Within the tumor microenvironment (TME), cancer cells use mechanotransduction pathways to convert biophysical forces to biochemical signals. However, the underlying mechanisms and functional... Show moreWithin the tumor microenvironment (TME), cancer cells use mechanotransduction pathways to convert biophysical forces to biochemical signals. However, the underlying mechanisms and functional significance of these pathways remain largely unclear. The upregulation of mechanosensitive pathways from biophysical forces such as interstitial flow (IF), leads to the activation of various cytokines, including transforming growth factor-β (TGF-β). TGF-β promotes in part via a Smad-dependent signaling pathway the epithelial–mesenchymal transition (EMT) in cancer cells. The latter process is linked to increased cancer cell motility and invasion. Current research models have limited ability to investigate the combined effects of biophysical forces (such as IF) and cytokines (TGF-β) in a 3D microenvironment. We used a 3D-matrix based microfluidic platform to demonstrate the potentiating effect of IF on exogenous TGF-β induced upregulation of the Smad-signaling activity and the expression of mesenchymal marker vimentin in A549 lung cancer spheroids. To monitor this, we used stably integrated fluorescent based reporters into the A549 cancer cell genome. Our results demonstrate that IF enhances exogenous TGF-β induced Smad-signaling activity in lung cancer spheroids embedded in a matrix microenvironment. In addition, we observed an increased cell motility for A549 spheroids when exposed to IF and TGF-β. Our 3D-microfluidic model integrated with real-time imaging provides a powerful tool for investigating cancer cell signaling and motility associated with invasion characteristics in a physiologically relevant TME. Show less
Mehta, P.; Rahman, Z.; Dijke, P. ten; Boukany, P.E. 2022
An early step of metastasis requires a complex and coordinated migration of invasive tumor cells into the surrounding tumor microenvironment (TME), which contains extracellular matrix (ECM). It is... Show moreAn early step of metastasis requires a complex and coordinated migration of invasive tumor cells into the surrounding tumor microenvironment (TME), which contains extracellular matrix (ECM). It is being appreciated that 3D matrix -based microfluidic models have an advantage over conventional in vitro and animal models to study tumor progression events. Recent microfluidic models have enabled recapitulation of key mechanobiological features present within the TME to investigate collective cancer cell migration and invasion. Microfluidics also allows for functional interrogation and therapeutic manipulation of specific steps to study the dynamic aspects of tumor progression. In this review, we focus on recent developments in cancer cell migration and how microfluidic strategies have evolved to address the physiological complexities of the TME to visualize migration modes adapted by various tumor cells. Show less
In the research of cancer cell invasion and metastasis, recreation of physiologically relevant and faithful three-dimensional (3D) tumor models that recapitulate spatial architecture,... Show moreIn the research of cancer cell invasion and metastasis, recreation of physiologically relevant and faithful three-dimensional (3D) tumor models that recapitulate spatial architecture, spatiotemporal control of cell communication and signaling pathways, and integration of extracellular cues remains an open challenge. Here, a programmable multifunctional 3D cancer cell invasion microbuckets-hydrogel (Mb-H) platform is developed by integrating various function-variable microbuckets and extracellular matrix (ECM)-like hydrogels. Based on this Mb-H micro platform, the aggregation of multi-cancer cells is well controlled to form cancer cell spheroids, and the guiding relationship of single-cell migration and collective cell migration during the epithelial-mesenchymal transition (EMT) of cancer cell invasion are demonstrated. By programming and precisely assembling multiple functions in one system, the Mb-H platform with spatial-temporal controlled release of cytokine transforming growth factor beta (TGF-beta) and various functionalized Mb-H platforms with intelligent adjustment of cell-matrix interactions are engineered to coordinate the 3D invasive migration of cancer cell spheroids. This programmable and adaptable 3D cancer cell invasion micro platform takes a new step toward mimicking the dynamically changing (localized) tumor microenvironment and exhibits wide potential applications in cancer research, bio-fabrication, cell signaling, and drug screening. Show less
Size of DNA molecules governs their interaction with the cell membrane during electroporation and their subsequent transport inside the cell. In order to investigate the effect of DNA size on DNA... Show moreSize of DNA molecules governs their interaction with the cell membrane during electroporation and their subsequent transport inside the cell. In order to investigate the effect of DNA size on DNA-membrane interaction during electroporation, cells are electro-pulsed with DNA molecules; 15 bp, 25 bp, 50 bp, 100 bp and 1000 bp (bp = base pairs). Within the experimental parameter space, DNA-membrane complexes or DNA aggregates are observed at the cell membrane for DNA molecules containing 25 or more base pairs. No aggregates are observed for DNA molecules containing 15 bp. For all DNA sizes, direct access to the cytoplasm is observed, however the amount translocated decays with the size. The observed dependency of DNA aggregate formation on the size of the DNA molecules is consistent with the Onsager's theory of condensation of anisotropic rod-like molecules. Show less
Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro... Show moreDelivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague. To investigate the mechanism of DNA translocation across the cell membrane, giant unilamellar vesicles (GUVs) were electroporated in the presence of DNA molecules keeping the size of the DNA molecules as a variable parameter. We experimentally determined the translocation efficiency for each size of the DNA molecule, to compare the results with the existing and conflicting theories of the translocation mechanism i.e. stochastic threading and bulk electrophoresis. We observed that the translocation efficiency is independent of DNA size (ranging from 25-20 000 bp, bp = base pairs), implying that DNA molecules translocate freely across the electro-pores in the lipid membrane in their native polymer conformation, as opposed to unravelling and threading through the electro-pore. Bulk electrophoretic mobility determines the relationship between translocation efficiency and the size of the DNA molecule. This research provides experimental evidence of the mechanistic understanding of DNA translocation across lipid membranes which is essential for devising efficient and predictable protocols for electric field mediated naked DNA delivery. Show less
We study the role of a biomimetic actin network during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model... Show moreWe study the role of a biomimetic actin network during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic network provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV. Show less
Transient permeabilisation of the cell membrane is a critical step to introduce drugs or DNA into living cells, yet challenging for both biological research and therapeutic applications. To achieve... Show moreTransient permeabilisation of the cell membrane is a critical step to introduce drugs or DNA into living cells, yet challenging for both biological research and therapeutic applications. To achieve this, electroporation (or electropermeabilisation) has become a widely used method due to its simplicity to deliver almost any biomolecule to any cell type. Although this method demonstrates promise in the field of drug/gene delivery, the underlying physical mechanisms of the response of the heterogeneous cell membrane to strong electric pulses is still unknown. In this study, we have investigated the role of gel-phase lipids in the electroporation of binary giant unilamellar vesicles (GUVs), composed from DPPC (gel-phase) and DPhPC (fluid-phase) lipids (molar ratio 8:2 and 2:8). We have observed that the exposure to electric pulses leads to expel of fluid-phase lipids and concomitant decrease in GUV size, whereas the gel-phase domains become buckled. Based on experiments on pure fluid-phase and gel-phase GUVs, we have found that fluid-phase lipids can be expelled by electrical forces and the highly viscous gel-phase lipids cannot. Moreover, our analyses suggest that pore formation occurs primarily in fluid-phase domains and that the pore size is similar in all GUVs containing fluid-phase lipids, irrespective of the gel-phase percentage. Show less
Molecular conformations of individual polymers during flow through porous media are directly observed by single-DNA imaging in microfluidics. As the Weissenberg number increases during flow (Wi >... Show moreMolecular conformations of individual polymers during flow through porous media are directly observed by single-DNA imaging in microfluidics. As the Weissenberg number increases during flow (Wi > 1), we observe two types of elastic instabilities: (a) stationary dead-zone and (b) time-dependant dead-zone washing. When stretched polymer chains enter a dead-zone, they first re-coil and, once inside the dead-zone, they rotate and re-stretch again. The probability distribution of DNA chains under the stretched condition inside the dead-zone is found to be heterogeneous with a broad distribution. Show less
We experimentally investigate the flow of hydrolyzed polyacrylamide (HPAM) solution with and without salt in model porous media at high Weissenberg numbers (Wi > 1.0). The effect of pore shapes... Show moreWe experimentally investigate the flow of hydrolyzed polyacrylamide (HPAM) solution with and without salt in model porous media at high Weissenberg numbers (Wi > 1.0). The effect of pore shapes on the flow pattern and pressure drop is explored by using periodic arrays of circular and square pillars in aligned and staggered layouts. In the apparent shear-thinning regime, we observe stationary dead zones upstream of the pillars. In addition, we confirm that the size of stationary dead zones correlates with the level of shear-thinning, by varying the amount of salt in HPAM solution. At higher shear rates (or Wi), these dead zones are periodically washed away. We present the mechanism of this elastic instability and characterize it based on the pressure drop fluctuation spectral density. Show less
Here, we investigate separately the dependence of the mixing time on the size and velocity of micro-droplets moving through serpentine channels. We find that the mixing time scales linearly with... Show moreHere, we investigate separately the dependence of the mixing time on the size and velocity of micro-droplets moving through serpentine channels. We find that the mixing time scales linearly with droplet size. All experimental data collapse on a master-line, when the convective time scale is multiplied by the dimensionless droplet size. Show less
