RESUMO
In this work, we developed a method that allows precise control over changes in the size of a cell via hydrostatic pressure changes in the medium. Specifically, we show that a sudden increase, or reduction, in the surrounding pressure, in the physiologically relevant range, triggers cross-membrane fluxes of sodium and potassium ions in leukemia cell lines K562 and HL60, resulting in reversible volumetric deformation with a characteristic time of around 30 min. Interestingly, healthy leukocytes do not respond to pressure shocks, suggesting that the cancer cells may have evolved the ability to adapt to pressure changes in their microenvironment. A model is also proposed to explain the observed cell deformation, which highlights how the apparent viscoelastic response of cells is governed by the microscopic cross-membrane transport.
Assuntos
Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Biológicos , Citometria de Fluxo , Células HL-60 , Humanos , Pressão Hidrostática , Células K562 , Potássio/química , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Sódio/química , Sódio/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismoRESUMO
A cancer cell changes its state from being epithelial- to mesenchymal-like in a dynamic manner during tumor progression. For example, it is well known that mesenchymal-to-epithelial transition (MET) is essential for cancer cells to regain the capability of seeding on and then invading secondary/tertiary regions. However, there is no fast yet reliable method for detecting this transition. Here, we showed that membrane undulation of invasive cancer cells could be used as a novel marker for MET detection, both in invasive model cell lines and repopulated circulating tumor cells (rCTCs) from non-small cell lung cancer (NSCLC) patients. Specifically, using atomic force microscopy (AFM), it was found that the surface oscillation spectra of different cancer cells, after undergoing MET, all exhibited two distinct peaks from 0.001 to 0.007 Hz that are absent in the spectra before MET. In addition, by adopting the long short-term memory (LSTM) based recurrent neural network learning algorithm, we showed that the positions of recorded membrane undulation peaks can be used to predict the occurrence of MET in invasive NSCLC cells with high accuracy (>90% for model cell lines and >80% for rCTCs when benchmarking against the conventional bio-marker vimentin). These findings demonstrate the potential of our approach in achieving rapid MET detection with a much reduced cell sample size as well as quantifying changes in the mesenchymal level of tumor cells.
RESUMO
We have developed a novel electro-osmotic microfluidic system to apply precisely controlled osmolarity gradients to cancer cells in micro-channels. We observed that albeit adhesion is not required for cells to migrate in such a confined microenvironment, the migrating velocity of cells is strongly influenced by the interactions between the cells and the channel wall, with a stronger adhesion leading to diminished cell motility. Furthermore, through examining more than 20 different types of cancer cells, we found a linear positive correlation between the protein concentration of the aquaporin-4 (AQP4) and the cell migrating speed. Knockdown of AQP4 in invasive re-populated cancer stem cells reduced their migration capability down to the level that is comparable to their parental cancer cells. Interestingly, these observations can all be quantitatively explained by the osmotic engine model where the cell movement is assumed to be driven by cross-membrane ion/water transport, while adhesion acts as a frictional resistance against the cell motility. By providing versatile and controllable features in regulating and characterizing the migration capability of cells, our system may serve as a useful tool in quantifying how cell motility is influenced by different physical and biochemical factors, as well as elucidating the mechanisms behind, in the future.
Assuntos
Aquaporina 4/metabolismo , Movimento Celular , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Osmose , Matriz Extracelular , Humanos , Transporte de Íons , Técnicas Analíticas Microfluídicas , Neoplasias/patologiaRESUMO
During tumor development, cancer cells constantly confront different types of extracellular barriers. However, fundamental questions like whether tumor cells will continue to grow against confinement or away from it and what key factors govern this process remain poorly understood. To address these issues, here we examined the growth dynamics of human lung epithelial carcinoma A549 cells partially confined in micrometer-sized cylindrical pores with precisely controlled wall stiffness. It was found that, after reaching confluency, the cell monolayer enclosed by a compliant wall was able to keep growing and pushing the boundary, eventually leading to a markedly enlarged pore. In contrast, a much reduced in-plane growth and elevated strain level among cells were observed when the confining wall becomes stiff. Furthermore, under such circumstance, cells switched their growth from within the monolayer to along the out-of-plane direction, resulting in cell stacking. We showed that these observations can be well explained by a simple model taking into account the deformability of the wall and the threshold stress for inhibiting cell growth. Interestingly, cadherins were found to play an important role in the proliferation and stress buildup within the cell monolayer by aggregating at cell-cell junctions. The stiff confinement led to an elevated expression level of cadherins. Furthermore, inhibition of N-cadherin resulted in a significantly suppressed cell growth under the same confining conditions.
RESUMO
Although accumulating evidence has demonstrated the important role of membrane blebbing in various cellular processes, the fundamental question of how the initiation/evolution of blebs are influenced by physical factors like membrane-cortex interactions and intracellular pressure remains unclear. Here, we report a combined modeling and experimental study to address this outstanding issue. Specifically, boundary integral method was used to track the motion of membrane (in 3D) during blebbing while possible rupture of the bilayer-cortex adhesion has also been taken into account. We showed that, for a given differential pressure across the cell membrane, the size of the weakened cortex must be over a critical value for blebbing to occur and the steady-state volume of a bleb is proportional to its initial growth rate, all in good agreement with recent experiments. The predicted shape evolution of blebs also matches well with our observations. Finally, a blebbing map, summarizing the essential physics involved, was obtained which exhibits three distinct regimes: no bleb formation corresponding to a low intracellular pressure or a small weakened cortex region; bleb formed with a fixed width when the disrupted cortex zone is very large; and a growing bleb resulted from progressive membrane-cortex detachment under intermediate weakened cortex size.
RESUMO
A number of growth factor receptor tyrosine kinases have been implicated in angiogenesis, including epidermal growth factor receptor, fibroblast growth factor receptor, platelet-derived growth factor receptor, Flk-1/KDR, Flt-1, Tie-1, and Tek/Tie-2. Flk-1/KDR, a receptor for vascular endothelial growth factor (VEGF), is expressed exclusively in endothelial cells. Using dominant-negative methods, Flk-1 was shown to play a role in angiogenesis and the growth of a variety of tumor types. Because of this, a drug discovery effort was established to identify Flk-1 kinase inhibitors. For initial screening, an ELISA in, a 96-well format was used to measure VEGF-induced Flk-1 tyrosine phosphorylation in whole cells. Compounds that inhibited ligand-induced receptor autophosphorylation were confirmed by antiphosphotyrosine immunoblotting. Inhibition of VEGF-stimulated DNA synthesis in human endothelial cells was also assessed. Inhibitors were further evaluated for their effects on vessel formation using the chorioallantoic membrane assay. Using these methods, antiangiogenesis compounds that inhibit Flk-1 tyrosine kinase activity, endothelial cell mitogenesis, and blood vessel formation in the chorioallantoic membrane assay have been found.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Células 3T3/efeitos dos fármacos , Células 3T3/enzimologia , Animais , Moléculas de Adesão Celular/análise , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Estimulação Química , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
In this report, we showed that two tumor cell characteristics, namely the malignancy and drug-resistance status can be evaluated by their membrane resealing response. Specifically, membrane pores in a number of pairs of cancer and normal cell lines originated from nasopharynx, lung and intestine were introduced by nano-mechanical puncturing. Interestingly, such nanometer-sized holes in tumor cells can reseal ~2-3 times faster than those in the corresponding normal cells. Furthermore, the membrane resealing time in cancer cell lines exhibiting resistance to several leading chemotherapeutic drugs was also found to be substantially shorter than that in their drug-sensitive counterparts, demonstrating the potential of using this quantity as a novel marker for future cancer diagnosis and drug resistance detection. Finally, a simple model was proposed to explain the observed resealing dynamics of cells which suggested that the distinct response exhibited by normal, tumor and drug resistant cells is likely due to the different tension levels in their lipid membranes, a conclusion that is also supported by direct cortical tension measurement.