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 cell’s 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.
Calcium mobilization: Kinetic assays spanning seconds
Dysregulation of cellular signaling is a significant foundation for most of the aforementioned “hallmarks of cancer”4. 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 µM 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.
Scratch assays: Kinetic assays spanning hours
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 µm 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.
Cell proliferation: Kinetic assays spanning days
Cell proliferation assays are a cornerstone of oncology research, as sustained chronic cell proliferation is among the most fundamental traits of cancer3. When incorporating kinetic live cell imaging using high contrast brightfield, these assays enable visual inspection and detailed analysis of subtle to gross biological changes over incubation periods lasting days to weeks. The automated live cell imager provides consistent environmental control, including temperature, gas and humidity levels, to maintain optimal cell health and subsequent behavior. Additionally, high contrast brightfield imaging allows for label-free cell counting, while autofocus capabilities keep the cells in focus over the long-term culture period without manual intervention. In the provided cell proliferation example, HT-1080 fibrosarcoma cells were treated with different concentrations of doxorubicin and incubated at 37 °C, 5 percent CO2, >80 percent humidity, while kinetic images were captured over four days. Object masks (Figure 3A) were automatically placed around the cells to accurately assess the proliferation and measurement of doubling time via cell counting. Additionally, full kinetic profiles (Figure 3B) were created of proliferating HT-1080 cells under various doses of doxorubicin spanning four days.
Summary
As we look forward to new cancer research inroads, live cell methodologies will provide critical insights to support and enhance the cancer hallmark data collected through other means. The broad variety of kinetic live cell imaging applications equally benefits from a single, automated live cell imager with robust capabilities that extend throughout all live cell assay requirements, and advanced software features to facilitate data collection and interpretation. Through examples spanning seconds to days, we show that the automated live cell imager is sufficiently responsive towards a wide range of dynamic processes, and enables assay choice flexibility.
References
- NIH Curriculum Supplement Series: Biological Sciences Curriculum Study; National Institutes of Health: Bethesda, MD, 2007.
- Hanahan, D.; Weinberg, R.A. The Hallmarks of Cancer. Cell. 2000, 100(1), 57-70.
- Hanahan, D.; Weinberg, R.A. Hallmarks of Cancer: The Next Generation. Cell. 2011, 144(5), 646-674.
- Sever, R.; Brugge, J.S. Signal Transduction in Cancer. CSH Perspect Med. 2015.
- Antonio, N.; BØnnelykke-Behrndtz, M.L.; Ward, L.C.; Collin, J.; Christensen, I.J.; Steiniche, T.; Schmidt, H.; Feng, Y. Martin, P. The wound inflammatory response exacerbates growth of pre-neoplastic cells and progression to cancer. EMBO J. 2015, 34(17), 2219-2236.
- Arnold, K.M.; Opdenaker, L.M.; Flynn, D.; Sims-Mourtada, J. Wound Healing and Cancer Stem Cells: Inflammation as a Driver of Treatment Resistance in Breast Cancer. Cancer Growth Metastasis. 2015, 8, 1-13.
