cells in mice with Bet v 1-induced asthma. Further analysis revealed that treatment with ferrostatin-1 reduced TH2/TH17-related inflammation and alleviated epithelial damage in mice with Bet v 1-induced asthma. In addition, ACSL4-knocked-down A549 cells are more resistant to Bet v 1-induced ferroptosis. Analysis of clinical samples verified higher serum MDA and 4-HNE concentrations compared to healthy individuals. We demonstrate that birch pollen allergen Bet v 1 induces ferroptosis underlaid TH2 and TH17 hybrid asthma. Lipid peroxidation levels can be considered as a biomarker of asthma severity, and treatment with a specific ferroptosis inhibitor could be a novel therapeutic strategy.

Contribution of Pseudomonas aeruginosa - mediated club cell necroptosis to the bias of type 17 inflammation and steroid insensitivity in asthma.

INTRODUCTION: Opportunistic pathogen infection is one of the important inducements for asthma exacerbation. Pseudomonas aeruginosa (PA) is a kind of dominant pathogenic bacteria in the respiratory tract that is associated with severe asthma, but the underlying mechanisms still remains unclear. OBJECTIVES: To examine the role of PA infection in the bias of the inflammatory endotype in asthma and its effect on the sensitivity to steroid therapy. METHODS: An adjusted HDM (House Dust Mite) -induced asthma model with PA inoculation in the airway was utilized to mimic the process of opportunistic PA infection in asthma, focusing on the interaction between bacteria and epithelium. Dexamethasone administration in vivo was used to test the sensitivity to steroid therapy. RESULTS: It was uncovered that PA could promote the loss of club cells in the necroptosis pattern through cellular CYP450 activation, leading to an imbalance of inflammatory response and steroid insensitivity. Club cell loss results in the activation of cellular E-cadherin/ß-catenin axis in the rest of club cells for goblet metaplasia and mucus hypersecretion, as well as epithelial damage and GR downregulation for steroid resistance. For clinical applications, the necroptosis inhibitor Nec-1 can effectively relieve the pathological symptoms of asthma in vivo. Meanwhile, CCSP administration in the airway can regulate the pulmonary inflammation and restore the steroid sensitivity in asthma. CONCLUSION: These experiments provide a novel mechanism of concurrent PA infection in asthma through club cell necroptosis and the pathological consequences. Nec-1 treatment and CCSP supplementation may be possible therapeutic strategies for asthma treatment.

Monolithic integrated micro-supercapacitors with ultra-high systemic volumetric performance and areal output voltage.

Monolithic integrated micro-supercapacitors (MIMSCs) with high systemic performance and cell-number density are important for miniaturized electronics to empower the Internet of Things. However, fabrication of customizable MIMSCs in an extremely small space remains a huge challenge considering key factors such as materials selection, electrolyte confinement, microfabrication and device-performance uniformity. Here, we develop a universal and large-throughput microfabrication strategy to address all these issues by combining multistep lithographic patterning, spray printing of MXene microelectrodes and controllable 3D printing of gel electrolytes. We achieve the monolithic integration of electrochemically isolated micro-supercapacitors in close proximity by leveraging high-resolution micropatterning techniques for microelectrode deposition and 3D printing for precise electrolyte deposition. Notably, the MIMSCs obtained demonstrate a high areal-number density of 28 cells cm-2 (340 cells on 3.5 × 3.5 cm2), a record areal output voltage of 75.6 V cm-2, an acceptable systemic volumetric energy density of 9.8 mWh cm-3 and an unprecedentedly high capacitance retention of 92% after 4000 cycles at an extremely high output voltage of 162 V. This work paves the way for monolithic integrated and microscopic energy-storage assemblies for powering future microelectronics.

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.

Integrated microdevice for long-term automated perfusion culture without shear stress and real-time electrochemical monitoring of cells.

Electrochemical techniques based on ultramicroelectrodes (UMEs) play a significant role in real-time monitoring of chemical messengers' release from single cells. Conversely, precise monitoring of cells in vitro strongly depends on the adequate construction of cellular physiological microenvironment. In this paper, we developed a multilayer microdevice which integrated high aspect ratio poly(dimethylsiloxane) (PDMS) microfluidic device for long-term automated perfusion culture of cells without shear stress and an independently addressable microelectrodes array (IAMEA) for electrochemical monitoring of the cultured cells in real time. Novel design using high aspect ratio between circular "moat" and ring-shaped micropillar array surrounding cell culture chamber combined with automated "circular-centre" and "bottom-up" perfusion model successfully provided continuous fresh medium and a stable and uniform microenvironment for cells. Two weeks automated culture of human umbilical endothelial cell line (ECV304) and neuronal differentiation of rat pheochromocytoma (PC12) cells have been realized using this device. Furthermore, the quantal release of dopamine from individual PC12 cells during their culture or propagation process was amperometrically monitored in real time. The multifunctional microdevice developed in this paper integrated cellular microenvironment construction and real-time monitoring of cells during their physiological process, and would possibly provide a versatile platform for cell-based biomedical analysis.

Interfacing droplet microfluidics with antibody barcodes for multiplexed single-cell protein secretion profiling.

Multiplexed protein secretion analysis of single cells is important to understand the heterogeneity of cellular functions and processes in healthy and disease states. However, current single-cell platforms, such as microwell-, microchamber-, or droplet-based assays, suffer from low single-cell occupancy, waste of reagents, limited sensitivity, or inability to perform necessary operations, etc. To overcome these drawbacks, we present an integrated droplet microfluidic device that interfaces with spatially patterned antibody barcodes for multiplexed single-cell secretome analysis. The trapping array of 100 picoliter-sized isolation chambers could achieve >80% single-cell capture efficiency with >90% viability. The single-cell analysis microchip was validated by the detection of four-plexed cytokines, including IL-8, MCP-1, MIP-1b, and TNF-a/IL-10, from unstimulated and lipopolysaccharide (LPS)-stimulated individual human macrophages. We also successfully applied the platform to profile protein secretions of human tumor cell lines and primary/metastatic cancer cells dissociated from cancer patients to observe the secretion heterogeneity among cells. This unique microfluidic platform enables multiplexed secretion assays for static droplet microfluidics, provides a reliable and straightforward workflow for protein secretion assays based on a low number of single cells in a short incubation time (∼4 h), and could have widespread applications for studying secretion-mediated cellular heterogeneity.