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Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I â KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.
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Neoplasias de la Mama , Epigénesis Genética , Histona Demetilasas , Interferón Tipo I , Antraciclinas/metabolismo , Antraciclinas/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Histona Demetilasas/metabolismo , Humanos , Interferón Tipo I/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patologíaRESUMEN
This paper reports an impedance-based system for the quantitative assessment of dielectrophoretic (DEP) focusing of single particles flowing in a microchannel. Particle lateral positions are detected in two electrical sensing zones placed before and after a DEP-focusing region, respectively. In each sensing zone, particle lateral positions are estimated using the unbalance between the opposite pulses of a differential current signal obtained with a straightforward coplanar electrode configuration. The system is used to monitor the focusing of polystyrene beads of 7 or 10 µm diameter, under various conditions of DEP field intensities and flow rates that produce different degrees of focusing. This electrical approach represents a simple and valuable alternative to optical methods for monitoring of particle focusing systems.
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Electroforesis/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Electrodos , Electroforesis/instrumentación , Diseño de Equipo , Poliestirenos , Procesamiento de Señales Asistido por ComputadorRESUMEN
Inkjet printing is here employed for the first time as a method to produce femtoliter-scale oil droplets dispersed in water. In particular, picoliter-scale fluorinated oil (FC40) droplets are printed in the presence of perfluoro-1-octanol surfactant at a velocity higher than 5 m/s. Femtoliter-scale oil droplets in water are spontaneously formed through a fragmentation process at the water/air interface using minute amounts of nonionic surfactant (down to 0.003% v/v of Tween 80). This fragmentation occurs by a Plateau-Rayleigh mechanism at a moderately high Weber number (101). A microfluidic chip with integrated microelectrodes allows droplets characterization in terms of number and diameter distribution (peaked at about 3 µm) by means of electrical impedance measurements. These results show an unprecedented possibility to scale oil droplets down to the femtoliter scale, which opens up several perspectives for a tailored oil-in-water emulsion fabrication for drug encapsulation, pharmaceutic preparations, and cellular biology.
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In this work we present an integrated biosensor that enables FTIR (Fourier Transform-Infrared) detection of analytes contained in diluted solutions. The fabricated nanosensor allows for the detection of proteins through the identification of the fine structure of their amide I and II bands, up to the nanomolar concentration range. We exploited two distinct effects to enhance the sensitivity: (i) the concentration effect due to the presence of the superhydrophobic surface that conveys molecules dispersed in solution directly inside the focus of a FTIR spectromicroscope; (ii) the plasmonic resonance of the nanoantenna array that provides electromagnetic field enhancement in the amide I and II spectral region (1500-1700 cm(-1)). We demonstrate the detection of ferritin in the nanomolar concentration range, a blood protein that is usually available in small amounts in typical blood samples.
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Técnicas Biosensibles/métodos , Proteínas/análisis , Espectroscopía Infrarroja por Transformada de Fourier , Técnicas Biosensibles/instrumentación , Interacciones Hidrofóbicas e Hidrofílicas , Nanoestructuras/química , Análisis por Matrices de ProteínasRESUMEN
Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.
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Técnicas de Cocultivo , Citometría de Flujo , Técnicas de Cocultivo/métodos , Ratones , Animales , Citometría de Flujo/métodos , Línea Celular Tumoral , Dispositivos Laboratorio en un Chip , Neoplasias/inmunología , Neoplasias/patología , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentación , HumanosRESUMEN
We investigate for the first time the compatibility of nanovials with microfluidic impedance cytometry (MIC). Nanovials are suspendable crescent-shaped single-cell microcarriers that enable specific cell adhesion, the creation of compartments for undisturbed cell growth and secretion, as well as protection against wall shear stress. MIC is a label-free single-cell technique that characterizes flowing cells based on their electrical fingerprints and it is especially targeted to cells that are naturally in suspension. Combining nanovial technology with MIC is intriguing as it would represent a robust framework for the electrical analysis of single adherent cells at high throughput. Here, as a proof-of-concept, we report the MIC analysis of mesenchymal stromal cells loaded in nanovials. The electrical analysis is supported by numerical simulations and validated by means of optical analysis. We demonstrate that the electrical diameter can discriminate among free cells, empty nanovials, cell-loaded nanovials, and clusters, thus grounding the foundation for the use of nanovials in MIC. Furthermore, we investigate the potentiality of MIC to assess the electrical phenotype of cells loaded in nanovials and we draw directions for future studies.
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Células Madre Mesenquimatosas , Técnicas Analíticas Microfluídicas , Análisis de la Célula Individual , Células Madre Mesenquimatosas/citología , Análisis de la Célula Individual/instrumentación , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Impedancia Eléctrica , Nanoestructuras/química , Citometría de Flujo/instrumentaciónRESUMEN
The development of advanced biological models like microphysiological systems, able to rebuild the complexity of the physiological and/or pathological environments at a single-cell detail level in an in-vivo-like approach, is proving to be a promising tool to understand the mechanisms of interactions between different cell populations and main features of several diseases. In this frame, the tumor-immune microenvironment on a chip represents a powerful tool to profile key aspects of cancer progression, immune activation, and response to therapy in several immuno-oncology applications. In the present chapter, we provide a protocol to identify and characterize the time evolution of apoptosis by time-lapse fluorescence and confocal imaging in a 3D microfluidic coculture murine model including cancer and spleen cells.
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Neoplasias , Animales , Humanos , Ratones , Caspasa 3 , Neoplasias/patología , Microfluídica/métodos , Apoptosis , Dispositivos Laboratorio en un Chip , Microambiente TumoralRESUMEN
Immune cell-derived extracellular vesicles (EV) affect tumor progression and hold promise for therapeutic applications. Eosinophils are major effectors in Th2-related pathologies recently implied in cancer. Here, we evaluated the anti-tumor activities of eosinophil-derived EV following activation with the alarmin IL-33. We demonstrate that IL-33-activated mouse and human eosinophils produce higher quantities of EV with respect to eosinophils stimulated with IL-5. Following incorporation of EV from IL-33-activated eosinophils (Eo33-EV), but not EV from IL-5-treated eosinophils (Eo5-EV), mouse and human tumor cells increased the expression of cyclin-dependent kinase inhibitor (CDKI)-related genes resulting in cell cycle arrest in G0/G1, reduced proliferation and inhibited tumor spheroid formation. Moreover, tumor cells incorporating Eo33-EV acquired an epithelial-like phenotype characterized by E-Cadherin up-regulation, N-Cadherin downregulation, reduced cell elongation and migratory extent in vitro, and impaired capacity to metastasize to lungs when injected in syngeneic mice. RNA sequencing revealed distinct mRNA signatures in Eo33-EV and Eo5-EV with increased presence of tumor suppressor genes and enrichment in pathways related to epithelial phenotypes and negative regulation of cellular processes in Eo33-EV compared to Eo5-EV. Our studies underscore novel IL-33-stimulated anticancer activities of eosinophils through EV-mediated reprogramming of tumor cells opening perspectives on the use of eosinophil-derived EV in cancer therapy.
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Eosinófilos , Vesículas Extracelulares , Interleucina-33 , Animales , Interleucina-33/metabolismo , Ratones , Eosinófilos/metabolismo , Humanos , Vesículas Extracelulares/metabolismo , Línea Celular Tumoral , Proliferación Celular , Reprogramación CelularRESUMEN
The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. The DZ and LZ differ significantly in their immune composition despite the lack of a physical barrier, yet the determinants of this polarization are poorly understood. This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features that underlie DZ T-cell exclusion. The DZ spatial transcriptional signature linked to T-cell immune evasion clustered aggressive Diffuse Large B-cell Lymphomas (DLBCL) for differential T cell infiltration. We reveal the dependence of the DZ transcriptional core signature on the ATR kinase and dissect its role in restraining inflammatory responses contributing to establishing an immune-repulsive imprint in DLBCL. These insights may guide ATR-focused treatment strategies bolstering immunotherapy in tumors marked by DZ transcriptional and chromatin-associated features.
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BACKGROUND: Microglia possess an elevated grade of plasticity, undergoing several structural changes based on their location and state of activation. The first step towards the comprehension of microglia's biology and functional responses to an extremely mutable extracellular milieu, consists in discriminating the morphological features acquired by cells maintained in vitro under diverse environmental conditions. Previous work described neither primary microglia grown on artificially patterned environments which impose physical cues and constraints, nor long distance migration of microglia in vitro. To this aim, the present work exploits artificial bio-mimetic microstructured substrates with pillar-shaped or line-grating geometries fabricated on poly(dimethylsiloxane) by soft lithography, in addition to microfluidic devices, and highlights some morphological/functional characteristics of microglia which were underestimated or unknown so far. RESULTS: We report that primary microglia selectively adapt to diverse microstructured substrates modifying accordingly their morphological features and behavior. On micropatterned pillar-shaped geometries, microglia appear multipolar, extend several protrusions in all directions and form distinct pseudopodia. On both micropatterned line-grating geometries and microfluidic channels, microglia extend the cytoplasm from a roundish to a stretched, flattened morphology and assume a filopodia-bearing bipolar structure. Finally, we show that in the absence of any applied chemical gradient, primary microglia spontaneously moves through microfluidic channels for a distance of up to 500 µm in approximately 12 hours, with an average speed of 0.66 µm/min. CONCLUSIONS: We demonstrate an elevated grade of microglia plasticity in response to a mutable extracellular environment, thus making these cells an appealing population to be further exploited for lab on chip technologies. The development of microglia-based microstructured substrates opens the road to novel hybrid platforms for testing drugs for neuroinflammatory diseases.
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Movimiento Celular/fisiología , Microfluídica , Microglía/citología , Animales , Técnica del Anticuerpo Fluorescente , Ratones , Microscopía Confocal , RatasRESUMEN
OBJECTIVE: Deformability is an essential feature of red blood cells (RBCs), enabling them to undergo significant shape change in response to external forces. Impaired erythrocyte deformability is associated with several pathologic conditions, and quantitative measurement of RBC deformability is critical to understanding and diagnosing RBC related diseases. Whereas traditional approaches to cell mechanical characterization generally have limited throughput, emerging microscale technologies are opening new opportunities for high-throughput deformability cytometry at the single-cell level. METHODS: In this work, we propose an innovative microfluidic system based on (i) a hyperbolic microchannel to induce erythrocyte deformation by extensional flow, and (ii) an electrical sensing zone with coplanar electrodes to evaluate the deformed cell shape. RESULTS: RBC deformation under extensional flow is achieved, and the deformed cell shape is quantified by means of an electrical anisotropy index, at a throughput of 300 cell/s. Measurements of healthy and chemically stiffened RBCs demonstrate that the anisotropy index can be used to characterize RBC deformability, as an alternative to deformation indices based on high-speed image processing. CONCLUSION: A contactless and optics-free approach for RBC deformability analysis has been presented. SIGNIFICANCE: Due to its simplicity and potential for integration, the proposed approach holds promises for fast and low-cost erythrocyte deformability assays, especially in point-of-care and resource-limited settings.
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Deformación Eritrocítica , Eritrocitos , Impedancia Eléctrica , Microfluídica , ElectrodosRESUMEN
Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells in a rapid manner and with single-cell resolution. Impedance measurements are particularly well-suited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in the presence of both cell types.
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Péptidos Catiónicos Antimicrobianos , Péptidos Antimicrobianos , Humanos , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Impedancia Eléctrica , Bacterias , EritrocitosRESUMEN
Microfluidic impedance cytometry is a label-free technique for high-throughput single-cell analysis. Multi-frequency impedance measurements provide data that allows full characterisation of cells, linking electrical phenotype to individual biophysical properties. To efficiently extract the information embedded in the electrical signals, potentially in real-time, tailored signal processing is needed. Artificial intelligence approaches provide a promising new direction. Here we demonstrate the ability of neural networks to decipher impedance cytometry signals in two challenging scenarios: (i) to determine the intrinsic dielectric properties of single cells directly from raw impedance data streams, (ii) to capture single-cell signals that are hidden in the measured signals of coincident cells. The accuracy of the results and the high processing speed (fractions of ms per cell) demonstrate that neural networks can have an important role in impedance-based single-cell analysis.
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Inteligencia Artificial , Microfluídica , Impedancia Eléctrica , Citometría de Flujo/métodos , Redes Neurales de la ComputaciónRESUMEN
OBJECTIVE: In aerobiological monitoring and agriculture there is a pressing need for accurate, label-free and automated analysis of pollen grains, in order to reduce the cost, workload and possible errors associated to traditional approaches. METHODS: We propose a new multimodal approach that combines electrical sensing and optical imaging to classify pollen grains flowing in a microfluidic chip at a throughput of 150 grains per second. Electrical signals and synchronized optical images are processed by two independent machine learning-based classifiers, whose predictions are then combined to provide the final classification outcome. RESULTS: The applicability of the method is demonstrated in a proof-of-concept classification experiment involving eight pollen classes from different taxa. The average balanced accuracy is 78.7% for the electrical classifier, 76.7% for the optical classifier and 84.2% for the multimodal classifier. The accuracy is 82.8% for the electrical classifier, 84.1% for the optical classifier and 88.3% for the multimodal classifier. CONCLUSION: The multimodal approach provides better classification results with respect to the analysis based on electrical or optical features alone. SIGNIFICANCE: The proposed methodology paves the way for automated multimodal palynology. Moreover, it can be extended to other fields, such as diagnostics and cell therapy, where it could be used for label-free identification of cell populations in heterogeneous samples.
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Aprendizaje Automático , Microfluídica , PolenRESUMEN
BACKGROUND: Poor infiltration of functioning T cells renders tumors unresponsive to checkpoint-blocking immunotherapies. Here, we identified a combinatorial in situ immunomodulation strategy based on the administration of selected immunogenic drugs and immunotherapy to sensitize poorly T-cell-infiltrated neuroblastoma (NB) to the host antitumor immune response. METHODS: 975A2 and 9464D NB cell lines derived from spontaneous tumors of TH-MYCN transgenic mice were employed to study drug combinations able of enhancing the antitumor immune response using in vivo and ex vivo approaches. Migration of immune cells towards drug-treated murine-derived organotypic tumor spheroids (MDOTS) were assessed by microfluidic devices. Activation status of immune cells co-cultured with drug-treated MDOTS was evaluated by flow cytometry analysis. The effect of drug treatment on the immune content of subcutaneous or orthotopic tumors was comprehensively analyzed by flow-cytometry, immunohistochemistry and multiplex immunofluorescence. The chemokine array assay was used to detect soluble factors released into the tumor microenvironment. Patient-derived organotypic tumor spheroids (PDOTS) were generated from human NB specimens. Migration and activation status of autologous immune cells to drug-treated PDOTS were performed. RESULTS: We found that treatment with low-doses of mitoxantrone (MTX) recalled immune cells and promoted CD8+ T and NK cell activation in MDOTS when combined with TGFß and PD-1 blockade. This combined immunotherapy strategy curbed NB growth resulting in the enrichment of a variety of both lymphoid and myeloid immune cells, especially intratumoral dendritic cells (DC) and IFNγ- and granzyme B-expressing CD8+ T cells and NK cells. A concomitant production of inflammatory chemokines involved in remodelling the tumor immune landscape was also detected. Interestingly, this treatment induced immune cell recruitment against PDOTS and activation of CD8+ T cells and NK cells. CONCLUSIONS: Combined treatment with low-dose of MTX and anti-TGFß treatment with PD-1 blockade improves antitumor immunity by remodelling the tumor immune landscape and overcoming the immunosuppressive microenvironment of aggressive NB.
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Neuroblastoma , Receptor de Muerte Celular Programada 1 , Humanos , Ratones , Animales , Mitoxantrona/farmacología , Linfocitos T CD8-positivos , Factor de Crecimiento Transformador beta , Línea Celular Tumoral , Neuroblastoma/tratamiento farmacológico , Ratones Transgénicos , Microambiente TumoralRESUMEN
OBJECTIVE: Cell counting and characterization is fundamental for medicine, science and technology. Coulter-type microfluidic devices are effective and automated systems for cell/particle analysis, based on the electrical sensing zone principle. However, their throughput and accuracy are limited by coincidences (i.e., two or more particles passing through the sensing zone nearly simultaneously), which reduce the observed number of particles and may lead to errors in the measured particle properties. In this work, a novel approach for coincidence resolution in microfluidic impedance cytometry is proposed. METHODS: The approach relies on: (i) a microchannel comprising two electrical sensing zones and (ii) a model of the signals generated by coinciding particles. Maximum a posteriori probability (MAP) estimation is used to identify the model parameters and therefore characterize individual particle properties. RESULTS: Quantitative performance assessment on synthetic data streams shows a counting sensitivity of 97% and a positive predictive value of 99% at concentrations of 2×106 particles/ml. An application to red blood cell analysis shows accurate particle characterization up to a throughput of about 2500 particles/s. An original formula providing the expected number of coinciding particles is derived, and good agreement is found between experimental results and theoretical predictions. CONCLUSION: The proposed cytometer enables the decomposition of signals generated by coinciding particles into individual particle contributions, by using a Bayesian approach. SIGNIFICANCE: This system can be profitably used in applications where accurate counting and characterization of cell/particle suspensions over a broad range of concentrations is required.
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Técnicas Analíticas Microfluídicas , Microfluídica , Teorema de Bayes , Impedancia Eléctrica , Eritrocitos , Citometría de Flujo , Dispositivos Laboratorio en un ChipRESUMEN
Oncoimmunology represents a biomedical research discipline coined to study the roles of immune system in cancer progression with the aim of discovering novel strategies to arm it against the malignancy. Infiltration of immune cells within the tumor microenvironment is an early event that results in the establishment of a dynamic cross-talk. Here, immune cells sense antigenic cues to mount a specific anti-tumor response while cancer cells emanate inhibitory signals to dampen it. Animals models have led to giant steps in this research context, and several tools to investigate the effect of immune infiltration in the tumor microenvironment are currently available. However, the use of animals represents a challenge due to ethical issues and long duration of experiments. Organs-on-chip are innovative tools not only to study how cells derived from different organs interact with each other, but also to investigate on the crosstalk between immune cells and different types of cancer cells. In this review, we describe the state-of-the-art of microfluidics and the impact of OOC in the field of oncoimmunology underlining the importance of this system in the advancements on the complexity of tumor microenvironment.
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Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle ''co-cultures/microscopy/advanced data analysis" sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.
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Técnicas de Cocultivo , Técnicas Analíticas Microfluídicas , Microambiente Tumoral/inmunología , Línea Celular Tumoral , Humanos , Leucocitos Mononucleares/inmunologíaRESUMEN
Understanding the interactions between immune and cancer cells occurring within the tumor microenvironment is a prerequisite for successful and personalized anti-cancer therapies. Microfluidic devices, coupled to advanced microscopy systems and automated analytical tools, can represent an innovative approach for high-throughput investigations on immune cell-cancer interactions. In order to study such interactions and to evaluate how therapeutic agents can affect this crosstalk, we employed two ad hoc fabricated microfluidic platforms reproducing advanced 2D or 3D tumor immune microenvironments. In the first type of chip, we confronted the capacity of tumor cells embedded in Matrigel containing one drug or Matrigel containing a combination of two drugs to attract differentially immune cells, by fluorescence microscopy analyses. In the second chip, we investigated the migratory/interaction response of naïve immune cells to danger signals emanated from tumor cells treated with an immunogenic drug, by time-lapse microscopy and automated tracking analysis. We demonstrate that microfluidic platforms and their associated high-throughput computed analyses can represent versatile and smart systems to: (i) monitor and quantify the recruitment and interactions of the immune cells with cancer in a controlled environment, (ii) evaluate the immunogenic effects of anti-cancer therapeutic agents and (iii) evaluate the immunogenic efficacy of combinatorial regimens with respect to single agents.
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Comunicación Celular , Dispositivos Laboratorio en un Chip , Neoplasias/inmunología , Microambiente Tumoral , Animales , Antineoplásicos Inmunológicos/farmacología , Comunicación Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Ensayos de Selección de Medicamentos Antitumorales/métodos , Diseño de Equipo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Neoplasias/tratamiento farmacológico , Microambiente Tumoral/efectos de los fármacosRESUMEN
The study and the characterization of cell death mechanisms are fundamental in cell biology research. Traditional death/viability assays usually involve laborious sample preparation and expensive equipment or reagents. In this work, we use electrical impedance spectroscopy as a label-free methodology to characterize viable, necrotic and apoptotic human lymphoma U937 cells. A simple three-electrode coplanar layout is used in a differential measurement scheme and thousands of cells are measured at high-throughput (≈200 cell/s). Tailored signal processing enables accurate and robust cell characterization without the need for cell focusing systems. The results suggest that, at low frequency (0.5 MHz), signal magnitude enables the discrimination between viable/necrotic cells and cell fragments, whereas phase information allows discriminating between viable cells and necrotic cells. At higher frequency (10 MHz) two subpopulations of cell fragments are distinguished. This work substantiates the prominent role of electrical impedance spectroscopy for the development of next-generation cell viability assays.