Associate Professor University of Georgia, United States
Introduction:: Detection of metastatic breast cancer will not occur until after the cancer has spread to other parts of the body, resulting in delayed treatment and often, more advanced/detrimental cancer cases. Thus, it is critical to understand the fundamental mechanisms underlying the metastatic process and the complex interactions that occur between the tumor and the host during disease progression for detection of metastatic cells.
While the mechanosensing abilities of breast cancer cells (mediated by integrin adhesion sites) are well known, the role that extracellular matrix (ECM) composition plays on breast cancer aggressiveness, separate from stiffness, is still a subject of study. In the present work, five breast cancer cell lines and two non-cancerous cell lines were cultured and evaluated using a bioelectronic impedance-based assay system (Fig. 1A) to elucidate the relationship between ECM composition, cell phenotype (e.g. proliferation, cell-cell barrier integrity), and 2D migration. Bioelectronic assays, like the one used in this work, offer a sensitive, label-free, and non-destructive method to continuously monitor cancer cells in real-time, allowing the assessment of multiple cancer behaviors simultaneously.
The long-term goal of this work is to improve current cancer assessment methods and determine a correlation between quantifiable impedance-based characteristics, 2D cell morphology, and cancer subtype.
Materials and Methods:: Cells were cultured using the Maestro Tray Z Impedance Assay System (Axion Biosystems). Collagen I, collagen IV, fibronectin, and laminin were chosen as ECM coatings since they make up a significant portion of breast tissue ECM, while Matrigel is a commonly used basement membrane matrix in breast cancer modeling that is comprised of multiple ECM proteins. A 20 µg/mL sample of each ECM coating was prepared and wells of 96-well CytoView plates (Axion Biosystems) were coated immediately prior to cell seeding. Cell proliferation was monitored via high-frequency impedance. After 24 hours of cell proliferation, confluent monolayers formed, and cell mediums were switched to a nutrient-reduced formula. Wells were scratched at 36 hours to create a wound and cell migration was monitored for an additional 36 hours (Fig. 1B-C). Selected cells included two non-cancerous cell lines (184B5, normal mammary epithelial; MCF10A, fibrocystic disease epithelial) and five breast cancer cell lines, including MCF-7 cells (non-invasive adenocarcinoma, ER+, PR+), and MDA-MB-231 cells (ER-/PR-/HER2-, Caucasian donor). Four of the selected breast cancer cell lines are triple negative (ER-, PR-, HER2-), a highly aggressive and often metastatic subtype (not all data shown here). Given the significant health disparities and inequities that exist in biomedical research and medical practice, the broader goals of this project are to have a better fundamental understanding of cancer cell processes within triple-negative breast cancer, of which African American women are disproportionately affected.
Results, Conclusions, and Discussions:: A distinct difference in impedance was observed across all cell lines that can be linked to single-cell morphology and growth patterns. For example, MCF7 cells exhibited a much higher impedance overall compared to the MDA-MB-231 cells (Fig. 1B-C). This correlates with known phenotypic characteristics for each cell type, where MCF7 cells are epithelial-like in morphology and grow tightly packed, compared to the spindly MDA-MB-231 cells. While the non-cancerous cell lines exhibited little or no recordable difference in cell growth/morphology as a response to ECM composition (not shown in figure), all tested breast cancer cell lines did. The more aggressive MDA-MB-231 cells showed more distinctions in response to ECM composition as compared to the less aggressive MCF7 cells (Fig. 1D). This could indicate that MDA-MB-231 cells have higher phenotypic plasticity, commonly exhibited in more aggressive cancers. Current work is ongoing to support this finding via cell imaging and morphological analysis.
While the MCF7 cells only exhibited significant wound closure within the fibronectin coating condition, MDA-MB-231 cells showed wound closure under multiple ECM conditions (collagen I, collagen IV, laminin). This not only confirms cancer aggressiveness based on reported cell line characterization but demonstrates the efficacy of bioelectronic assays to evaluate multiple cancer cell parameters in real-time. ECM composition was not shown to affect wound closure over 36 hours for any cell line evaluated, suggesting that the specific processes underpinning breast cancer outgrowth and epithelial-to-mesenchymal-transition are found outside of the cell-ECM protein relationship.
Our hypothesis that ECM composition affects cell behavior was supported, although ECM proteins did not affect 2D migration specifically in this work. These findings demonstrate the feasibility of using an impedance-based assay for characterizing cancer cell migration, proliferation, and response to microenvironment using quantitative metrics. Ongoing work includes performing additional studies to determine a correlation between cell response to ECM and morphological features. This will strengthen our knowledge on the relationship between tumor microenvironment and breast cancer cell phenotypic plasticity. Overall, this work creates a foundation for our long-term goal of improving current assessment methods to yield a more detailed and objective evaluation of cancer metastatic behavior.
