Comparison of dose selection based on target engagement versus inhibition of receptor-ligand interaction for checkpoint inhibitors.

Immune checkpoint inhibitors block the interaction between a receptor on one cell and its ligand on another cell, thus preventing the transduction of an immunosuppressive signal. While inhibition of the receptor-ligand interaction is key to the pharmacological activity of these drugs, it can be technically challenging to measure these intercellular interactions directly. Instead, target engagement (or receptor occupancy) is commonly measured, but may not always be an accurate predictor of receptor-ligand inhibition, and can be misleading when used to inform clinical dose projections for this class of drugs. In this study, a mathematical model explicitly representing the intercellular receptor-ligand interaction is used to compare dose prediction based on target engagement or receptor-ligand inhibition for two checkpoint inhibitors, atezolizumab and magrolimab. For atezolizumab, there is little difference between target engagement and receptor-ligand inhibition, but for magrolimab, the model predicts that receptor-ligand inhibition is significantly less than target engagement. The key variables explaining the difference between these two drugs are the relative concentrations of the target receptors and their ligands. Drug-target affinity and receptor-ligand affinity can also have divergent effects on target engagement and inhibition. These results suggest that it is important to consider ligand-receptor inhibition in addition to target engagement and demonstrate the impact of using modeling for efficacious dose estimation.

Quantitative modeling of approved and emerging therapeutics for modifying TTR levels in patients with Transthyretin Amyloid Cardiomyopathy.

Transthyretin amyloidosis is a rare yet lethal disease caused by an increase in the destabilization of transthyretin tetramer into monomers, leading to amyloid fibril aggregates in tissues. Multiple therapeutics have been developed to limit or halt disease progression by altering tetramer kinetics. Small molecules, like Tafamidis and AG10, stabilize the tetrameric structure while genetic therapies, like Patisiran and NTLA-002, limit the production of TTR protein by silencing genetic expression. Both of these interventions slow the accumulation of fibrils by intervening at different points in the tetramer-monomer-fibril pathway. We developed a mathematical model to compare the pharmacological efficacies of these modalities by comparing each drug's ability to reduce the rate of tetramer to monomer formation, or "tetrameric flux." The model was trained on in vitro tetramer data as well as clinical measurements of tetramer concentration in humans. Overall, genetic silencers reduced tetrameric flux more than small molecule stabilizers. Properties that led to an improvement in small molecule stabilizer function and potential benefit of gene therapy - small molecule combination were explored. This study exemplifies how modeling can be used to compare modalities with differing mechanisms of action.

Transfer of Cellular Content from the Allogeneic Cell-Based Cancer Vaccine DCP-001 to Host Dendritic Cells Hinges on Phosphatidylserine and Is Enhanced by CD47 Blockade.

DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell-cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.

Quantifying the efficacy of checkpoint inhibitors on CD8+ cytotoxic T cells for immunotherapeutic applications via single-cell interaction.

The inhibition of the PD1/PDL1 pathway has led to remarkable clinical success for cancer treatment in some patients. Many, however, exhibit little to no response to this treatment. To increase the efficacy of PD1 inhibition, additional checkpoint inhibitors are being explored as combination therapy options. TSR-042 and TSR-033 are novel antibodies for the inhibition of the PD1 and LAG3 pathways, respectively, and are intended for combination therapy. Here, we explore the effect on cellular interactions of TSR-042 and TSR-033 alone and in combination at the single-cell level. Utilizing our droplet microfluidic platform, we use time-lapse microscopy to observe the effects of these antibodies on calcium flux in CD8+ T cells upon antigen presentation, as well as their effect on the cytotoxic potential of CD8+ T cells on human breast cancer cells. This platform allowed us to investigate the interactions between these treatments and their impacts on T-cell activity in greater detail than previously applied in vitro tests. The novel parameters we were able to observe included effects on the exact time to target cell killing, contact times, and potential for serial-killing by CD8+ T cells. We found that inhibition of LAG3 with TSR-033 resulted in a significant increase in calcium fluctuations of CD8+ T cells in contact with dendritic cells. We also found that the combination of TSR-042 and TSR-033 appears to synergistically increase tumor cell killing and the single-cell level. This study provides a novel single-cell-based assessment of the impact these checkpoint inhibitors have on cellular interactions with CD8+ T cells.

Machine learning-aided quantification of antibody-based cancer immunotherapy by natural killer cells in microfluidic droplets.

Natural killer (NK) cells have emerged as an effective alternative option to T cell-based immunotherapies, particularly against liquid (hematologic) tumors. However, the effectiveness of NK cell therapy has been less than optimal for solid tumors, partly due to the heterogeneity in target interaction leading to variable anti-tumor cytotoxicity. This paper describes a microfluidic droplet-based cytotoxicity assay for quantitative comparison of immunotherapeutic NK-92 cell interaction with various types of target cells. Machine learning algorithms were developed to assess the dynamics of individual effector-target cell pair conjugation and target death in droplets in a semi-automated manner. Our results showed that while short contacts were sufficient to induce potent killing of hematological cancer cells, long-lasting stable conjugation with NK-92 cells was unable to kill HER2+ solid tumor cells (SKOV3, SKBR3) significantly. NK-92 cells that were engineered to express FcγRIII (CD16) mediated antibody-dependent cellular cytotoxicity (ADCC) selectively against HER2+ cells upon addition of Herceptin (trastuzumab). The requirement of CD16, Herceptin and specific pre-incubation temperature served as three inputs to generate a molecular logic function with HER2+ cell death as the output. Mass proteomic analysis of the two effector cell lines suggested differential changes in adhesion, exocytosis, metabolism, transport and activation of upstream regulators and cytotoxicity mediators, which can be utilized to regulate specific functionalities of NK-92 cells in future. These results suggest that this semi-automated single cell assay can reveal the variability and functional potency of NK cells and may be used to optimize immunotherapeutic efficacy for preclinical analyses.

Interaction kinetics with transcriptomic and secretory responses of CD19-CAR natural killer-cell therapy in CD20 resistant non-hodgkin lymphoma.

We investigated the cytolytic and mechanistic activity of anti-CD19 chimeric antigen receptor natural killer (CD19.CAR.NK92) therapy in lymphoma cell lines (diffuse large B-cell, follicular, and Burkitt lymphoma), including rituximab- and obinutuzumab-resistant cells, patient-derived cells, and a human xenograft model. CD19.CAR.NK92 therapy significantly increased cytolytic activity at E:T ratios (1:1-10:1) via LDH release and prominent induction of apoptosis in all cell lines, including in anti-CD20 resistant lymphoma cells. The kinetics of CD19.CAR.NK92 cell death measured via droplet-based single cell microfluidics analysis showed that most lymphoma cells were killed by single contact, with anti-CD20 resistant cell lines requiring significantly longer contact duration with NK cells. In addition, systems biology transcriptomic analyses of flow-sorted lymphoma cells co-cultured with CD19.CAR.NK92 revealed conserved activation of IFNγ signaling, execution of apoptosis, ligand binding, and immunoregulatory and chemokine signaling pathways. Furthermore, a 92-plex cytokine panel analysis showed increased secretion of granzymes, increased secretion of FASL, CCL3, and IL10 in anti-CD20 resistant SUDHL4 cells with induction of genes relevant to mTOR and G2/M checkpoint activation, which were noted in all anti-CD20 resistant cells co-cultured with CD19.CAR.NK92 cells. Collectively, CD19.CAR.NK92 was associated with potent anti-lymphoma activity across a host of sensitive and resistant lymphoma cells that involved distinct immuno-biologic mechanisms of cell death.

Anti-myeloma activity and molecular logic operation by Natural Killer cells in microfluidic droplets.

Immune-targeted therapies that activate effector lymphocytes such as Natural Killer (NK) cells are currently being investigated for the treatment of Multiple myeloma (MM), the second most common form of hematological cancer. However, individual NK cells are highly heterogeneous in their cytolytic potential, making it difficult to detect, quantify and correlate the outcome of dynamic effector-target cell interactions at single cell resolution. Here, we present a microfluidic bioassay platform capable of activity-based screening of cellular and molecular immunotherapies. We identified distinct functional signatures associated with NK-MM cell interaction. The addition of immunomodulatory drug lenalidomide altered responses of NK-susceptible MM cells but not that of NK-tolerant MM cells. Antitumor cytotoxicity was significantly increased by the blockade of PD1/PDL1 axis as well as the clinically relevant cell line NK92, which were used to construct molecular logic functions (AND and NOT gates). A predictive agent-based mathematical model was developed to simulate progressive disease states and drug efficacy. The findings of the current study validate the applicability of this microfluidic cytotoxicity assay for immunotherapy screening, biocomputation and for future employment in detection of patient-specific cell response for precision medicine.

Ultrafast Parallelized Microfluidic Platform for Antimicrobial Susceptibility Testing of Gram Positive and Negative Bacteria.

Antimicrobial susceptibility testing (AST) is an essential diagnostic procedure to determine the correct course of treatment for various types of pathogen infections. Patients are treated with broad spectrum antibiotics until AST results become available, which has contributed to the emergence of multidrug resistant bacteria worldwide. Conventional AST methods require 16-24 h to assess sensitivity of the bacteria to a given drug and establish its minimum inhibitory concentration (MIC). A rapid AST assay can assist clinicians in making an informed choice of targeted therapy and avoid unnecessary overprescription. Here, we have developed a highly parallelized droplet microfluidic platform that can screen four antibiotics/pathogens simultaneously and assess antibiotic sensitivity in 15-30 min. The device consists of four integrated microdroplet arrays, each hosting over 8000 docking sites, which can be operated individually or jointly for greater flexibility of operation. Small numbers (1-4) of bacterial cells were entrapped in droplets of 110 pL volume and monitored dynamically over 2 h. This imaging-based AST approach was used to determine the growth rates of four types of clinically relevant bacteria known to cause urinary tract infection (UTI) in millions of patients. We quantified doubling times of both Gram positive ( Staphylococcus aureus, Enterococcus faecalis) and Gram negative bacteria (e.g., Escherichia coli, Klebsiella pneumoniae) with varying levels of antibiotic resistance. Six concentrations of bactericidal and bacteriostatic antibiotics (oxacillin and tetracycline, respectively) were tested to determine the MIC of the strains as well as the heterogeneity in growth profiles of bacteria at single cell resolution. The MIC determined from phenotypic analysis in droplets matched the MIC obtained from broth microdilution method for all strains. The advantages of the proposed droplet-based AST, including rapid drug sensitivity response, morphological analysis, and heterogeneity in antibiotic-resistance profiles, make it an excellent alternative to standard phenotypic AST with potential applications in clinical diagnostics and point of care testing.

Self-assembly of surface functionalized amphiphilic carbon dots: tuning in morphological manifestations.

Despite the continuous surge of interest in supramolecular chemistry, the design and synthesis of building blocks to develop diverse examples of self-assemblies is still challenging. During the past decades, formation of self-assemblies such as micelles, vesicles, and gels with a fibril network using amphiphiles has been investigated at length. Considering the increasing applications of these self-aggregates across the scientific domain, it is crucial to adopt an alternative strategy for the preparation of self-aggregates using a new building block that has been applied in diverse domains. With this aim, surface functionalized carbon dots (CDs) with varying aliphatic/aromatic (cholesteryl, palmitoyl, naphthyl) substitutions linked with spacers such as ethylenediamine, p-phenylenediamine, 2,2'-(ethylenedioxy)bis(ethylamine) were developed. The surface passivated CDs formed self-assemblies in dimethylsulfoxide-water (DMSO-H2O, 2 : 1, v/v). The roles of surface functionalities and spacer units in the formation of self-assemblies using the synthesized CDs were investigated by microscopic and spectroscopic studies. Progressive morphological transition was found from vesicle-to-fiber in DMSO-H2O (2 : 1, v/v) which was dependent on surface passivating substitutions of the CDs from cholesteryl to naphthyl to palmitoyl. Together with the exclusive formation of self-assemblies using amphiphilic CDs, the present study enabled the tuning of self-organization behaviour of the CD by alteration of its surface functionality.

Microstructure characterization of biocompatible heterojunction hydrogen titanate-Ag2O nanocomposites for superior visible light photocatalysis and antibacterial activity.

Hydrogen trititanate (H2Ti3O7·2H2O) and hydrogen trititanate/Ag2O hybrid nanocomposites (NCs) with novel structure have been synthesized by a simple solvothermal route followed by Na+/H+ ion-exchange. Growths of hydrogen trititanate with nanofiber (HTNF) and nanotube (HTNT) morphologies and hydrogen trititanate-Ag2O (HTFAG and HTTAG) nanocomposites have been tailored by controlling the solvent media. Detailed microstructure characterization of all these samples have been carried out by Rietveld refinement of XRD data and analyzing FESEM/HRTEM micrographs and FTIR spectra. Band gap energies of all these semiconducting samples are obtained from UV-Vis absorption spectra. Both HTFAG and HTTAG NCs exhibit enhanced photocatalytic degradation of organic pollutant (Congo red dye) under visible light, in comparison to HTNF and HTNT respectively due to the formation of a heterojunction between H2Ti3O7·2H2O and Ag2O, which is supported by photoluminescence spectroscopy. HTFAG and HTTAG NCs also show superior antibacterial activity against both gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacteria compared to their pure counterparts. MTT assay reflects a sufficiently high percentage of cell viability and confirms the significant cytocompatibility of all the samples.

Photosensitizer Tailored Surface Functionalized Carbon Dots for Visible Light Induced Targeted Cancer Therapy.

Herein, a photosensitizer (riboflavin) tailored surface functionalized carbon dot (RCD1s) was designed to utilize it in visible light induced targeted cancer therapy. At first, phenylboronic acid appended biotinylated blue emitting carbon dot (CD1s) was synthesized. Riboflavin having "diol" moiety was covalently linked with this CD1s to prepare RCD1s by using complementary boronate-diol linkage. Lewis acid-base interaction facilitated the covalent linkage formation between the surface functionalizing agent of CD1s and riboflavin to develop water-soluble, green emitting RCD1s. Interestingly, this newly synthesized RCD1s has the ability to produce reactive oxygen species (ROS) such as hydroxyl and superoxide radicals under exposure of visible light (wavelength: 460-490 nm). These ROS also can destroy the structure of DNA by oxidative pathway. Thus, under irradiation of visible light (wavelength: 460-490 nm), RCD1s was found to kill HeLa and B16F10 melanoma cells over noncancer cell NIH3T3 by ∼5-fold higher efficacy through ROS induced oxidative DNA damage. The presence of biotin on the surface of the riboflavin tethered carbon dot is essential for the selective killing of cancer cells over normal cells. In the presence of UV light (340-420 nm), RCD1s showed no notable killing of cancer cells as well as normal cells. Besides, RCD1s in the presence of visible light selectively stained HeLa and B16F10 cells over noncancerous cell NIH3T3 by exploiting its fluorescence and cancer cell targeting moiety, biotin. Hence, the newly developed RCD1s can be utilized in theranostic applications including bioimaging and selective killing of cancer cells in the presence of visible light (460-490 nm).

Microfluidic assembly of hydrogel-based immunogenic tumor spheroids for evaluation of anticancer therapies and biomarker release.

Diffuse large B cell lymphoma (DLBCL), the most common subtype of Non-Hodgkin lymphoma, exhibits pathologic heterogeneity and a dynamic immunogenic tumor microenvironment (TME). However, the lack of preclinical in vitro models of DLBCL TME hinders optimal therapeutic screening. This study describes the development of an integrated droplet microfluidics-based platform for high-throughput generation of immunogenic DLBCL spheroids. The spheroids consist of three cell types (cancer, fibroblast and lymphocytes) in a novel hydrogel combination of alginate and puramatrix, which promoted cell adhesion and aggregation. This system facilitates dynamic analysis of cellular interaction, proliferation and therapeutic efficacy via spatiotemporal monitoring and secretome profiling. The immunomodulatory drug lenalidomide had direct anti-proliferative effect on activated B-cell like DLBCL spheroids and reduced several cytokines and other markers (e.g., CCL2, CCL3, CCL4, CD137 and ANG-1 levels) compared with untreated spheroids. Collectively, this novel spheroid platform will enable high-throughput anti-cancer therapeutic screening in a semi-automated manner.

Tunable Aggregation-Induced Multicolor Emission of Organic Nanoparticles by Varying the Substituent in Naphthalene Diimide.

In this article, we have designed l-aspartic acid-linked naphthalene diimide (NDI)-based amphiphilic molecules having a benzyl ester group at both the terminals with varying substituents (NAB-1-5). The substituent was judiciously modified from an electron-withdrawing group (EWG) like nitrobenzene to an electron-donating group (EDG), methoxybenzene, and finally to an extended aromatic residue (naphthalene) to regulate the π-electron density at the terminal of NDI derivatives. All of the synthesized NDI derivatives were molecularly dissolved in dimethyl sulfoxide (DMSO), and with an increase in the water content within the DMSO solution, the NDI derivative starts to get self-assembled through J-aggregation at and above 40% water content. Self-assembled spherical organic nanoparticles formed in 99% water in DMSO ( fw = 99%) were characterized by microscopic studies. All of the NDI derivatives showed very weak emission in the molecularly dissolved state (DMSO). Aggregation-induced emission (AIE) was observed for the NDI derivatives (except NAB-1) at the self-assembled state through excimer formation. Upon excitation at 350 nm, the emission maxima of these NDI-based AIE luminogens (AIE-gens) (NAB-2-5) get red shifted from 463 to 588 nm upon altering the substitution from EWG to EDG at the donor site. Inclusion of proper donor-acceptor moieties in the molecular backbone of the self-assembling unit can govern the AIE in combination with the intramolecular charge-transfer process. Consequently, the emission color of these AIE-gens (NAB-2-5) gets tuned from cyan blue to faint green to strong green and finally to bright orange. The tunable aggregation-induced multicolor emission was investigated by different spectroscopic techniques. These cytocompatible, multicolor-emitting fluorescent organic nanoparticles were utilized for bioimaging applications.

Glucose oxidase mediated targeted cancer-starving therapy by biotinylated self-assembled vesicles.

Herein, we demonstrate glucose oxidase (GOx) mediated targeted cancer-starving therapy by self-assembled vesicle of trimesic acid based biotinylated amphiphile (TMB). The TMB vesicles entrapped GOx and selectively killed cancer cells (HeLa, B16F10), with ∼6-fold higher efficiency compared to non-cancer cells (CHO, NIH3T3), by blocking the energy supply to tumors through the oxidation of intracellular glucose.

Tailor-Made Self-Assemblies from Functionalized Amphiphiles: Diversity and Applications.

The objective of this feature article is to coalesce our recent advancements on different expressions of tailor-made supramolecular self-assemblies and to explore them as a function of molecular architecture. In the last decade, we have developed a library of elegant and simple functional amphiphilic small molecules, which have very interesting abilities to form diverse manifestations of supramolecular self-assemblies such as micelles, reverse micelles, vesicles, fibers, supramolecular gels, and so on. Each of the expressions of the self-aggregated structures has its individual prominence and finds important applications in the fields of chemistry, physics, biology, and others. In this feature article, the major emphasis is mostly on how to attain precise control over the development of various well-defined supramolecular self-assemblies through the judicious design of low-molecular-weight amphiphiles. By tuning only the functional moieties of the amphiphilic structure, diverse supramolecular architectures can be constructed with task-specific applications. We expect that this article will provide a general and conceptual demonstration of various approaches to the development of different functional supramolecular systems and their prospective applications in numerous domains.

Self-Assembled Vesicle-Carbon Nanotube Conjugate Formation through a Boronate-Diol Covalent Linkage.

A vesicle-single walled carbon nanotube (CNT) conjugate was developed by a boronic acid-diol covalent linkage between a self-assembled vesicle and dispersed CNT. Trimesic acid based phenylboronic acid appended triple-tailed amphiphiles (T1 and T1S) were synthesized that formed monolayered vesicles through H-aggregation in DMSO-water (2:1 v/v) and pure water, respectively. Aqueous CNT dispersion was prepared with cholesterol-based glucose-functionalized amphiphile (D1). These two supramolecular self-assemblies were covalently linked by using a boronic acid-diol interaction between a phenylboronic acid based T1S vesicle and 1,2-diol moieties of glucose tethered dispersing agent (D1) to develop a vesicle-CNT conjugate. Lewis acid-base chemistry was exploited to form this boronate-diol adduct between two supramolecular self-assemblies. The formation of vesicles, CNT dispersion, and the vesicle-CNT conjugate was characterized by microscopic and spectroscopic techniques. Anticancer drug doxorubicin was encapsulated within this T1S-vesicle-D1-CNT conjugate with a higher loading capacity compared to the individual cargo carrier (vesicle or CNT). This cytocompatible T1S-vesicle-D1-CNT conjugate successfully delivered loaded doxorubicin within a B16F10 melanoma cell and also exhibited better cellular transportation ability compared to the drug-loaded vesicle or CNT. This was further reflected in an enhanced killing efficiency of the cancer cells by the vesicle-CNT conjugate compared to the drug-loaded vesicle or CNT.

Quantification of intercellular adhesion forces measured by fluid force microscopy.

The mechanics of cancer cell adhesion to its neighboring cells, homotypic or heterotypic, have significant impact on tumor progression and metastasis. Intercellular adhesion has been quantified previously using atomic force microscopy-based methods. Here we show the feasibility of the recently developed fluidic force microscopy (FluidFM) to measure adhesive forces exerted by breast cancer cells. Multiple cell pairs were assessed at precisely controlled, increasing contact durations by pressure-dependent immobilization of a cell at the probe tip. Eliminating chemical fixation of the cell at the tip ensured repeated use of the same probe and also minimized changes in cell physiology. Our data indicates distinct trends of adhesion forces between homotypic breast cancer cells compared to heterotypic adhesion between cancer-fibroblast and cancer-epithelial cell pairs. Adhesion forces were similar for all three cell pairs at short contact duration (< 1min) but differed at longer contact period (30min). Our study suggests that FluidFM is a rapid efficient technique that could be used to assess heterogeneity in cellular adhesion at various stages of malignant transformation.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

Isothermal Amplification Strategies for Detection in Microfluidic Devices.

Isothermal rolling circle amplification (RCA) is used to detect nucleic and non-nucleic acid biomarkers with high sensitivity. Immuno-RCA, the specific detection of proteins via antigen-antibody recognition, has been miniaturized for microfluidic platforms to reduce reagent and sample consumption, accelerate reaction kinetics, and enhance the sensitivity and specificity of detection.

A droplet-merging platform for comparative functional analysis of m1 and m2 macrophages in response to e. coli-induced stimuli.

Microfluidic droplets are used to isolate cell pairs and prevent crosstalk with neighboring cells, while permitting free motility and interaction within the confined space. Dynamic analysis of cellular heterogeneity in droplets has provided insights in various biological processes. Droplet manipulation methods such as fusion and fission make it possible to precisely regulate the localized environment of a cell in a droplet and deliver reagents as required. Droplet fusion strategies achieved by passive mechanisms preserve cell viability and are easier to fabricate and operate. Here, we present a simple and effective method for the co-encapsulation of polarized M1 and M2 macrophages with Escherichia coli (E. coli) by passive merging in an integrated droplet generation, merging, and docking platform. This approach facilitated live cell profiling of effector immune functions in situ and quantitative functional analysis of macrophage heterogeneity. Biotechnol. Bioeng. 2017;114: 705-709. © 2016 Wiley Periodicals, Inc.