The World Health Organization stated that as of 2020, nearly 8 million women alive were diagnosed with breast cancer in the last 5 years, making it the world’s most prevalent cancer (Retrieved from WHO). Globally, 1 in 8 women will be affected at some point during their lives with breast cancer and must undergo surgery or radiation treatment options that still include invasive and damaging methods developed over 50 years ago.
Aiding in the development of new treatment modalities that are noninvasive with fewer side effects leads to the evolution of breast cancer cell models. These new methods shine a light of hope on a very dark past for individuals who have undergone radiation treatment only to fail treatment or relapse. While these treatment options offer substantial promise, they often take nearly a decade or longer to complete all phases of clinical trials before full FDA approval.
To begin the process for a clinical trial, cancer models are created from cell lines to understand the in vitro and in vivo effects of the drugs before being tested in people. Only after being cleared from having any toxic effects will the drug be moved further into clinical research with humans. After that, the FDA will review and determine a drug to be safe and effective against its intended use and offer recommendation for approval.
One aggressive subtype of breast cancer is HER2 positive disease. HER2 is a human epidermal growth factor receptor that normally acts to maintain cell functions for growth and development of breast cells. When this gene is overexpressed, it leads to aberrant expression of the receptor and tumor formation. Approximately 10-20% of breast cancers are traced back to HER2 receptors (Figure 1).
Figure 1: Distribution of the breast cancer subtypes by ER/HER2 status and their age-specific incidence in Scotland for 2009–2016 (N = 31,099). a Shows a pie chart and b shows age-specific incidence on the log scale by subtype. b Data are for 31,099 breast cancer cases with ER/HER2 missing status imputed for analysis. Dotted lines in the graph denote ages 50–70 years, the age group invited for screening in Scotland every 3 years. (Retrieved from Nature, 2020)
Cellular models provide consistency and reliability for in vitro and in vivo testing pre-clinical trials. To ensure stability and genetic reproducibility, it is important to derive a cellular model from a monoclonal population. Without uniform models, results can be unreliable and discredit potential treatment methods that may be successful and provide improved outcomes for cancer patients.
Despite the importance of such models, the actual development of cell lines in the lab is a time-consuming hurdle that necessitates substantial effort. These efforts are often met with failure due to technical challenges and limitations.
To study aggressive HER2+ breast cancer, a cell line was engineered to overexpress HER2 and blue fluorescent protein (EBFP2). The researchers generating this line leveraged a traditional method for deriving a stable monoclonal cell population, fluorescence activated cell sorting (FACS), which involves using laser excitation and fluidics to recover single cells. However, FACS often results in an unstable cell population with varying degrees of expression of the gene of interest. This inevitability dilutes or ultimately obscures downstream analysis, making reliability and reproducibility impossible. After two separate rounds of FACS to enrich the double positive population (HER2+/EBFP2+), the cells nonetheless remained heterogeneous. This caused an inherent competition for survival in the population and the genetic edits became diluted and unstable.
Realizing the cellular population was quickly fading and the correct edits were being lost in the bulk population, a monoclonal and stable cell line needed to be generated quickly. By seeding the population on a CellRaft ArrayConsumables containing thousands of microwells (containing CellRafts) for spatial segregation, imaging, and isolation of single cells, monoclonal colonies, or 3D cultures. More, clonality was able to be tracked and traced from day 0, as well as expression of the EBFP2 marker. This ensured chain of custody in the development of each cell colony (Figure 2). In addition to clonality, it was also necessary to identify cells expressing high levels of HER2. Using a live stain for HER2 on the CellRaft AIR SystemAn all-in-one platform for imaging, identifying, and isolating viable single cells, colonies, or organoids using CellRaft Arrays and software-guided selection with CellRaft Cytometry. More, monoclonal populations that were HER2 positive as well as EBFP2 positive were discovered. When interrogating the population of cells on the CellRaft ArrayConsumables containing thousands of microwells (containing CellRafts) for spatial segregation, imaging, and isolation of single cells, monoclonal colonies, or 3D cultures. More, it was clear that the actual population of double expressing cells was extremely rare compared to the data generated from FACS. Approximately 1.5% of the population of cells retained the correct gene edits compared to 89% reported from sorting. The AIR System was able to isolate populations that met all criteria, and they were expanded for cryopreservation.
Figure 2: Time course images of a CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More containing an EBFP2+/HER2+ clone. The CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More arrays were imaged in brightfield and fluorescence 4 hours post-seeding and every 24 hours until colony formation. On day 6, the array was stained with Anti-HER2-FITC to visualize HER2 expression (green). 10X Magnification.
This workflow enabled thousands of cells to be screened using a single consumable to identify the rare double positive clones in a matter of weeks, not months. In total, the time from cell seeding to clonal outgrowth was only 16 days, and viable cell banks of the verified clones were cryopreserved in 27 days. Thus, the CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More AIR Technology enables successful monoclonal cell line development for even rare or challenging cells faster and more efficiently than traditional methods.
For an in-depth look at this case study, download: Identification of a rare double positive clone.
Learn more about CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More Technology.
Lexi Land contributes to developing scientific workflows that are compatible with the CellRaft® AIR System. Her focus at Cell Microsystems involves stem cell research, 3D organoid work, as well as adherent and suspension cell culture. Lexi received a Bachelor of Science in Biological Sciences from North Carolina State University with a focus in molecular, cellular, and developmental biology.
In the ongoing battle against antibiotic-resistant bacteria, or “superbugs,” scientists are constantly seeking innovative tools to understand the mechanisms of resistance and develop effective treatments. One such groundbreaking technology is shear flow systems, which have emerged as game-changers in microbiological research. By simulating the dynamic conditions of bacteria’s natural environments, such as the human body or food processing, shear flow systems provide unique insights into bacterial behavior and resistance mechanisms that traditional static methods simply cannot achieve. In this blog post, we’ll explore the benefits of using shear flow systems to combat antibiotic resistance and develop next-generation therapies.
Antibiotic resistance is a complex phenomenon influenced by a variety of factors, including genetic mutations, horizontal gene transfer, and the formation of biofilms. Traditional laboratory methods often fail to capture the dynamic nature of these processes, leading to incomplete or misleading results. Shear flow systems address this limitation by providing a more realistic environment for bacterial growth and interaction. Here’s how:
1. Mimicking Real-World Conditions
In the human body, bacteria are rarely in a static state. They are constantly exposed to fluid flow, such as blood circulation or urine flow, which influences their behavior and resistance mechanisms. Shear flow systems can replicate these conditions, allowing researchers to study how bacteria respond to antibiotics under realistic physiological conditions. This is critical for understanding how resistance develops and persists in vivo. This benefit was recently highlighted in a webinar presented by Dr. Katharina Richter, a microbiology researcher from the University of Adelaide in Australia. Using a high-throughput BioFlux Shear Flow System, Dr. Richter and her team were able to test 3 different methods of superbug treatments under physiological conditions.
2. Studying Biofilm Formation and Resistance
Biofilms—structured communities of bacteria encased in a protective matrix—are a major contributor to antibiotic resistance. Biofilms are notoriously difficult to treat because they shield bacteria from antibiotics and the immune system. Shear flow systems enable researchers to study biofilm formation in real time, observing how bacteria adhere to surfaces, form microcolonies, and develop resistance under flow conditions. For example, using a BioFlux, Dr. Richter was able to leverage high-resolution imaging to obtain unprecedented insight into the biofilm killing and prevention efficacy of a copper and diethyldithiocarbamate (DDC) combination nanoparticles¹. The group is currently investigating the use of a Cu-DDC infused gel that can be injected into wounds that are at high risk for chronic infection, such as hernia.
The study of biofilm communities presents unique challenges in experimental design and analysis. While mixed bacterial populations offer fascinating insights into microbial interactions and community dynamics, accurately characterizing and investigating these complex systems has previously required sophisticated and tedious technical approaches. Here, we will discuss advances in technology that have helped to simplify and streamline polymicrobial investigations.
Throughout the last decade, a trend for growing and assaying biofilms under shear flow has gained traction among microbiologists. The rationale for investigating biofilms under flow is multi-dimensional. A recent Cell Microsystems survey asked why microbiologists are using shear flow found that the top responses were:
Of the scientists surveyed, 40% indicated that they use a BioFlux Shear Flow System. This system provides unique advantages over other methods to induce shear stress, such as slide-based parallel plate flow chambers and benchtop rockers.
BioFlux uses microfluidic channels embedded onto the bottom of standard-sized 6, 24, or 48-well plates to create isolated and independent flow chambers where user-defined wall shear stress is initiated and maintained by digitally controlled air pressure. This not only simplifies the setup and experimental processes but also ensures that experiments are contained and do not contaminate the shear flow system.
Although shear flow has been thoroughly demonstrated to improve the biological relevance of biofilm investigations1, an often overlooked aspect of biofilm physiology is the significant role that multicellular aggregates play in biofilm formation2. When investigating Pseudomonas aeruginosa, it has been demonstrated that multicellular aggregates lead to faster biofilm formation and enhanced resistance to antimicrobials and immune responses3. In addition, multispecies microbial aggregates have been shown to dominate single cells in human saliva4. Together, these data show that aggregates are important areas of investigation because they are more likely to represent physiological biofilm formation and growth.
