3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches

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 micro uidic platform in which leukocytes can move through suitably built channels toward their target. Leukocyte behavior is recorded by standard time lapse imaging. In particular, we analyze three groups of human peripheral blood mononuclear cells (PBMC): heterozygous mutants (in which only one copy of the FPR1 gene is normal), homozygous mutants (in which both alleles encoding FPR1 are loss-of-function variants) and cells from ‘wild type’ donors (with normal expression of FPR1). We characterize the migration of these cells providing a quantitative con rmation of the essential role of FPR1 in cancer chemotherapy response. Indeed wild type PBMC perform biased random walks toward chemotherapy-treated cancer cells establishing persistent interactions with them. Conversely, heterozygous mutants present a weaker bias in their motion and homozygous mutants perform rather uncorrelated random walks, both failing to engage with their targets. We next focus on wild type cells and study the interactions of leukocytes with cancerous cells developing a novel heuristic procedure, inspired by Lyapunov stability in dynamical systems

Numerical and experimental characterization of a piezoelectric actuator for microfluidic cell sorting

Piezoelectric actuators offer great opportunities for precise and low-cost control of fluids at the microscale. Microfluidic systems with integrated piezoelectric actuators find application as droplet generators, micropumps, and microsorters. To accelerate device design and optimization, modeling and simulation approaches represent an attractive tool, but there are challenges arising from the multiphysics nature of the problem. Simple, potentially real-time approaches to experimentally characterize the fluid response to piezoelectric actuation are also highly desirable. In this work, we propose a strategy for the numerical and experimental characterization of a piezoelectric microfluidic cell sorter. Specifically, we present a 3D coupled multiphysics finite-element model of the system and an easy image-based approach for flow monitoring. Sinusoidal and pulse actuation are considered as case studies to test the proposed methodology. The results demonstrate the validity of the approach as well as the suitability of the system for cell sorting applications

Spontaneous Interfacial Fragmentation of Inkjet Printed Oil Droplets and Their electrical characterization

This work presents the fabrication of femtoliter-scale oil droplets by inkjet printing based on a novel mechanism for the spontaneous fragmentation at the interface with an immiscible water phase and the electrical characterization of the resulting immersed “daughter” droplets. [1] In particular, picoliter-scale fluorinated oil droplets impact on surfactant laden water phase at moderately high Weber number (101), and are subjected to spreading and capillary instabilities at the water/air interface which ultimately lead to rupture in smaller sized droplets, according to reported models for macroscale droplets systems - [2] the emerging fragmentation results in “daughter” droplets having volumes of about 10-30 % with respect to the initial droplet volume. Remarkably, the picoliter scale downscaling leads to a novel surfactant-driven fragmentation due to the low Bond number (around 10-4-10-5), meaning that droplet immersion is dependent on surface tension forces and not on gravitational forces. In fact, the non-ionic Polyoxyethylene (20) sorbitan monolaurate was observed to permit the droplet immersion in the water phase only if spiked in the water phase at concentrations equal or higher than its critical micellar concentration (i.e. around 0.003% v/v). The resulting oil “daughter” droplets are characterized by a chip with integrated microelectrodes, permitting to extract number, velocities and diameter distribution (peaked at about 3 m) employing electrical impedance measurements. In accordance with reported models, the electrical characterizations show that the droplets have volumes in the femtoliter scale and are subjected to inertial focusing. [3] This work can be considered an important advancement for understanding the effects of downscaling on fragmentation phenomena at immiscible interfaces, leading to a knowledge platform for a tailored oil droplets fabrication applicable for drug encapsulation, pharmaceutic preparations, and thin-film wrapping around droplets. [4] Bibliography 1. D. Spencer, F. Caselli, P. Bisegna and H. Morgan., Lab Chip, 2016, 16, 2467. 2. H. Lhuissier, C. Sun, A. Prosperetti, and D. Lohse, Phys. Rev. Lett., 2013, 110, 3. G. Arrabito, V. Errico, A. De Ninno, F. Cavaleri, V. Ferrara, B. Pignataro, and F.Caselli, Langmuir, 2019, 35, 4936. 4. D. Kumar, J. D. Paulsen, T. P. Russell, N. Menon, Science, 2018, 359, 775

On the compatibility of single-cell microcarriers (nanovials) with microfluidic impedance cytometry

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

Cancer-driven dynamics of immune cells in a microfluidic environment

Scope of the present work is to frame into a rigorous, quantitative scaffold - stemmed from stochastic process theory - two sets of experiments designed to infer the spontaneous organization of leukocytes against cancer cells, namely mice splenocytes vs. B16 mouse tumor cells, and embedded in an "ad hoc" microfluidic environment developed on a LabOnChip technology. In the former, splenocytes from knocked out (KO) mice engineered to silence the transcription factor IRF-8, crucial for the development and function of several immune populations, were used. In this case lymphocytes and cancer cells exhibited a poor reciprocal exchange, resulting in the inability of coordinating or mounting an effective immune response against melanoma. In the second class of tests, wild type (WT) splenocytes were able to interact with and to coordinate a response against the tumor cells through physical interaction. The environment where cells moved was built of by two different chambers, containing respectively melanoma cells and splenocytes, connected by capillary migration channels allowing leucocytes to migrate from their chamber toward the melanoma one. We collected and analyzed data on the motility of the cells and found that the first ensemble of IRF-8 KO cells performed pure uncorrelated random walks, while WT splenocytes were able to make singular drifted random walks, that, averaged over the ensemble of cells, collapsed on a straight ballistic motion for the system as a whole. At a finer level of investigation, we found that IRF-8 KO splenocytes moved rather uniformly since their step lengths were exponentially distributed, while WT counterpart displayed a qualitatively broader motion as their step lengths along the direction of the melanoma were log-normally distributed

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

Antimicrobial peptides (AMPs) represent a promising classof compoundsto fight antibiotic-resistant infections. In most cases, they killbacteria by making their membrane permeable and therefore exhibitlow propensity to induce bacterial resistance. In addition, they areoften selective, killing bacteria at concentrations lower than thoseat which they are toxic to the host. However, clinical applicationsof AMPs are hindered by a limited understanding of their interactionswith bacteria and human cells. Standard susceptibility testing methodsare based on the analysis of the growth of a bacterial populationand therefore require several hours. Moreover, different assays arerequired to assess the toxicity to host cells. In this work, we proposethe use of microfluidic impedance cytometry to explore the actionof AMPs on both bacteria and host cells in a rapid manner and withsingle-cell resolution. Impedance measurements are particularly well-suitedto detect the effects of AMPs on bacteria, due to the fact that themechanism of action involves perturbation of the permeability of cellmembranes. 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-PMAP23bactericidal activity and toxicity to RBCs. The impedance-based characterizationis validated by comparison with standard antibacterial activity assaysand absorbance-based hemolytic activity assays. Furthermore, we demonstratethe applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the wayto study AMP selectivity for bacterial versus eukaryotic cells inthe presence of both cell types

A multidisciplinary study using in vivo tumor models and microfluidic cell-on-chip approach to explore the cross-talk between cancer and immune cells

A full elucidation of events occurring inside the cancer microenvironment is fundamental for the optimization of more effective therapies. In the present study, the cross-talk between cancer and immune cells was examined by employing mice deficient (KO) in interferon regulatory factor (IRF)-8, a transcription factor essential for induction of competent immune responses. The in vivo results showed that IRF-8 KO mice were highly permissive to B16.F10 melanoma growth and metastasis due to failure of their immune cells to exert proper immunosurveillance. These events were found to be dependent on soluble factors released by cells of the immune system capable of shaping the malignant phenotype of melanoma cells. An on-chip model was then generated to further explore the reciprocal interactions between the B16.F10 and immune cells. B16.F10 and immune cells were co-cultured in a microfluidic device composed of three culturing chambers suitably inter-connected by an array of microchannels; mutual interactions were then followed using time-lapse microscopy. It was observed that WT immune cells migrated through the microchannels towards the B16.F10 cells, establishing tight interactions that in turn limited tumor spread. In contrast, IRF-8 KO immune cells poorly interacted with the melanoma cells, resulting in a more invasive behavior of the B16.F10 cells. These results suggest that IRF-8 expression plays a key role in the cross-talk between melanoma and immune cells, and under-score the value of cell-on-chip approaches as useful in vitro tools to reconstruct complex in vivo microenvironments on a microscale level to explore cell interactions such as those occurring within a cancer immunoenvironment

Estimation Algorithm for a Hybrid PDE–ODE Model Inspired by Immunocompetent Cancer-on-Chip Experiment

The present work is motivated by the development of a mathematical model mimicking the mechanisms observed in lab-on-chip experiments, made to reproduce on microfluidic chips the in vivo reality. Here we consider the Cancer-on-Chip experiment where tumor cells are treated with chemotherapy drug and secrete chemical signals in the environment attracting multiple immune cell species. The in silico model here proposed goes towards the construction of a “digital twin” of the experimental immune cells in the chip environment to better understand the complex mechanisms of immunosurveillance. To this aim, we develop a tumor-immune microfluidic hybrid PDE–ODE model to describe the concentration of chemicals in the Cancer-on-Chip environment and immune cells migration. The development of a trustable simulation algorithm, able to reproduce the immunocompetent dynamics observed in the chip, requires an efficient tool for the calibration of the model parameters. In this respect, the present paper represents a first methodological work to test the feasibility and the soundness of the calibration technique here proposed, based on a multidimensional spline interpolation technique for the time-varying velocity field surfaces obtained from cell trajectories