Patient-derived tumoroids and proteomic signatures: tools for early drug discovery.

Onco-virotherapy is an emergent treatment for cancer based on viral vectors. The therapeutic activity is based on two different mechanisms including tumor-specific oncolysis and immunostimulatory properties. In this study, we evaluated onco-virotherapy in vitro responses on immunocompetent non-small cell lung cancer (NSCLC) patient-derived tumoroids (PDTs) and healthy organoids. PDTs are accurate tools to predict patient's clinical responses at the in vitro stage. We showed that onco-virotherapy could exert specific antitumoral effects by producing a higher number of viral particles in PDTs than in healthy organoids. In the present work, we used multiplex protein screening, based on proximity extension assay to highlight different response profiles. Our results pointed to the increase of proteins implied in T cell activation, such as IFN-γ following onco-virotherapy treatment. Based on our observation, oncolytic viruses-based therapy responders are dependent on several factors: a high PD-L1 expression, which is a biomarker of greater immune response under immunotherapies, and the number of viral particles present in tumor tissue, which is dependent to the metabolic state of tumoral cells. Herein, we highlight the use of PDTs as an alternative in vitro model to assess patient-specific responses to onco-virotherapy at the early stage of the preclinical phases.

Using a micro-device with a deformable ceiling to probe stiffness heterogeneities within 3D cell aggregates.

Recent advances in the field of mechanobiology have led to the development of methods to characterise single-cell or monolayer mechanical properties and link them to their functional behaviour. However, there remains a strong need to establish this link for three-dimensional (3D) multicellular aggregates, which better mimic tissue function. Here we present a platform to actuate and observe many such aggregates within one deformable micro-device. The platform consists of a single polydimethylsiloxane piece cast on a 3D-printed mould and bonded to a glass slide or coverslip. It consists of a chamber containing cell spheroids, which is adjacent to air cavities that are fluidically independent. Controlling the air pressure in these air cavities leads to a vertical displacement of the chamber's ceiling. The device can be used in static or dynamic modes over time scales of seconds to hours, with displacement amplitudes from a fewµm to several tens of microns. Further, we show how the compression protocols can be used to obtain measurements of stiffness heterogeneities within individual co-culture spheroids, by comparing image correlations of spheroids at different levels of compression with finite element simulations. The labelling of the cells and their cytoskeleton is combined with image correlation methods to relate the structure of the co-culture spheroid with its mechanical properties at different locations. The device is compatible with various microscopy techniques, including confocal microscopy, which can be used to observe the displacements and rearrangements of single cells and neighbourhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behaviour with the global mechanical response of the aggregates.

In vitro vascularized immunocompetent patient-derived model to test cancer therapies.

This work describes a patient-derived tumoroid model (PDTs) to support precision medicine in lung oncology. The use of human adipose tissue-derived microvasculature and patient-derived peripheral blood mononuclear cells (PBMCs) permits to achieve a physiologically relevant tumor microenvironment. This study involved ten patients at various stages of tumor progression. The vascularized, immune-infiltrated PDT model could be obtained within two weeks, matching the requirements of the therapeutic decision. Histological and transcriptomic analyses confirmed that the main features from the original tumor were reproduced. The 3D tumor model could be used to determine the dynamics of response to antiangiogenic therapy and platinum-based chemotherapy. Antiangiogenic therapy showed a significant decrease in vascular endothelial growth factor (VEGF)-A expression, reflecting its therapeutic effect in the model. In an immune-infiltrated PDT model, chemotherapy showed the ability to decrease the levels of lymphocyte activation gene-3 protein (LAG-3), B and T lymphocyte attenuator (BTLA), and inhibitory receptors of T cells functions.

High resolution microfluidic assay and probabilistic modeling reveal cooperation between T cells in tumor killing.

Cytotoxic T cells are important components of natural anti-tumor immunity and are harnessed in tumor immunotherapies. Immune responses to tumors and immune therapy outcomes largely vary among individuals, but very few studies examine the contribution of intrinsic behavior of the T cells to this heterogeneity. Here we show the development of a microfluidic-based in vitro method to track the outcome of antigen-specific T cell activity on many individual cancer spheroids simultaneously at high spatiotemporal resolution, which we call Multiscale Immuno-Oncology on-Chip System (MIOCS). By combining parallel measurements of T cell behaviors and tumor fates with probabilistic modeling, we establish that the first recruited T cells initiate a positive feedback loop to accelerate further recruitment to the spheroid. We also provide evidence that cooperation between T cells on the spheroid during the killing phase facilitates tumor destruction. Thus, we propose that both T cell accumulation and killing function rely on collective behaviors rather than simply reflecting the sum of individual T cell activities, and the possibility to track many replicates of immune cell-tumor interactions with the level of detail our system provides may contribute to our understanding of immune response heterogeneity.

Cell Culture in Microfluidic Droplets.

Cell manipulation in droplets has emerged as one of the great successes of microfluidic technologies, with the development of single-cell screening. However, the droplet format has also served to go beyond single-cell studies, namely by considering the interactions between different cells or between cells and their physical or chemical environment. These studies pose specific challenges linked to the need for long-term culture of adherent cells or the diverse types of measurements associated with complex biological phenomena. Here we review the emergence of droplet microfluidic methods for culturing cells and studying their interactions. We begin by characterizing the quantitative aspects that determine the ability to encapsulate cells, transport molecules, and provide sufficient nutrients within the droplets. This is followed by an evaluation of the biological constraints such as the control of the biochemical environment and promoting the anchorage of adherent cells. This first part ends with a description of measurement methods that have been developed. The second part of the manuscript focuses on applications of these technologies for cancer studies, immunology, and stem cells while paying special attention to the biological relevance of the cellular assays and providing guidelines on improving this relevance.

Mechanical plasticity in collective cell migration.

Collective cell migration is crucial to maintain epithelium integrity during developmental and repair processes. It requires a tight regulation of mechanical coordination between neighboring cells. This coordination embraces different features including mechanical self-propulsion of individual cells within cellular colonies and large-scale force transmission through cell-cell junctions. This review discusses how the plasticity of biomechanical interactions at cell-cell contacts could help cellular systems to perform coordinated motions and adapt to the properties of the external environment.

The role of single cell mechanical behavior and polarity in driving collective cell migration.

The directed migration of cell collectives is essential in various physiological processes, such as epiboly, intestinal epithelial turnover, and convergent extension during morphogenesis as well as during pathological events like wound healing and cancer metastasis. Collective cell migration leads to the emergence of coordinated movements over multiple cells. Our current understanding emphasizes that these movements are mainly driven by large-scale transmission of signals through adherens junctions. In this study, we show that collective movements of epithelial cells can be triggered by polarity signals at the single cell level through the establishment of coordinated lamellipodial protrusions. We designed a minimalistic model system to generate one-dimensional epithelial trains confined in ring shaped patterns that recapitulate rotational movements observed in vitro in cellular monolayers and in vivo in genitalia or follicular cell rotation. Using our system, we demonstrated that cells follow coordinated rotational movements after the establishment of directed Rac1-dependent polarity over the entire monolayer. Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front-back polarity within each single cell but does not require the maintenance of cell-cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations can arise from single cell behavior through a sustained memory of cell polarity.

Inference of Internal Stress in a Cell Monolayer.

We combine traction force data with Bayesian inversion to obtain an absolute estimate of the internal stress field of a cell monolayer. The method, Bayesian inversion stress microscopy, is validated using numerical simulations performed in a wide range of conditions. It is robust to changes in each ingredient of the underlying statistical model. Importantly, its accuracy does not depend on the rheology of the tissue. We apply Bayesian inversion stress microscopy to experimental traction force data measured in a narrow ring of cohesive epithelial cells, and check that the inferred stress field coincides with that obtained by direct spatial integration of the traction force data in this quasi one-dimensional geometry.

Terpenoid and flavonoid spectrum of in vitro cultures of Glycyrrhiza glabra revealed high chemical heterogeneity: platform to understand biosynthesis.

Simultaneous qualitative and quantitative assessment of eight flavonoids and two terpenoids were performed in fourteen in vitro raised morphogenic cultures of Glycyrrhiza glabra. Our study revealed that the spectrum and production of ten compounds, under investigation, were higher in organized tissue than the undifferentiated mass, however, aerial portions of the in vitro raised plants (leaf and stem) were found to be devoid of therapeutically relevant triterpenoid, glycyrrhizin. A correlation was observed between cell maturation, morphological differentiation and glycyrrhizin accumulation. Mature stolons (4 months) were characterized by the maximum accumulation of glycyrrhizin (8.60 µg/mg) in in vitro plantlets. The cytotoxic effect of the extracts evaluated against a panel of human cancer cell lines (in vitro) indicated that the pancreatic cell line (MIA-PaCa-2) were sensitive to all the fourteen extracts investigated. To the best of our knowledge this is the first comprehensive report relating plant growth regulators to metabolite spectrum and cytotoxic assessment in in vitro raised G. glabra cultures. Overall, our findings demonstrated that the metabolite spectrum of in vitro raised morphogenetic lines, under different stages of maturation, might offer a platform to understand the regulatory aspects of the concerned metabolite pathway and their consequent role in differentiation.