December 1, 2015 (Vol. 35, No. 21)
Philip Lee Director of Global Marketing for Cell Culture Systems EMD Millipore
Cindy Chen Scientist EMD Millipore
Fen Xu Research Associate EMD Millipore
Terry Gaige Systems/Design Engineer EMD Millipore
Paul Hung R&D Senior Manager EMD Millipore
Microfluidic Platform Allows Real-Time Observation
Cancer cell transformation represents the epitome of dynamic cell behavior. During transformation, once-quiescent cells can exhibit genomic changes, metabolic reprogramming, microenvironment alterations, unchecked replication, and transmigration.
The CellASIC® ONIX microfluidic platform from EMD Millipore enables monitoring of these dynamic cellular processes from start to finish. By allowing precise control of the cancer cell microenvironment, this powerful platform creates new opportunities to study proliferation, motility, differentiation, or viability circuits. Growth factors, inhibitors, and mimetics can be added, the hypoxic environment can be altered, and autophagic, apoptotic, or transmigration events can be observed without disrupting the culture environment to change experimental conditions.
The CellASIC ONIX platform enables cancer biologists to:
- Discover translocation mechanisms not resolvable by end-point assays
- Simulate conditions of pulse exposure to drug compounds
- Quantify the rate of protein translocation
- Provide key parameter values for therapeutic compound profiling
Below, we summarize three applications of the CellASIC ONIX microfluidic platform: transcription factor translocation, autophagy, and cell migration and chemotaxis.
Classical biochemical techniques to measure transcription factor localization are semi-quantitiative and do not provide per-cell translocation measurements. Manual microscopy allows visual identification of nuclear translocation on a per-cell basis, but an objective and statistically rigorous assessment is difficult to obtain.
We performed live-cell analysis using the CellASIC ONIX microfluidic platform to create a dynamic assay for translocation of the Forkhead transcription factor, FOXO4.
Forkhead transcription factors are altered in several types of cancers, interacting with an array of downstream targets and partners that are involved in the regulation of the PI3L-Akt pathway, leading to cell survival or cell death. In this study, changes in nuclear translocation were observed and measured in reporter cells cultured, wortmannin-treated, and flushed all within the controlled microenvironment created and maintained by the CellASIC ONIX system while continuously sitting on a standard inverted fluorescent microscope stage (Figure 1).
With the use of microfluidic live-cell analysis, the intracellular relocation of FOXO4 in reporter cells in response to 150 nM wortmannin treatment can be instantaneously monitored. We not only observed the temporal features of translocation in real time, but also dissected the dynamics of the FOXO4 translocation process at the single-cell level.
By performing live-cell analysis using the CellASIC ONIX microfluidic platform, we created a dynamic assay that has the potential to simultaneously monitor multiple intracellular components throughout the entire protein translocation process without disruption. The platform also enables the researcher to precisely manipulate culture parameters (media flow, inducer/inhibitor concentration, gas content) and discover translocation mechanisms that the end-point assays cannot offer. Consequently, this platform may be capable of simulating conditions of pulse exposure to drug compounds, and could potentially provide novel and vital information for compound profiling by enabling quantification of the rate of protein translocation.
Visualize Autophagy in Real Time
Experimental manipulation of the cellular microenvironment is important when studying cancer cell behavior under stressed conditions, such as nutrient deprivation, hypoxia, or drug exposure. Stressed tumor cells may use the autophagic pathway to promote survival.
Live-cell imaging assays can be used to monitor both the rate of autophagosome formation and changes in lysosomal degradative processes during autophagy. The CellASIC ONIX microfluidic platform allows control and measurement precision in autophagy assays.
Cell lines stably expressing fluorescently tagged markers specific for autophagosomes (LC3-GFP) were used in combination with microfluidic control of media and gas exchange to create starvation or hypoxia. Changes in LC3 levels (as measured by autophagosome counts) were monitored and quantified throughout culture duration by fluorescence microscopy. Also, two-color imaging of transduced LAMP1-RFP/LC3-GFP CHO reporter cells was used to monitor autophagosomes (green) and lysosomes (red) throughout the entire hypoxia-induced autophagy assay (Figure 2).
Visualize Migration and Chemotaxis
Migration dynamics are critical to understanding tumor metastasis and evasion behavior. Microfluidic control of chemical gradients by the CellASIC ONIX platform allows precise manipulation of classical chemotaxis/migration assays. With the platform, researchers can:
- Create defined diffusion gradients
- Control stability, direction, and composition
- Do multiday observations without moving culture
- Interrogate cells with antimigration compounds
- Measure induced morphology and protein changes
The most widely accepted cell migration assay is the Boyden chamber assay, a two-chamber multiwell plate in which a membrane in each well provides a porous interface between two chambers. Chemoattractant is placed in the lower chamber and the system is allowed to equilibrate, with the expectation that a gradient will form between the upper and lower wells.
However, in reality, very steep gradients can form along a single axis perpendicular to the surface of the membrane, resulting in a lower-than-expected difference in chemoattractant concentration between upper and lower wells. As a result, this method is unsuitable for correlating specific cell responses with particular gradient characteristics (i.e., slope, concentration, temporal evolution, etc.), and for studying multigradient signal integration. Furthermore, gradients are not very stable under “static” cell culture conditions, precluding live-cell analysis.
In order to create a quantitatively defined diffusion gradient that is stable enough for long-term, live-cell analysis over the course of days, we developed a microfluidic gradient plate. This plate, designed for use on the CellASIC ONIX microfluidic platform, enables precision-controlled chemoattractant diffusion across perfusion barriers to create a spatial gradient in the culture area.
In the study shown in Figure 3, the microfluidic gradient plate was used to create a defined, stable, and controllable chemoattractant gradient. Migratory behavior of MDA-MB-231 human breast cancer cells in response to FBS was measured in real-time and time-lapse imaging.
Microfluidic control of cell culture conditions and chemoattractant gradient formation enables precise, dynamic quantitation of cell migration in response to stimuli. The ability to control gradient parameters and culture conditions while maintaining an uninterrupted optical path to the cells enables researchers to draw conclusions regarding the molecular mechanisms of cell migration. This study provides a reference point from which to build upon future research comparing the effects of signaling molecules and growth factors on the migration propensities of cells in tumors, wounds, developing tissues, immune responses, and other biological systems defined by active cell migration.
Find out how the dynamic CellASIC ONIX cell culture system can revolutionize cancer studies and reveal more of cancer’s complexities by visiting www.emdmillipore.com/cellASIC.
Philip Lee ([email protected]) is director of global marketing for cell culture systems, Shin-Yi Cindy Chen is research scientist, Fen Xu is research associate, Terry Gaige is systems/design engineer, and Paul J. Hung is R&D manager, all at EMD Millipore.