Decades of research into characterization, prevention, detection and treatment have substantially expanded our collective understanding of cancer biology. However, these insights elicit a new generation of unanswered questions about the complexity of this group of often-deadly diseases. Historical investigations yielded the key understandings that cancer cells arise from indigenous cells, and most, if not all, tumors are derived from a single parent cell1. In 2000, Hanahan and Weinberg simplified the many aspects of transformation from normal human cells into cancerous ones through six essential cell physiology alterations. These so-called hallmarks of cancer include self-sufficiency in growth signals, insensitivity to anti-growth or inhibitory signals, evasion of apoptosis, unlimited replication capability, sustained angiogenesis, and tissue invasion and metastasis2. They later added deregulating cellular energetics and avoiding immune destruction as emerging hallmarks; genome instability and mutation and tumor-promoting inflammation as enabling characteristics3. The impact of external stimuli, interactions with neighboring cells and the extracellular matrix (ECM), heterogeneity, inherited traits, and other factors further complicate the elucidation of cancer biology.
Along with the expanding scope of research interests, methodologies have evolved to include live cell studies in addition to conventional biochemical and fixed cell assays. Live cell assays allow researchers to dynamically study a cells function in an environment that better represents in vivo conditions. Kinetic imaging of live cells provides a useful framework in which to gather meaningful details of cellular dynamics in real time, however, applications are often constrained by the limited versatility of available instruments. Most imaging systems are not suitable for capturing the widely ranging timelines in which cellular events occur from sub-second responses to events manifesting over days or weeks. Thus, multiple, dedicated instruments or bulky external accessories are often required, taking up precious bench space. Similarly, integrated image processing and data analysis is frequently limited or requires additional software to properly quantify the captured information.
Here, we describe an automated live cell imager designed for a wide range of temporal dynamics in live cell assays. Specifically, we demonstrate its capabilities for short-, medium- and long-term kinetic assays typically used when investigating cancer hallmarks. The integrated design of this system precludes the use of multiple instruments, while the advanced image capture and data analysis features deliver powerful and actionable insights.
Dysregulation of cellular signaling is a significant foundation for most of the aforementioned hallmarks of cancer4. Capturing a rapid, short-lived signaling event, such as calcium flux following GPCR activation, requires high temporal resolution. The automated live cell imager provides image capture rates of up to 20 frames per second, while in-line injectors enable reagent addition with continuous monitoring of cellular response. In the provided calcium mobilization example, we characterize the ATP-induced activation of endogenously expressed P2Y receptors in HeLa cells, using the cell membrane permeable calcium indicator dye Fluo-4 AM. Binding of calcium ions to Fluo-4 causes a structural change that results in a significant increase in fluorescence quantum yield and more than a hundred-fold increase in fluorescence relative to the unbound state. Per Figure 1, ATP (10 M final) was injected at t=5 seconds, an increase in intracellular calcium was detected approximately 3 seconds after the addition, and peak calcium mobilization for the entire field of cells was reached 13 seconds post-ATP addition. Image preprocessing and object masking tools reduced background fluorescence and a generated a larger assay window compared to total fluorescence measurements, resulting in a seven-fold increase in relative Fluo-4 fluorescence following ATP addition.
The relationship between wound healing and tumorigenesis is well-established5,6. Additionally, although migration is a function of normal cells, it is considered one of the hallmarks of cancer when dysregulated signals lead to cancer metastasis. Scratch assays are widely used to investigate in vitro cell migration and wound healing, where a monolayer cell culture is manually scratched to generate an area free of cells into which surrounding cells can migrate and proliferate. The imaging chamber of the automated live cell imager maintains cell health through consistent environmental conditions, including temperature, gas and humidity levels, over the entire incubation period, which is cell-dependent, but typically lasts no more than twenty-four hours. Automated phase contrast (label-free) imaging tracks the migratory characteristics of the cell model at pre-determined time points, while advanced software automatically places object masks to track parameters such as object size, area and total signal over the incubation period. In the provided scratch assay example, an approximately 500 m wide wound was created using HT-1080 fibrosarcoma cells and ibidi culture inserts. Per Figure 2A, the wound was treated with different concentrations of the migration inhibitor, cytochalasin D; and kinetic images were captured over twenty-four hours, while the cells were incubated under controlled conditions of 37 C, 5 percent CO2. Percent confluency was calculated (Figure 2B), showing that wound closure inhibition is proportional to cytochalasin D concentration, to the point where cytotoxicity begins to affect the cells neighboring the wound.
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Revealing New Details of Cancer Biology with Automated Kinetic Live Cell Imaging - Bioscience Technology