Assessing personalized responses to anti-PD-1 treatment using patient-derived lung tumor-on-chip.

There is a compelling need for approaches to predict the efficacy of immunotherapy drugs. Tumor-on-chip technology exploits microfluidics to generate 3D cell co-cultures embedded in hydrogels that recapitulate simplified tumor ecosystems. Here, we present the development and validation of lung tumor-on-chip platforms to quickly and precisely measure ex vivo the effects of immune checkpoint inhibitors on T cell-mediated cancer cell death by exploiting the power of live imaging and advanced image analysis algorithms. The integration of autologous immunosuppressive FAP+ cancer-associated fibroblasts impaired the response to anti-PD-1, indicating that tumors-on-chips are capable of recapitulating stroma-dependent mechanisms of immunotherapy resistance. For a small cohort of non-small cell lung cancer patients, we generated personalized tumors-on-chips with their autologous primary cells isolated from fresh tumor samples, and we measured the responses to anti-PD-1 treatment. These results support the power of tumor-on-chip technology in immuno-oncology research and open a path to future clinical validations.

A continuum model for magnetic particle flows in microfluidics applicable from dilute to packed suspensions.

The manipulation of magnetic microparticles has always been pivotal in the development of microfluidic devices, as it encompasses a broad range of applications, such as drug delivery, bioanalysis, on-chip diagnostics, and more recently organ-on-chip development. However, predicting the behavior and trajectory of these particles remains a recurring and partly unresolved question. Magnetic particle-laden flows can display intricate collective behaviors, such as packed plugs, column-shaped aggregates, or fluidization, which are difficult to predict. In this study, we introduce a finite-element model to simulate highly dense flows of magnetic microparticles. Our method relies on an interpenetrating continuum approach, where both the liquid and particle phases are described by the Navier-Stokes equations, in which the magnetic force, interphase friction, and interparticle forces were included. We demonstrate its applicability across the entire range of particle packing densities and compare the results with experimental data from real microfluidic application cases. The model successfully replicates complex behaviors, such as particle aggregation, plug formation and fluidization. This approach has potential to accelerate microfluidic device development by reducing the need for costly and time-consuming experimental optimization.

Cancer-associated fibroblasts actively compress cancer cells and modulate mechanotransduction.

During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce an excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule acts as a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP and a decrease in proliferation. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.

Multiomic Droplet-Based Assay for Ultralow Input Samples.

The extraction and separation of cellular compounds are crucial steps in numerous biological protocols, particularly in multiomics studies, where several cellular modalities are examined simultaneously. While magnetic particle extraction is commonly used, it may not be applicable for ultralow input samples. Microfluidics has made possible the analysis of rare or low-materiality samples such as circulating tumor cells or single cells through miniaturization of numerous protocols. In this study, a microfluidics workflow for separating different cellular modalities from ultralow input samples is presented. This approach is based on magnetic tweezers technology, allowing the extraction and resuspension of magnetic particles between consecutive nanoliter droplets to perform multistep assays on small volumes. The ability to separate and recover mRNA and gDNA in samples containing less than 10 cells is demonstrated, achieving separation efficiency comparable to the one obtained with conventional pipetting but with a significantly lower amount of starting material, typically 1-2 orders of magnitude less.

Tunnelling nanotube formation is driven by Eps8/IRSp53-dependent linear actin polymerization.

Tunnelling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy, and optical tweezer-based approaches, we demonstrate that TNTs formed through the outward extension of actin achieve distances greater than the mean length of filopodia and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, thus limiting their occurrence. Proteomic analysis using epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that, upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form tunnelling nanotubes, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour tunnelling nanotube formation.

High-throughput extraction on a dynamic solid phase for low-abundance biomarker isolation from biological samples.

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum.

Bridging the gap between tumor-on-chip and clinics: a systematic review of 15 years of studies.

Over the past 15 years, the field of oncology research has witnessed significant progress in the development of new cell culture models, such as tumor-on-chip (ToC) systems. In this comprehensive overview, we present a multidisciplinary perspective by bringing together physicists, biologists, clinicians, and experts from pharmaceutical companies to highlight the current state of ToC research, its unique features, and the challenges it faces. To offer readers a clear and quantitative understanding of the ToC field, we conducted an extensive systematic analysis of more than 300 publications related to ToC from 2005 to 2022. ToC offer key advantages over other in vitro models by enabling precise control over various parameters. These parameters include the properties of the extracellular matrix, mechanical forces exerted on cells, the physico-chemical environment, cell composition, and the architecture of the tumor microenvironment. Such fine control allows ToC to closely replicate the complex microenvironment and interactions within tumors, facilitating the study of cancer progression and therapeutic responses in a highly representative manner. Importantly, by incorporating patient-derived cells or tumor xenografts, ToC models have demonstrated promising results in terms of clinical validation. We also examined the potential of ToC for pharmaceutical industries in which ToC adoption is expected to occur gradually. Looking ahead, given the high failure rate of clinical trials and the increasing emphasis on the 3Rs principles (replacement, reduction, refinement of animal experimentation), ToC models hold immense potential for cancer research. In the next decade, data generated from ToC models could potentially be employed for discovering new therapeutic targets, contributing to regulatory purposes, refining preclinical drug testing and reducing reliance on animal models.

Construction of a Multitubular Perfusable Kidney-on-Chip for the Study of Renal Diseases.

The organ-on-chip model offers versatility and modularity of in vitro models while approaching the biological fidelity of in vivo models. We propose a method to build a perfusable kidney-on-chip aiming at reproducing key features of the densely packed segments of nephrons in vitro; such as their geometry, their extracellular matrix, and their mechanical properties. The core of the chip is made of parallel tubular channels molded into collagen I that are as small as 80 µm in diameter and as close as 100 µm apart. These channels can further be coated with basement membrane components and seeded by perfusion of a suspension of cells originating from a given segment of the nephron. We optimized the design of our microfluidic device to achieve high reproducibility regarding the seeding density of the channels and high fluidic control of the channels. This chip was designed as a versatile tool to study nephropathies in general, contributing to building ever better in vitro models. It could be particularly interesting for pathologies such as polycystic kidney diseases where mechanotransduction of the cells and their interaction with adjacent extracellular matrix and nephrons may play a key role.

On-line dual-stage enrichment via magneto-extraction and electrokinetic preconcentration: A new concept and instrumentation for capillary electrophoresis.

This study reports on the development of a new concept of on-line dual preconcentration stages for capillary electrophoresis (CE), in which two completely different preconcentration approaches can be realized in the same capillary. In the first stage, a dynamic magneto-extraction of target analytes on circulating magnetic beads is implemented within the capillary. In the second one, electrokinetic preconcentration of eluted analytes via large volume sample stacking is carried out to focus them into a nano band, prior to CE separation of enriched analytes. To implement the dual-stage preconcentration operation, a purpose-made instrument was designed, combining electrophoretic and microfluidic modules to allow precise control of the movement of magnetic beads and analyte's flow. The potential of this new enrichment principle and its associated instrument was demonstrated for CE separation with light-emitting-diode-induced fluorescent (LEDIF) detection of target double-stranded DNA (ds-DNA). The workflow consists of purification and preconcentration of a target DNA fragment (300 bp) on negatively charged magnetic beads, followed by in-capillary elution and fluorescent labelling of the enriched DNA. Large volume sample stacking of the DNA eluent was then triggered to further preconcentrate the labelled DNA before its analysis by CE-LEDIF. An enrichment factor of 125 was achieved for the target DNA fragment. With our new approach, dual-stage sample pretreatment and CE separation can now be performed in-capillary without any mismatch of working volumes, nor any waste of pretreated samples.

Correction: Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions.

Correction for 'Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions' by Marine Verhulsel et al., Lab Chip, 2021, 21, 365-377, https://doi.org/10.1039/d0lc00672f.

Correction to 'Mathematical modelling of oxygen transport in a muscle-on-chip device' (2022) by Hardman et al.

[This corrects the article DOI: 10.1098/rsfs.2022.0020.][This corrects the article DOI: 10.1098/rsfs.2022.0020.].

Precise and fast control of the dissolved oxygen level for tumor-on-chip.

In vitro cell cultures are most often performed in unphysiological hyperoxia since the oxygen partial pressure of conventional incubators is set at 141 mmHg (18.6%, close to ambient air oxygen 20.1%). This value is higher than human tissue oxygen levels, as the in vivo oxygen partial pressures range from 104 mmHg (lung alveoli) to 8 mmHg (skin epidermis). Importantly, under pathological conditions such as cancer, cells can experience oxygen pressure lower than the healthy tissue. Although hypoxic incubators can regulate gas oxygen, they do not take into account the dissolved oxygen concentration in the cell culture medium. In the context of organ on chip and micro-physiological system development, we present here a new system, called Oxalis (OXygen ALImentation System) that allows fine control of the dissolved oxygen level in the cell culture medium. Oxalis regulates simultaneously the gas composition and the inlet reservoir pressure by modulating the pneumatic valve opening. This dual regulation allows both the pressure driven liquid flowrate and the level of oxygen dissolved in the chip to be controlled independently. Oxalis offers unprecedented features such as an oxygen equilibration time lower than 3 minutes and an accuracy of 3 mmHg. These performances can be reached for chip perfusion flow as low as 1 µL min-1. This low flow rate allows the shear stress experienced by the cells in the chip to be accurately controlled. In addition, the system enables modulation of the pH in the cell culture medium through the modulation of CO2. The fine control and monitoring of both O2 and pH pave the way for new precise investigations on physiological and pathological biological processes. Using Oxalis in the context of tumor-on-chip, we demonstrate the capacity of the system to recapitulate hypoxia-induced gene expression, offering an innovative strategy for future studies on the role of hypoxia in malignant progression and drug resistance.

Mathematical modelling of oxygen transport in a muscle-on-chip device.

Muscle-on-chip devices aim to recapitulate the physiological characteristics of in vivo muscle tissue and so maintaining levels of oxygen transported to cells is essential for cell survival and for providing the normoxic conditions experienced in vivo. We use finite-element method numerical modelling to describe oxygen transport and reaction in a proposed three-dimensional muscle-on-chip bioreactor with embedded channels for muscle cells and growth medium. We determine the feasibility of ensuring adequate oxygen for muscle cell survival in a device sealed from external oxygen sources and perfused via medium channels. We investigate the effects of varying elements of the bioreactor design on oxygen transport to optimize muscle tissue yield and maintain normoxic conditions. Successful co-culturing of muscle cells with motor neurons can boost muscle tissue function and so we estimate the maximum density of seeded neurons supported by oxygen concentrations within the bioreactor. We show that an enclosed bioreactor can provide sufficient oxygen for muscle cell survival and growth. We define a more efficient arrangement of muscle and perfusion chambers that can sustain a predicted 50% increase in maximum muscle volume per perfusion vessel. A study of simulated bioreactors provides functions for predicting bioreactor designs with normoxic conditions for any size of perfusion vessel, muscle chamber and distance between chambers.

Direct imaging and automatic analysis in tumor-on-chip reveal cooperative antitumoral activity of immune cells and oncolytic vaccinia virus.

Organ-on-chip and tumor-on-chip microfluidic cell cultures represent a fast-growing research field for modelling organ functions and diseases, for drug development, and for promising applications in personalized medicine. Still, one of the bottlenecks of this technology is the analysis of the huge amount of bio-images acquired in these dynamic 3D microenvironments, a task that we propose to achieve by exploiting the interdisciplinary contributions of computer science and electronic engineering. In this work, we apply this strategy to the study of oncolytic vaccinia virus (OVV), an emerging agent in cancer immunotherapy. Infection and killing of cancer cells by OVV were recapitulated and directly imaged in tumor-on-chip. By developing and applying appropriate image analysis strategies and advanced automatic algorithms, we uncovered synergistic cooperation of OVV and immune cells to kill cancer cells. Moreover, we observed that the kinetics of immune cells were modified in presence of OVV and that these immune modulations varied during the course of infection. A correlation between cancer cell infection and cancer-immune interaction time was pointed out, strongly supporting a cause-effect relationship between infection of cancer cells and their recognition by the immune cells. These results shed new light on the mode of action of OVV, and suggest new clinical avenues for immunotherapy developments.

Modular microfluidic system for on-chip extraction, preconcentration and detection of the cytokine biomarker IL-6 in biofluid.

The cytokine interleukin 6 (IL-6) is involved in the pathogenesis of different inflammatory diseases, including cancer, and its monitoring could help diagnosis, prognosis of relapse-free survival and recurrence. Here, we report an innovative microfluidic approach that uses the fluidization of magnetic beads to specifically extract, preconcentrate and fluorescently detect IL-6 directly on-chip. We assess how the physical properties of the beads can be tuned to improve assay performance by enhancing mass transport, reduce non-specific binding and multiply the detection signal threefold by transitioning between packed and fluidization states. With the integration of a full ELISA protocol in a single microfluidic chamber, we show a twofold reduction in LOD compared to conventional methods along with a large dynamic range (10 pg/mL to 2 ng/mL). We additionally demonstrate its application to IL-6 detection in undiluted serum samples.

Deciphering HER2-HER3 Dimerization at the Single CTC Level: A Microfluidic Approach.

Microfluidics has provided clinicians with new technologies to detect and analyze circulating tumor biomarkers in order to further improve their understanding of disease mechanism, as well as to improve patient management. Among these different biomarkers, circulating tumor cells have proven to be of high interest for different types of cancer and in particular for breast cancer. Here we focus our attention on a breast cancer subtype referred as HER2-positive breast cancer, this cancer being associated with an amplification of HER2 protein at the plasma membrane of cancer cells. Combined with therapies targeting the HER2 protein, HER2-HER3 dimerization blockade further improves a patient's outcome. In this work, we propose a new approach to CTC characterization by on-chip integrating proximity ligation assay, so that we can quantify the HER2-HER3 dimerization event at the level of single CTC. To achieve this, we developed a microfluidic approach combining both CTC capture, identification and HER2-HER3 status quantification by Proximity Ligation Assay (PLA). We first optimized and demonstrated the potential of the on-chip quantification of HER2-HER3 dimerization using cancer cell lines with various levels of HER2 overexpression and validated its clinical potential with a patient's sample treated or not with HER2-targeted therapy.

A Hybrid In Silico and Tumor-on-a-Chip Approach to Model Targeted Protein Behavior in 3D Microenvironments.

To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm2·s-1 and 2 µm2 ·s-1 for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models.

A Multitubular Kidney-on-Chip to Decipher Pathophysiological Mechanisms in Renal Cystic Diseases.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major renal pathology provoked by the deletion of PKD1 or PKD2 genes leading to local renal tubule dilation followed by the formation of numerous cysts, ending up with renal failure in adulthood. In vivo, renal tubules are tightly packed, so that dilating tubules and expanding cysts may have mechanical influence on adjacent tubules. To decipher the role of this coupling between adjacent tubules, we developed a kidney-on-chip reproducing parallel networks of tightly packed tubes. This original microdevice is composed of cylindrical hollow tubes of physiological dimensions, parallel and closely packed with 100-200 µm spacing, embedded in a collagen I matrix. These multitubular systems were properly colonized by different types of renal cells with long-term survival, up to 2 months. While no significant tube dilation over time was observed with Madin-Darby Canine Kidney (MDCK) cells, wild-type mouse proximal tubule (PCT) cells, or with PCT Pkd1 +/- cells (with only one functional Pkd1 allele), we observed a typical 1.5-fold increase in tube diameter with isogenic PCT Pkd1 -/- cells, an ADPKD cellular model. This tube dilation was associated with an increased cell proliferation, as well as a decrease in F-actin stress fibers density along the tube axis. With this kidney-on-chip model, we also observed that for larger tube spacing, PCT Pkd1 -/- tube deformations were not spatially correlated with adjacent tubes whereas for shorter spacing, tube deformations were increased between adjacent tubes. Our device reveals the interplay between tightly packed renal tubes, constituting a pioneering tool well-adapted to further study kidney pathophysiology.

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy.

The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing this aim within reach. Here, we developed a mathematical model specifically built for the evaluation of approaches towards cytosolic protein delivery, involving all steps from systemic administration to translocation into the cytosol and target engagement. Focusing on solid cancer tissues, we utilized the model to investigate the effects of microvascular permeability, receptor affinity, the cellular density of targeted receptors, as well as the mode of activity (blocking/degradation) on therapeutic potential. Our analyses provide guidance for the rational optimization of protein design for enhanced activity and highlight the importance of tuning the receptor affinity as a function of receptor density as well as the receptor internalization rate. Furthermore, we provide quantitative insights into how enzymatic cargoes can enhance the distribution, extent, and duration of therapeutic activity, already at very low catalytic rates. Our results illustrate that with current protein engineering approaches, the goal of delivery of cytosolic delivery of proteins for therapeutic effects is well within reach.

Apoptosis mapping in space and time of 3D tumor ecosystems reveals transmissibility of cytotoxic cancer death.

The emerging tumor-on-chip (ToC) approaches allow to address biomedical questions out of reach with classical cell culture techniques: in biomimetic 3D hydrogels they partially reconstitute ex vivo the complexity of the tumor microenvironment and the cellular dynamics involving multiple cell types (cancer cells, immune cells, fibroblasts, etc.). However, a clear bottleneck is the extraction and interpretation of the rich biological information contained, sometime hidden, in the cell co-culture videos. In this work, we develop and apply novel video analysis algorithms to automatically measure the cytotoxic effects on human cancer cells (lung and breast) induced either by doxorubicin chemotherapy drug or by autologous tumor-infiltrating cytotoxic T lymphocytes (CTL). A live fluorescent dye (red) is used to selectively pre-stain the cancer cells before co-cultures and a live fluorescent reporter for caspase activity (green) is used to monitor apoptotic cell death. The here described open-source computational method, named STAMP (spatiotemporal apoptosis mapper), extracts the temporal kinetics and the spatial maps of cancer death, by localizing and tracking cancer cells in the red channel, and by counting the red to green transition signals, over 2-3 days. The robustness and versatility of the method is demonstrated by its application to different cell models and co-culture combinations. Noteworthy, this approach reveals the strong contribution of primary cancer-associated fibroblasts (CAFs) to breast cancer chemo-resistance, proving to be a powerful strategy to investigate intercellular cross-talks and drug resistance mechanisms. Moreover, we defined a new parameter, the 'potential of death induction', which is computed in time and in space to quantify the impact of dying cells on neighbor cells. We found that, contrary to natural death, cancer death induced by chemotherapy or by CTL is transmissible, in that it promotes the death of nearby cancer cells, suggesting the release of diffusible factors which amplify the initial cytotoxic stimulus.