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Probing the mechanical properties of brain cancer cells using a microfluidic cell squeezer device.

ABSTRACT: Despite being invasive within surrounding brain tissues and the central nervous system, little is known about the mechanical properties of brain tumor cells in comparison with benign cells. Here, we present the first measurements of the peak pressure drop due to the passage of benign and cancerous brain cells through confined microchannels in a "microfluidic cell squeezer" device, as well as the elongation, speed, and entry time of the cells in confined channels. We find that cancerous and benign brain cells cannot be differentiated based on speeds or elongation. We have found that the entry time into a narrow constriction is a more sensitive indicator of the differences between malignant and healthy glial cells than pressure drops. Importantly, we also find that brain tumor cells take a longer time to squeeze through a constriction and migrate more slowly than benign cells in two dimensional wound healing assays. Based on these observations, we arrive at the surprising conclusion that the prevailing notion of extraneural cancer cells being more mechanically compliant than benign cells may not apply to brain cancer cells.

SUBMITTER: Khan ZS

PROVIDER: S-EPMC3555914 | biostudies-other | 2013

REPOSITORIES: biostudies-other

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Project description:Tumors tissues house a diverse array of cell types, requiring powerful cell-based analysis methods to characterize cellular heterogeneity and identify rare cells. Tumor tissue is dissociated into single cells by treatment with proteolytic enzymes, followed by mechanical disruption using vortexing or pipetting. These procedures can be incomplete and require significant time, and the latter mechanical treatments are poorly defined and controlled. Here, we present a novel microfluidic device to improve mechanical dissociation of digested tissue and cell aggregates into single cells. The device design includes a network of branching channels that range in size from millimeters down to hundreds of microns. The channels also contain flow constrictions that generate well-defined regions of high shear force, which we refer to as "hydrodynamic micro-scalpels", to progressively disaggregate tissue fragments and clusters into single cells. We show using in vitro cancer cell models that the microfluidic device significantly enhances cell recovery in comparison to mechanical disruption by pipetting and vortexing after digestion with trypsin or incubation with EDTA. Notably, the device enabled superior results to be obtained after shorter proteolytic digestion times, resulting in fully viable cells in less than ten minutes. The device could also be operated under enzyme-free conditions that could better maintain expression of certain surface markers. The microfluidic format is advantageous because it enables application of well-defined mechanical forces and rapid processing times. Furthermore, it may be possible to directly integrate downstream processing and detection operations to create integrated cell-based analysis platforms. The enhanced capabilities enabled by our novel device may help promote applications of single cell detection and purification techniques to tumor tissue specimens, advancing the current understanding of cancer biology and enabling molecular diagnostics in clinical settings.

Project description:IntroductionIn response to external stress, cells alter their morphology, metabolic activity, and functions to mechanically adapt to the dynamic, local environment through cell allostasis. To explore mechanotransduction in cellular allostasis, we applied an integrated micromechanical system that combines an 'ultrasound tweezers'-based mechanical stressor and a Förster resonance energy transfer (FRET)-based molecular force biosensor, termed "actinin-sstFRET," to monitor in situ single-cell allostasis in response to transient stimulation in real time.MethodsThe ultrasound tweezers utilize 1 Hz, 10-s transient ultrasound pulses to acoustically excite a lipid-encapsulated microbubble, which is bound to the cell membrane, and apply a pico- to nano-Newton range of forces to cells through an RGD-integrin linkage. The actinin-sstFRET molecular sensor, which engages the actin stress fibers in live cells, is used to map real-time actomyosin force dynamics over time. Then, the mechanosensitive behaviors were examined by profiling the dynamics in Ca2+ influx, actomyosin cytoskeleton (CSK) activity, and GTPase RhoA signaling to define a single-cell mechanical allostasis.ResultsBy subjecting a 1 Hz, 10-s physical stress, single vascular smooth muscle cells (VSMCs) were observed to remodeled themselves in a biphasic mechanical allostatic manner within 30 min that caused them to adjust their contractility and actomyosin activities. The cellular machinery that underscores the vital role of CSK equilibrium in cellular mechanical allostasis, includes Ca2+ influx, remodeling of actomyosin CSK and contraction, and GTPase RhoA signaling. Mechanical allostasis was observed to be compromised in VSMCs from patients with type II diabetes mellitus (T2DM), which could potentiate an allostatic maladaptation.ConclusionsBy integrating tools that simultaneously permit localized mechanical perturbation and map actomyosin forces, we revealed distinct cellular mechanical allostasis profiles in our micromechanical system. Our findings of cell mechanical allostasis and maladaptation provide the potential for mechanophenotyping cells to reveal their pathogenic contexts and their biophysical mediators that underlie multi-etiological diseases such as diabetes, hypertension, or aging.

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Probing the mechanical properties of brain cancer cells using a microfluidic cell squeezer device.

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