Crosstalk and communication of cancer-associated fibroblasts with natural killer and dendritic cells: New frontiers and unveiled opportunities for cancer immunotherapy.

Natural killer (NK) cells and dendritic cells (DCs) are critical mediators of anti-cancer immune responses. In addition to their individual roles, NK cells and DCs are involved in intercellular crosstalk which is essential for the initiation and coordination of adaptive immunity against cancer. However, NK cell and DC activity is often compromised in the tumor microenvironment (TME).

Kinetic Detection of Apoptosis Events Via Caspase 3/7 Activation in a Tumor-Immune Microenvironment on a Chip.

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.

Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry.

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.

Combined mitoxantrone and anti-TGFβ treatment with PD-1 blockade enhances antitumor immunity by remodelling the tumor immune landscape in neuroblastoma.

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.

Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B.

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.

Electro-Optical Classification of Pollen Grains via Microfluidics and Machine Learning.

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.

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments.

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.

Oncoimmunology Meets Organs-on-Chip.

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.

Accelerating the experimental responses on cell behaviors: a long-term prediction of cell trajectories using Social Generative Adversarial Network.

The incremented uptake provided by time-lapse microscopy in Organ-on-a-Chip (OoC) devices allowed increased attention to the dynamics of the co-cultured systems. However, the amount of information stored in long-time experiments may constitute a serious bottleneck of the experimental pipeline. Forward long-term prediction of cell trajectories may reduce the spatial-temporal burden of video sequences storage.

A Bayesian Approach for Coincidence Resolution in Microfluidic Impedance Cytometry.

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.

Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea.

Prior work supports the hypothesis that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge (GER) in the developing cochlea; however, direct proof is lacking. To address this issue, we plated cochlear organotypic cultures (COCs) and whole cell-based biosensors with nM ATP sensitivity (ATP-WCBs) at the bottom and top of an ad hoc designed transparent microfluidic chamber, respectively. By performing dual multiphoton Ca2+ imaging, we monitored the propagation of intercellular Ca2+ waves in the GER of COCs and ATP-dependent Ca2+ responses in overlying ATP-WCBs.

Discovering the hidden messages within cell trajectories using a deep learning approach for in vitro evaluation of cancer drug treatments.

We describe a novel method to achieve a universal, massive, and fully automated analysis of cell motility behaviours, starting from time-lapse microscopy images. The approach was inspired by the recent successes in application of machine learning for style recognition in paintings and artistic style transfer. The originality of the method relies i) on the generation of atlas from the collection of single-cell trajectories in order to visually encode the multiple descriptors of cell motility, and ii) on the application of pre-trained Deep Learning Convolutional Neural Network architecture in order to extract relevant features to be used for classification tasks from this visual atlas.

High-throughput analysis of cell-cell crosstalk in ad hoc designed microfluidic chips for oncoimmunology applications.

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.

High-throughput label-free characterization of viable, necrotic and apoptotic human lymphoma cells in a coplanar-electrode microfluidic impedance chip.

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.

IL-33 Promotes CD11b/CD18-Mediated Adhesion of Eosinophils to Cancer Cells and Synapse-Polarized Degranulation Leading to Tumor Cell Killing.

Eosinophils are major effectors of Th2-related pathologies, frequently found infiltrating several human cancers. We recently showed that eosinophils play an essential role in anti-tumor responses mediated by immunotherapy with the 'alarmin' intereukin-33 (IL-33) in melanoma mouse models. Here, we analyzed the mechanisms by which IL-33 mediates tumor infiltration and antitumor activities of eosinophils.

CaMKK2 in myeloid cells is a key regulator of the immune-suppressive microenvironment in breast cancer.

Tumor-associated myeloid cells regulate tumor growth and metastasis, and their accumulation is a negative prognostic factor for breast cancer. Here we find calcium/calmodulin-dependent kinase kinase (CaMKK2) to be highly expressed within intratumoral myeloid cells in mouse models of breast cancer, and demonstrate that its inhibition within myeloid cells suppresses tumor growth by increasing intratumoral accumulation of effector CD8 T cells and immune-stimulatory myeloid subsets. Tumor-associated macrophages (TAMs) isolated from Camkk2 mice expressed higher levels of chemokines involved in the recruitment of effector T cells compared to WT.

The influence of spatial and temporal resolutions on the analysis of cell-cell interaction: a systematic study for time-lapse microscopy applications.

Cell-cell interactions are an observable manifestation of underlying complex biological processes occurring in response to diversified biochemical stimuli. Recent experiments with microfluidic devices and live cell imaging show that it is possible to characterize cell kinematics via computerized algorithms and unravel the effects of targeted therapies. We study the influence of spatial and temporal resolutions of time-lapse videos on motility and interaction descriptors with computational models that mimic the interaction dynamics among cells.

High-throughput electrical position detection of single flowing particles/cells with non-spherical shape.

We present an innovative impedance cytometer for the measurement of the cross-sectional position of single particles or cells flowing in a microchannel. As predicted by numerical simulations and experimentally validated, the proposed approach is applicable to particles/cells with either spherical or non-spherical shape. In particular, the optics-free high-throughput position detection of individual flowing red blood cells (RBCs) is demonstrated and applied to monitor RBCs hydrodynamic focusing under different sheath flow conditions.

From Petri Dishes to Organ on Chip Platform: The Increasing Importance of Machine Learning and Image Analysis.

The increasing interest for microfluidic devices in medicine and biology has opened the way to new time-lapse microscopy era where the amount of images and their acquisition time will become crucial. In this optic, new data analysis algorithms have to be developed in order to extract novel features of cell behavior and cell-cell interactions. In this brief article, we emphasize the potential strength of a new paradigm arising in the integration of microfluidic devices (i.

A simple electrical approach to monitor dielectrophoretic focusing of particles flowing in a microchannel.

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.

Dissecting Effects of Anti-cancer Drugs and Cancer-Associated Fibroblasts by On-Chip Reconstitution of Immunocompetent Tumor Microenvironments.

A major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2 breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor.

Site-Controlled Single-Photon Emitters Fabricated by Near-Field Illumination.

Many of the most advanced applications of semiconductor quantum dots (QDs) in quantum information technology require a fine control of the QDs' position and confinement potential, which cannot be achieved with conventional growth techniques. Here, a novel and versatile approach for the fabrication of site-controlled QDs is presented. Hydrogen incorporation in GaAsN results in the formation of N-2H and N-2H-H complexes, which neutralize all the effects of N on GaAs, including the N-induced large reduction of the bandgap energy.

Organs on chip approach: a tool to evaluate cancer -immune cells interactions.

In this paper we discuss the applicability of numerical descriptors and statistical physics concepts to characterize complex biological systems observed at microscopic level through organ on chip approach. To this end, we employ data collected on a microfluidic platform in which leukocytes can move through suitably built channels toward their target. Leukocyte behavior is recorded by standard time lapse imaging.