Magnetically Actuated Cell Stretching Platform to Induce Phenotypic Changes in Metastatic Cells

Although metastasis is responsible for about 90% of cancer deaths, only few in vitro models can be used to evaluate dynamic behaviors of metastatic cancer cells. Many studies have shown that mechanical stimuli can trigger various cellular responses such as gene and protein expression, which could lead to changes in cellular phenotype. Similarly, metastasized breast cancer cells in the lung tissue are constantly stretched by cyclic mechanical stress due to breathing, which alters cellular morphology and proliferation state. Such transitions can make the secondary tumors resistant to the chemotherapy used to effectively treat the primary tumors. In this work, we developed an in vitro tumor microenvironment that simulates in vivo respiration to investigate the mechanism of the phenotypic changes of metastatic breast cancer cells due to mechanical stimulation. We designed and fabricated magnetic microactuators using maskless photolithography technique to stretch tumor cells. Next, we coated fibronectin fibrils over the gaps of microactuators to mimic natural ECM environment and seeded tumor cells on the fibronectin mesh to generate a tumor microenvironment. As a result, the amount of strain that our microdevice could apply on the fibronectin mesh corresponded to the amount of strain experienced during normal respiration. In conclusion, the magnetically actuated in vitro cell stretching platform can provide precise strain control over a large actuation range to mimic mechanical stimulation in the lung. In the future, we will evaluate potential changes in metastatic cell phenotype and provide additional insights on the mechanism of secondary tumor drug resistance

Measurement of Hydrogen Peroxide Influx Into Cells: Preparation For Measurement Using On-Chip Microelectrode Array

Hydrogen peroxide (H2O2) is commonly known as a toxic reactive oxidative species (ROS) for cells. Recent studies have found evidence that H2O2 is also an important cellular signalling molecule. Quantifying cellular influx of H2O2 will contribute to researchers’ understanding of the role H2O2 plays in healthy cells and cells involved in the progression of cancers and degenerative diseases. This work utilizes an assay kit and fluorescence techniques to evaluate cell lines and conditions to create a model biological system for measuring cellular H2O2 consumption. Pancreatic beta cells (MIN6), astrocytes, and glioblastoma cells (GBM43 and GBAM1) were placed in 10 μM and 20 μM H2O2 solutions for up to 5 hours. The consumption of H2O2 was measured using an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes/Invitrogen). GBAM1 cells exposed to 20 μM H2O2 displayed the fastest rate of H2O2 consumption (4.8 ± 1.2 nmol H2O2/min/106 cells), followed by GBM43 cells (1.5±0.46), astrocytes (1.1±0.24), and MIN6 cells (0.29±0.075). Additionally, the rate of consumption increased with increases in H2O2 concentration. In the future, an on-chip micro-electrode array (MEA) will be used for real-time electrochemical experiments to measure influx of H2O2 by astrocytes and GBAM1 cells with spatio-temporal resolution that the current techniques lack. The results from the electrochemical experiments will be compared to results from the assay kit to determine the ability of the MEA to accurately measure H2O2 concentration and flux. The MEA can be extended to a wide variety of cellular environments for analysis of additional real-time biological events

Cellular Model of Hydrogen Peroxide Release: In Preparation for On-Chip Sensor Measurements

Hydrogen peroxide is traditionally associated with cellular damage; however, recent studies show that low levels of H2O2 are released by cells as part of normal intercellular communication. The mechanisms of hydrogen peroxide transport, uptake and release, and biological effects are not yet well known but have important implications for cancer, stem cells, and aging. Standard H2O2 assays cannot make spatially or temporally resolved quantitative measurements at a cellular scale. Previously we developed a microelectrode array (MEA) and calibration methods for quantifying H2O2 gradients in space and time. The sensor was validated using artificial H2O2 gradients at subsecond and micrometer scale resolutions. The present study begins cellular work on H2O2 release to identify a cellular model system for MEA sensor testing. The morphology and H2O2 release from U937 human monocytes were analyzed after stimulation with ionomycin (1.2 ug/mL) and/or phorbol 12-myristate 13-acetate (PMA). Monocytes were stimulated with PMA (10 ng/mL to 150 ng/mL) for six hours. Hydrogen peroxide release was quantified over time using a traditional amplex red flurometric assay method. Mouse pancreatic beta (MIN6) cells were also tested as a negative control. Monocytes stimulated with PMA alone produced, on average, three times more H2O2 than those stimulated with ionomycin or a combination. Monocytes without ionomycin released H2O2 at 18.34 pmol/min/106 cells at 25 ng/mL of PMA. Ten, 25, and 100 ng/mL of PMA produced H2O2 significantly faster than the non-stimulated control. No significant difference was seen between PMA concentrations when ionomycin was added. These results indicate that PMA stimulated human monocytes may serve as a good model system for cellular validation of the H2O2 MEAs. In the future, biofunctionalization of the electrodes for additional molecular specificity will allow for the expansion of the method to other analytes, giving the sensor potential use in non-traditional lab environments with the ability to perform multiple assays autonomously

In Vitro Magnetic Techniques for Investigating Cancer Progression

Worldwide, there are currently around 18.1 million new cancer cases and 9.6 million cancer deaths yearly. Although cancer diagnosis and treatment has improved greatly in the past several decades, a complete understanding of the complex interactions between cancer cells and the tumor microenvironment during primary tumor growth and metastatic expansion is still lacking. Several aspects of the metastatic cascade require in vitro investigation. This is because in vitro work allows for a reduced number of variables and an ability to gather real-time data of cell responses to precise stimuli, decoupling the complex environment surrounding in vivo experimentation. Breakthroughs in our understanding of cancer biology and mechanics through in vitro assays can lead to better-designed ex vivo precision medicine platforms and clinical therapeutics. Multiple techniques have been developed to imitate cancer cells in their primary or metastatic environments, such as spheroids in suspension, microfluidic systems, 3D bioprinting, and hydrogel embedding. Recently, magnetic-based in vitro platforms have been developed to improve the reproducibility of the cell geometries created, precisely move magnetized cell aggregates or fabricated scaffolding, and incorporate static or dynamic loading into the cell or its culture environment. Here, we will review the latest magnetic techniques utilized in these in vitro environments to improve our understanding of cancer cell interactions throughout the various stages of the metastatic cascade

The Dynamic Relationship of Breast Cancer Cells and Fibroblasts in Fibronectin Accumulation at Primary and Metastatic Tumor Sites

In breast cancer (BC), tissue stiffening via fibronectin (FN) and collagen accumulation is associated with advanced disease progression at both the primary tumor and metastatic sites. Here, we evaluate FN production in 15 BC cell lines, representing a variety of subtypes, phenotypes, metastatic potentials, and chemotherapeutic sensitivities. We demonstrate that intracellular and soluble FN is initially lost during tumorigenic transformation but is rescued in all lines with epithelial-mesenchymal plasticity (EMP). Importantly, we establish that no BC cell line was able to independently organize a robust FN matrix. Non-transformed mammary epithelial cells were also unable to deposit FN matrices unless transglutaminase 2, a FN crosslinking enzyme, was overexpressed. Instead, BC cells manipulated the FN matrix production of fibroblasts in a phenotypic-dependent manner. In addition, varied accumulation levels were seen depending if the fibroblasts were conditioned to model paracrine signaling or endocrine signaling of the metastatic niche. In the former, fibroblasts conditioned by BC cultures with high EMP resulted in the largest FN matrix accumulation. In contrast, mesenchymal BC cells produced extracellular vesicles (EV) that resulted in the highest levels of matrix formation by conditioned fibroblasts. Overall, we demonstrate a dynamic relationship between tumor and stromal cells within the tumor microenvironment, in which the levels and fibrillarization of FN in the extracellular matrix are modulated during the particular stages of disease progression

Fibronectin-Expressing Mesenchymal Tumor Cells Promote Breast Cancer Metastasis

Tumor metastasis is connected to epithelial-mesenchymal heterogeneity (EMH) and the extracellular matrix (ECM) within the tumor microenvironment. Mesenchymal-like fibronectin (FN) expressing tumor cells enhance metastasis within tumors that have EMH. However, the secondary tumors are primarily composed of the FN null population. Interestingly, during tumor cell dissemination, the invasive front has more mesenchymal-like characteristics, although the outgrowths of metastatic colonies consist of a more epithelial-like population of cells. We hypothesize that soluble FN provided by mesenchymal-like tumor cells plays a role in supporting the survival of the more epithelial-like tumor cells within the metastatic niche in a paracrine manner. Furthermore, due to a lower rate of proliferation, the mesenchymal-like tumor cells become a minority population within the metastatic niche. In this study, we utilized a multi-parametric cell-tracking algorithm and immunoblotting to evaluate the effect of EMH on the growth and invasion of an isogenic cell series within a 3D collagen network using a microfluidic platform. Using the MCF10A progression series, we demonstrated that co-culture with FN-expressing MCF10CA1h cells significantly enhanced the survival of the more epithelial MCF10CA1a cells, with a two-fold increase in the population after 5 days in co-culture, whereas the population of the MCF10CA1a cells began to decrease after 2.5 days when cultured alone (p < 0.001). However, co-culture did not significantly alter the rate of proliferation for the more mesenchymal MCF10CA1h cells. Epithelial tumor cells not only showed prolonged survival, but migrated significantly longer distances (350 µm compared with 150 µm, respectively, p < 0.01) and with greater velocity magnitude (4.5 µm/h compared with 2.1 µm/h, respectively, p < 0.001) under co-culture conditions and in response to exogenously administered FN. Genetic depletion of FN from the MCF10CA1h cells resulted in a loss of survival and migration capacity of the epithelial and mesenchymal populations. These data suggest that mesenchymal tumor cells may function to support the survival and outgrowth of more epithelial tumor cells within the metastatic niche and that inhibition of FN production may provide a valuable target for treating metastatic disease