Quantitative screening of the effects of hyper-osmotic stress on cancer cells cultured in 2- or 3-dimensional settings.

The maintenance of precise cell volume is critical for cell survival. Changes in extracellular osmolarity affect cell volume and may impact various cellular processes such as mitosis, mitochondrial functions, DNA repair as well as cell migration and proliferation. Much of what we know about the mechanisms of cell osmoregulation comes from in vitro two-dimensional (2D) assays that are less physiologically relevant than three-dimensional (3D) in vitro or in vivo settings. Here, we developed a microfluidic model to study the impact of hyper-osmotic stress on the migration, proliferation and ion channel/transporter expression changes of three metastatic cell lines (MDA-MB-231, A549, T24) in 2D versus 3D environments. We observed a global decrease in cell migration and proliferation upon hyper-osmotic stress treatment, with similar responses between 2D and 3D conditions. Specific ion channels/aquaporins are over-expressed in metastatic cells and play a central role during osmo-regulation. Therefore, the effects of hyper-osmotic stress on two transporters, aquaporin 5 (AQP5) and the transient receptor potential cation channel (TRPV4), was investigated. While hyper-osmotic stress had no major impact on the transporters of cells cultured in 2D, cells embedded in collagen gel (3D) decreased their AQP5 expression and exhibited a reduction in intra-cellular translocation of TRPV4. Furthermore, cell dispersion from T24 aggregates embedded in 3D collagen gel decreased with higher levels of hyper-osmotic stress. In conclusion, this study provides evidence on the impact of hyper-osmotic stress on various aspects of metastatic cell progression and highlights the importance of having a 3D cell culture platform in investigating molecular players involved in cancer cell migration.

The fate of cells undergoing spontaneous DNA damage during development.

Embryonic development involves extensive and often rapid cell proliferation. An unavoidable side effect of cell proliferation is DNA damage. The consequences of spontaneous DNA damage during development are not clear. Here, we define an approach to determine the effects of DNA damage on cell fate choice. Using single cell transcriptomics, we identified a subpopulation of Dictyostelium cells experiencing spontaneous DNA damage. Damaged cells displayed high expression of rad51, with the gene induced by multiple types of genotoxic stress. Using live imaging, we tracked high Rad51 cells from differentiation onset until cell fate assignment. High Rad51 cells were shed from multicellular structures, excluding damaged cells from the spore population. Cell shedding resulted from impaired cell motility and defective cell-cell adhesion, with damaged cells additionally defective in activation of spore gene expression. These data indicate DNA damage is not insulated from other aspects of cell physiology during development and multiple features of damaged cells prevent propagation of genetic error. Our approach is generally applicable for monitoring rare subpopulations during development, and permits analysis of developmental perturbations occurring within a physiological dynamic range.

Transition state dynamics during a stochastic fate choice.

The generation of multiple fates from a uniform cell population via self-organisation is a recurring feature in development and regeneration. However, for most self-organising systems, we have little understanding of the processes that allow cells to become different. One of the clearest examples of developmental self-organisation is shown by Dictyostelium, with cells segregating into two major fates, stalk and spore, within multicellular aggregates. To characterise the gene expression decisions that underlie this cell fate bifurcation, we carried out single cell transcriptomics on Dictyostelium aggregates. Our data show the transition of progenitors into prespore and prestalk cells occurs via distinct developmental intermediates. Few cells were captured switching between states, with minimal overlap in fate marker expression between cell types, suggesting states are discrete and transitions rapid. Surprisingly, fate-specific transcript dynamics were a small proportion of overall gene expression changes, with transcript divergence coinciding precisely with large-scale remodelling of the transcriptome shared by prestalk and prespore cells. These observations suggest the stepwise separation of cell identity is temporally coupled to global expression transitions common to both fates.

A combined microfluidic-transcriptomic approach to characterize the extravasation potential of cancer cells.

The reciprocal interaction between circulating tumor cells (CTCs) and tissue-specific cells is influential for the progression of metastases. In particular, the process of extravasation relies on the complex cross-talk between cancer cells and other cellular players such as the endothelium and the secondary tissue. However, most in vitro studies only focus on one heterotypic cell-cell interaction and often lack of physiological relevance. In this project, we investigated both CTC-endothelium and CTC-secondary site interactions during cancer cell extravasation. We first used a microarray analysis of extravasated MDA-MB-231 breast cancer cells to identify key markers involved in extravasation. Then, we developed a tri-culture microfluidic platform combining cancer cells, endothelium and a bone-mimicking (BMi) microenvironment to assess how organ tropism influences the extravasation potential of cancer cells from different tissues. Through the microarray analyses of extravasated cancer cells we found that extravasation is associated with upregulation of late-metastatic markers along with specific proteases, such as matrix metalloprotease (MMP), a-disintegrin and metalloprotease (ADAM) and a-disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family members, which are all involved in endothelium glycocalyx shedding. Through the microfluidic extravasation assay, we found that the bone-like microenvironment increased invasion and motility of breast, bladder and ovarian cancer cell (MDA-MB-231, T24 and OVCAR-3). Among the three cell types, ovarian cancer cells presented the lowest migration rate and bladder cancer cells the highest, hence recapitulating their different level of bone tropism observed in vivo. Taken together, our results shed light on the importance of intercellular communication between CTCs and other non-tumor cells essential for promoting cancer cell extravasation.

A 3D microvascular network model to study the impact of hypoxia on the extravasation potential of breast cell lines.

Hypoxia is a common feature of the tumor microenvironment. Accumulating evidence has demonstrated hypoxia to be an important trigger of tumor cell invasion or metastasizes via hypoxia-signaling cascades, including hypoxia-inducible factors (HIFs). Microfluidic model can be a reliable in vitro tool for systematically interrogating individual factors and their accompanying downstream effects, which may otherwise be difficult to study in complex tumor tissues. Here, we used an in vitro model of microvascular networks in a microfluidic chip to measure the extravasation potential of breast cell lines subjected to different oxygen conditions. Through the use of HIF-1α knock-down cell lines, we also validated the importance of HIF-1α in the transmigration ability of human breast cell lines. Three human breast cell lines derived from human breast tissues (MCF10A, MCF-7 and MDA-MB-231) were used in this study to evaluate the role of hypoxia in promoting metastasis at different stages of cancer progression. Under hypoxic conditions, HIF-1α protein level was increased, and coincided with changes in cell morphology, viability and an elevated metastatic potential. These changes were accompanied by an increase in the rate of extravasation compared to normoxia (21% O2). siRNA knockdown of HIF-1α in hypoxic tumors significantly decreased the extravasation rates of all the cell lines tested and may have an effect on the function of metastatic and apoptotic-related cellular processes.

Generation of Single-Cell Transcript Variability by Repression.

Gene expression levels vary greatly within similar cells, even within clonal cell populations [1]. These spontaneous expression differences underlie cell fate diversity in both differentiation and disease [2]. The mechanisms responsible for generating expression variability are poorly understood. Using single-cell transcriptomics, we show that transcript variability emerging during Dictyostelium differentiation is driven predominantly by repression rather than activation. The increased variability of repressed genes was observed over a broad range of expression levels, indicating that variability is actively imposed and not a passive statistical effect of the reduced numbers of molecules accompanying repression. These findings can be explained by a simple model of transcript production, with expression controlled by the frequency, rather than the magnitude, of transcriptional firing events. Our study reveals that the generation of differences between cells can be a direct consequence of the basic mechanisms of transcriptional regulation.

Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity.

Much of development and disease concerns the generation of gene expression differences between related cells sharing similar niches. However, most analyses of gene expression only assess population and time-averaged levels of steady-state transcription. The mechanisms driving differentiation are buried within snapshots of the average cell, lacking dynamic information and the diverse regulatory history experienced by individual cells. Here, we use a quantitative imaging platform with large time series data sets to determine the regulation of developmental gene expression by cell cycle, lineage, motility and environment. We apply this technology to the regulation of the pluripotency gene Nanog in mouse embryonic stem cells. Our data reveal the diversity of cell and population-level interactions with Nanog dynamics and heterogeneity, and how this regulation responds to triggers of pluripotency. Cell cycles are highly heterogeneous and cycle time increases with Nanog reporter expression, with longer, more variable cycle times as cells approach ground-state pluripotency. Nanog reporter expression is highly stable over multiple cell generations, with fluctuations within cycles confined by an attractor state. Modelling reveals an environmental component to expression stability, in addition to any cell-autonomous behaviour, and we identify interactions of cell density with both cycle behaviour and Nanog. Rex1 expression dynamics showed shared and distinct regulatory effects. Overall, our observations of multiple partially overlapping dynamic heterogeneities imply complex cell and environmental regulation of pluripotent cell behaviour, and suggest simple deterministic views of stem cell states are inappropriate.

In silico control of biomolecular processes.

By implementing an external feedback loop one can tightly control the expression of a gene over many cell generations with quantitative accuracy. Controlling precisely the level of a protein of interest will be useful to probe quantitatively the dynamical properties of cellular processes and to drive complex, synthetically-engineered networks. In this chapter we describe a platform for real-time closed-loop control of gene expression in yeast that integrates microscopy for monitoring gene expression at the cell level, microfluidics to manipulate the cells environment, and original software for automated imaging, quantification, and model predictive control. By using an endogenous osmo-stress responsive promoter and playing with the osmolarity of the cells environment, we demonstrate that long-term control can indeed be achieved for both time-constant and time-varying target profiles, at the population level, and even at the single-cell level.

Severe osmotic compression triggers a slowdown of intracellular signaling, which can be explained by molecular crowding.

Regulation of the cellular volume is fundamental for cell survival and function. Deviations from equilibrium trigger dedicated signaling and transcriptional responses that mediate water homeostasis and volume recovery. Cells are densely packed with proteins, and molecular crowding may play an important role in cellular processes. Indeed, increasing molecular crowding has been shown to modify the kinetics of biochemical reactions in vitro; however, the effects of molecular crowding in living cells are mostly unexplored. Here, we report that, in yeast, a sudden reduction in cellular volume, induced by severe osmotic stress, slows down the dynamics of several signaling cascades, including the stress-response pathways required for osmotic adaptation. We show that increasing osmotic compression decreases protein mobility and can eventually lead to a dramatic stalling of several unrelated signaling and cellular processes. The rate of these cellular processes decreased exponentially with protein density when approaching stalling osmotic compression. This suggests that, under compression, the cytoplasm behaves as a soft colloid undergoing a glass transition. Our results shed light on the physical mechanisms that force cells to cope with volume fluctuations to maintain an optimal protein density compatible with cellular functions.

Long-term model predictive control of gene expression at the population and single-cell levels.

Gene expression plays a central role in the orchestration of cellular processes. The use of inducible promoters to change the expression level of a gene from its physiological level has significantly contributed to the understanding of the functioning of regulatory networks. However, from a quantitative point of view, their use is limited to short-term, population-scale studies to average out cell-to-cell variability and gene expression noise and limit the nonpredictable effects of internal feedback loops that may antagonize the inducer action. Here, we show that, by implementing an external feedback loop, one can tightly control the expression of a gene over many cell generations with quantitative accuracy. To reach this goal, we developed a platform for real-time, closed-loop control of gene expression in yeast that integrates microscopy for monitoring gene expression at the cell level, microfluidics to manipulate the cells' environment, and original software for automated imaging, quantification, and model predictive control. By using an endogenous osmostress responsive promoter and playing with the osmolarity of the cells environment, we show that long-term control can, indeed, be achieved for both time-constant and time-varying target profiles at the population and even the single-cell levels. Importantly, we provide evidence that real-time control can dynamically limit the effects of gene expression stochasticity. We anticipate that our method will be useful to quantitatively probe the dynamic properties of cellular processes and drive complex, synthetically engineered networks.

The Dynamical Systems Properties of the HOG Signaling Cascade.

The High Osmolarity Glycerol (HOG) MAP kinase pathway in the budding yeast Saccharomyces cerevisiae is one of the best characterized model signaling pathways. The pathway processes external signals of increased osmolarity into appropriate physiological responses within the yeast cell. Recent advances in microfluidic technology coupled with quantitative modeling, and techniques from reverse systems engineering have allowed yet further insight into this already well-understood pathway. These new techniques are essential for understanding the dynamical processes at play when cells process external stimuli into biological responses. They are widely applicable to other signaling pathways of interest. Here, we review the recent advances brought by these approaches in the context of understanding the dynamics of the HOG pathway signaling.