Advancing Point-of-Care Applications with Droplet Microfluidics: From Single-Cell to Multicellular Analysis.

Recent advances in single-cell and multicellular microfluidics technology have provided powerful tools for studying cancer biology and immunology. The ability to create controlled microenvironments, perform high-throughput screenings, and monitor cellular interactions at the single-cell level has significantly advanced our understanding of tumor biology and immune responses. We discuss cutting-edge multicellular and single-cell microfluidic technologies and methodologies utilized to investigate cancer-immune cell interactions and assess the effectiveness of immunotherapies. We explore the advantages and limitations of the wide range of 3D spheroid and single-cell microfluidic models recently developed, highlighting the various approaches in device generation and applications in immunotherapy screening for potential opportunities for point-of-care approaches.

Acoustofluidic Droplet Sorter Based on Single Phase Focused Transducers.

Droplet microfluidics has revolutionized the biomedical and drug development fields by allowing for independent microenvironments to conduct drug screening at the single cell level. However, current microfluidic sorting devices suffer from drawbacks such as high voltage requirements (e.g., >200 Vpp), low biocompatibility, and/or low throughput. In this article, a single-phase focused transducer (SPFT)-based acoustofluidic chip is introduced, which outperforms many microfluidic droplet sorting devices through high energy transmission efficiency, high accuracy, and high biocompatibility. The SPFT-based sorter can be driven with an input power lower than 20 Vpp and maintain a postsorting cell viability of 93.5%. The SPFT sorter can achieve a throughput over 1000 events per second and a sorting purity up to 99.2%. The SPFT sorter is utilized here for the screening of doxorubicin cytotoxicity on cancer and noncancer cells, proving its drug screening capability. Overall, the SPFT droplet sorting device shows great potential for fast, precise, and biocompatible drug screening.

Cyclic Thiosulfinates as a Novel Class of Disulfide Cleavable Cross-Linkers for Rapid Hydrogel Synthesis.

We recently reported that cyclic thiosulfinates are cysteine selective cross-linkers that avoid the "dead-end" modifications that contribute to other cross-linkers' toxicity. In this study, we generalize the chemistry of cyclic thiosulfinates to that of thiol selective cross-linking and apply them to the synthesis of hydrogels. Thiol-functionalized four-arm poly(ethylene glycol) and hyaluronic acid monomers were cross-linked with 1,2-dithiane-1-oxide to form disulfide cross-linked hydrogels within seconds. The synthesized hydrogel could be reduced with physiological concentrations of glutathione, which modulated hydrogel mechanical properties and degradation kinetics. Bovine serum albumin protein was successfully encapsulated in hydrogel, and diffusion-mediated release was demonstrated in vitro. Hep G2 cells grew in the presence of preformed hydrogel and during hydrogel synthesis, demonstrating acceptable cytotoxicity. We encapsulated cells within a hydrogel and demonstrated cell growth and recovery up to 10 days, with and without cell adhesion peptides. In summary, we report cyclic thiosulfinates as a novel class of cross-linkers for the facile synthesis of biodegradable hydrogels.

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.

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.

Innovative Tools and Technology for Analysis of Single Cells and Cell-Cell Interaction.

Heterogeneity in single-cell responses and intercellular interactions results from complex regulation of cell-intrinsic and environmental factors. Single-cell analysis allows not only detection of individual cellular characteristics but also correlation of genetic content with phenotypic traits in the same cell. Technological advances in micro- and nanofabrication have benefited single-cell analysis by allowing precise control of the localized microenvironment, cell manipulation, and sensitive detection capabilities. Additionally, microscale techniques permit rapid, high-throughput, multiparametric screening that has become essential for -omics research. This review highlights innovative applications of microscale platforms in genetic, proteomic, and metabolic detection in single cells; cell sorting strategies; and heterotypic cell-cell interaction. We discuss key design aspects of single-cell localization and isolation in microfluidic systems, dynamic and endpoint analyses, and approaches that integrate highly multiplexed detection of various intracellular species.

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.

Characterizing influence of rCHOP treatment on diffuse large B-cell lymphoma microenvironment through in vitro microfluidic spheroid model.

For over two decades, Rituximab and CHOP combination treatment (rCHOP) has remained the standard treatment approach for diffuse large B-cell lymphoma (DLBCL). Despite numerous clinical trials exploring treatment alternatives, few options have shown any promise at further improving patient survival and recovery rates. A wave of new therapeutic approaches have recently been in development with the rise of immunotherapy for cancer, however, the cost of clinical trials is prohibitive of testing all promising approaches. Improved methods of early drug screening are essential for expediting the development of the therapeutic approaches most likely to help patients. Microfluidic devices provide a powerful tool for drug testing with enhanced biological relevance, along with multi-parameter data outputs. Here, we describe a hydrogel spheroid-based microfluidic model for screening lymphoma treatments. We utilized primary patient DLBCL cells in combination with NK cells and rCHOP treatment to determine the biological relevance of this approach. We observed cellular viability in response to treatment, rheological properties, and cell surface marker expression levels correlated well with expected in vivo characteristics. In addition, we explored secretory and transcriptomic changes in response to treatment. Our results showed complex changes in phenotype and transcriptomic response to treatment stimuli, including numerous metabolic and immunogenic changes. These findings support this model as an optimal platform for the comparative screening of novel treatments.

An innovative single-cell approach for phenotyping and functional genotyping of CAR NK cells.

BACKGROUND: To accelerate the translation of novel immunotherapeutic treatment approaches, the development of analytic methods to assess their efficacy at early in vitro stages is necessary. Using a droplet-based microfluidic platform, we have established a method for multiparameter quantifiable phenotypic and genomic observations of immunotherapies. Chimeric antigen receptor (CAR) natural killer (NK) cells are of increased interest in the current immunotherapy landscape and thus provide an optimal model for evaluating our novel methodology. METHODS: For this approach, NK cells transduced with a CD19 CAR were compared with non-transduced NK cells in their ability to kill a lymphoma cell line. Using our microfluidic platform, we were able to quantify the increase in cytotoxicity and synaptic contact formation of CAR NK cells over non-transduced NK cells. We then optimized our droplet sorter and successfully used it to separate NK cells based on target cell killing to perform transcriptomic analyses. RESULTS: Our data revealed expected improvement in cytotoxicity with the CD19 CAR but more importantly, provided unique insights into the factors involved in the cytotoxic mechanisms of CAR NK cells. This demonstrates a novel, improved system for accelerating the pre-clinical screening of future immunotherapy treatments. CONCLUSIONS: This study provides a new potential approach for enhanced early screening of immunotherapies to improve their development, with a highly relevant cell model to demonstrate. Additionally, our validation studies provided some potential insights into transcriptomic determinants influencing CAR NK cytotoxicity.

Cellular immunity analysis by a modular acoustofluidic platform: CIAMAP.

The study of molecular mechanisms at the single-cell level holds immense potential for enhancing immunotherapy and understanding neuroinflammation and neurodegenerative diseases by identifying previously concealed pathways within a diverse range of paired cells. However, existing single-cell pairing platforms have limitations in low pairing efficiency, complex manual operation procedures, and single-use functionality. Here, we report a multiparametric cellular immunity analysis by a modular acoustofluidic platform: CIAMAP. This platform enables users to efficiently sort and collect effector-target (i.e., NK92-K562) cell pairs and monitor the real-time dynamics of immunological response formation. Furthermore, we conducted transcriptional and protein expression analyses to evaluate the pathways that mediate effector cytotoxicity toward target cells, as well as the synergistic effect of doxorubicin on the cellular immune response. Our CIAMAP can provide promising building blocks for high-throughput quantitative single-cell level coculture to understand intercellular communication while also empowering immunotherapy by precision analysis of immunological synapses.

The fusion of biology, computer science, and engineering: towards efficient and successful synthetic biology.

Synthetic biology is a nascent field that emerged in earnest only around the turn of the millennium. It aims to engineer new biological systems and impart new biological functionality, often through genetic modifications. The design and construction of new biological systems is a complex, multistep process, requiring multidisciplinary collaborative efforts from "fusion" scientists who have formal training in computer science or engineering, as well as hands-on biological expertise. The public has high expectations for synthetic biology and eagerly anticipates the development of solutions to the major challenges facing humanity. This article discusses laboratory practices and the conduct of research in synthetic biology. It argues that the fusion science approach, which integrates biology with computer science and engineering best practices, including standardization, process optimization, computer-aided design and laboratory automation, miniaturization, and systematic management, will increase the predictability and reproducibility of experiments and lead to breakthroughs in the construction of new biological systems. The article also discusses several successful fusion projects, including the development of software tools for DNA construction design automation, recursive DNA construction, and the development of integrated microfluidics systems.

Hypoxia-sensitive drug delivery to tumors.

Achievement of a high dose of drug in the tumor while minimizing its systemic side effects is one of the important features of an improved drug delivery system. Thus, developing responsive carriers for site-specific delivery of chemotherapeutic agents has become a main goal of research efforts. One of the known hallmarks of cancerous tumors is hypoxia, which offers a target for selective drug delivery. The stimuli-sensitive micellar system developed by us, (PEG-azobenzene-PEI-DOPE (PAPD) has proven to be effective in vitro. The proposed construct developed, PAPD, contains an azobenzene group as a hypoxia-sensitive moiety that triggers the shedding of the PEG layer from the nanoparticle surface under conditions of hypoxia to improve cellular uptake. Using microfluidics, we show significantly improved cellular association and penetration under hypoxia in both single cells and in a 3D tumor model. Employing an in vivo model, we demonstrate slower tumor growth that did not induce systemic side effects, including weight loss in an experimental animal model, when compared to the free drug treatment. This complex-in-nature but simple-in-design system for the simultaneous delivery of siRNA to silence the P-glycoprotein and doxorubicin with active tumor targeting and proven therapeutic efficacy represents a universal platform for the delivery of other hydrophobic chemotherapeutic agents and siRNA molecules which can be further modified.

Droplet microfluidics for functional temporal analysis and cell recovery on demand using microvalves: application in immunotherapies for cancer.

Most common methods of cellular analysis employ the top-down approach (investigating proteomics or genomics directly), thereby destroying the cell, which does not allow the possibility of using the same cell to correlate genomics with functional assays. Herein we describe an approach for single-cell tools that serve as a bottom-up approach. Our technology allows functional phenotyping to be conducted by observing the cytotoxicity of cells and then probe the underlying biology. We have developed a droplet microfluidic device capable of trapping droplets in the array and releasing the droplet of interest selectively using microvalves. Each droplet in the array encapsulates natural killer cells (NK cells) and tumour cells for real-time monitoring of burst kinetics and spatial coordination during killing by single NK cells. Finally, we use the microvalve actuation to selectively release droplets with the desired functional phenotype such as for fast and serial killing of target tumour cells by NK cells. From this perspective, our device allows for investigating first interactions and real-time monitoring of kinetics and later cell recovery on demand for single-cell omic analysis such as single-cell RNA sequencing (scRNA), which to date, is primarily based on in-depth analyses of the entire transcriptome of a relatively low number of cells.

A Novel Rolling Circle Amplification-Based Detection of SARS-CoV-2 with Multi-Region Padlock Hybridization.

SARS-CoV-2 has remained a global health burden, primarily due to the continuous evolution of different mutant strains. These mutations present challenges to the detection of the virus, as the target genes of qPCR, the standard diagnostic method, may possess sequence alterations. In this study, we develop an isothermal one-step detection method using rolling circle amplification (RCA) for SARS-CoV-2. This novel strategy utilizes a multi-padlock (MP-RCA) approach to detect viral-RNA via a simplified procedure with the reliable detection of mutated strains over other procedures. We designed 40 padlock-based probes to target different sequences across the SARS-CoV-2 genome. We established an optimal one-step isothermal reaction protocol utilizing a fluorescent output detected via a plate reader to test a variety of padlock combinations. This method was tested on RNA samples collected from nasal swabs and validated via PCR. S-gene target failure (SGTF)-mutated strains of SARS-CoV-2 were included. We demonstrated that the sensitivity of our assay was linearly proportional to the number of padlock probes used. With the 40-padlock combination the MP-RCA assay was able to correctly detect 45 out 55 positive samples (81.8% efficiency). This included 10 samples with SGTF mutations which we were able to detect as positive with 100% efficiency. We found that the MP-RCA approach improves the sensitivity of the MP-RCA assay, and critically, allows for the detection of SARS-CoV-2 variants with SGTF. Our method offers the simplicity of the reaction and requires basic equipment compared to standard qPCR. This method provides an alternative approach to overcome the challenges of detecting SARS-CoV-2 and other rapidly mutating viruses.

Ultrasensitive detection of low-abundance surface-marker protein using isothermal rolling circle amplification in a microfluidic nanoliter platform.

With advances in immunology and cancer biology, there is an unmet need for increasingly sensitive systems to monitor the expression of specific cell markers for the development of new diagnostic and therapeutic tools. To address this challenge, a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection events that can be greatly amplified via isothermal rolling circle amplification (RCA) is applied. By merging the single-molecule detection power of RCA reactions with microfluidic technology, it is demonstrated that the identification of specific protein markers can be achieved on tumor-cell surfaces in miniaturized nanoliter reaction droplets. Furthermore, this combined approach of signal amplification in a microfluidic format could extend the utility of existing methods by reducing sample and reagent consumption and enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer.

High throughput microfluidic system with multiple oxygen levels for the study of hypoxia in tumor spheroids.

Replication of physiological oxygen levels is fundamental for modeling human physiology and pathology inin vitromodels. Environmental oxygen levels, applied in mostin vitromodels, poorly imitate the oxygen conditions cells experiencein vivo, where oxygen levels average ∼5%. Most solid tumors exhibit regions of hypoxic levels, promoting tumor progression and resistance to therapy. Though this phenomenon offers a specific target for cancer therapy, appropriatein vitroplatforms are still lacking. Microfluidic models offer advanced spatio-temporal control of physico-chemical parameters. However, most of the systems described to date control a single oxygen level per chip, thus offering limited experimental throughput. Here, we developed a multi-layer microfluidic device coupling the high throughput generation of 3D tumor spheroids with a linear gradient of five oxygen levels, thus enabling multiple conditions and hundreds of replicates on a single chip. We showed how the applied oxygen gradient affects the generation of reactive oxygen species (ROS) and the cytotoxicity of Doxorubicin and Tirapazamine in breast tumor spheroids. Our results aligned with previous reports of increased ROS production under hypoxia and provide new insights on drug cytotoxicity levels that are closer to previously reportedin vivofindings, demonstrating the predictive potential of our system.

High-throughput microfluidic 3D biomimetic model enabling quantitative description of the human breast tumor microenvironment.

Cancer is driven by both genetic aberrations in the tumor cells and fundamental changes in the tumor microenvironment (TME). These changes offer potential targets for novel therapeutics, yet lack of in vitro 3D models recapitulating this complex microenvironment impedes such progress. Here, we generated several tumor-stroma scaffolds reflecting the dynamic in vivo breast TME, using a high throughput microfluidic system. Alginate (Alg) or alginate-alginate sulfate (Alg/Alg-S) hydrogels were used as ECM-mimics, enabling the encapsulation and culture of tumor cells, fibroblasts and immune cells (macrophages and T cells, of the innate and adaptive immune systems, respectively). Specifically, Alg/Alg-S was shown capable of capturing and presenting growth factors and cytokines with binding affinity that is comparable to heparin. Viability and cytotoxicity were shown to strongly correlate with the dynamics of cellular milieu, as well as hydrogel type. Using on-chip immunofluorescence, production of reactive oxygen species and apoptosis were imaged and quantitatively analyzed. We then show how macrophages in our microfluidic system were shifted from a proinflammatory to an immunosuppressive phenotype when encapsulated in Alg/Alg-S, reflecting in vivo TME dynamics. LC-MS proteomic profiling of tumor cells sorted from the TME scaffolds revealed upregulation of proteins involved in cell-cell interactions and immunomodulation in Alg/Alg-S scaffolds, correlating with in vivo findings and demonstrating the appropriateness of Alg/Alg-S as an ECM biomimetic. Finally, we show the formation of large tumor-derived vesicles, formed exclusively in Alg/Alg-S scaffolds. Altogether, our system offers a robust platform for quantitative description of the breast TME that successfully recapitulates in vivo patterns. STATEMENT OF SIGNIFICANCE: Cancer progression is driven by profound changes in both tumor cells and surrounding stroma. Here, we present a high throughput microfluidic system for the generation and analysis of dynamic tumor-stroma scaffolds, that mimic the complex in vivo TME cell proportions and compositions, constructing robust in vitro models for the study of the TME. Utilizing Alg/Alg-S as a bioinspired ECM, mimicking heparin's in vivo capabilities of capturing and presenting signaling molecules, we show how Alg/Alg-S induces complex in vivo-like responses in our models. Alg/Alg-S is shown here to promote dynamic protein expression patterns, that can serve as potential therapeutic targets for breast cancer treatment. Formation of large tumor-derived vesicles, observed exclusively in the Alg/Alg-S scaffolds suggests a mechanism for tumor survival.

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.