Tunable biomaterials that mimic selected features of the extracellular matrix (ECM) such as its stiffness, protein composition, and dimensionality are increasingly popular for studying how cells ...sense and respond to ECM cues. In the field, there exists a significant trade-off for how complex and how well these biomaterials represent the in vivo microenvironment versus how easy they are to make and how adaptable they are to automated fabrication techniques. To address this need to integrate more complex biomaterials design with high-throughput screening approaches, we present several methods to fabricate synthetic biomaterials in 96-well plates and demonstrate that they can be adapted to semiautomated liquid handling robotics. These platforms include (1) glass bottom plates with covalently attached ECM proteins and (2) hydrogels with tunable stiffness and protein composition with either cells seeded on the surface or (3) laden within the three-dimensional hydrogel matrix. This study includes proof-of-concept results demonstrating control over breast cancer cell line phenotypes via these ECM cues in a semiautomated fashion. We foresee the use of these methods as a mechanism to bridge the gap between high-throughput cell-matrix screening and engineered ECM-mimicking biomaterials.
Abstract
Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and clinical application. Many ...methods exist to create biomaterial platforms, including hydrogels, which we use to study cells in contexts more akin to what they experience in vivo. Our lab has multiple approaches to create such biomaterials, based on combinations of poly(ethylene glycol) (PEG) with peptides and zwitterions. In this presentation, I will discuss our findings in using these cell culture environments to understand the role of the extracellular matrix (ECM) in controlling cancer cell innate drug response via adaptive signaling. Specifically, I will present data comparing the behavior of breast, prostate, and ovarian cancer cells to chemotherapy and targeted drugs when cultured as 3D spheroids, on 2D gels, and as a function of the stiffness of the tumor microenvironment. This approach uncovered that cells on 2D hydrogels and spheroids encapsulated in 3D hydrogels were less responsive to receptor tyrosine kinase (RTK)-targeting drugs sorafenib and lapatinib, but not cytotoxic drugs, compared to single cells in hydrogels and cells on plastic. We found that transcriptomic differences between these in vitro models and tumor xenografts did not reveal mechanisms of ECM-mediated resistance to sorafenib. However, a systems biology analysis of phospho-kinome data uncovered that variation in MEK phosphorylation was associated with RTK-targeted drug resistance. Using sorafenib as a model drug, we found that co-administration with a MEK inhibitor decreased ECM-mediated resistance in vitro and reduced in vivo tumor burden compared to sorafenib alone. In sum, we provide a novel strategy for identifying and overcoming ECM-mediated resistance mechanisms by performing drug screening, phospho-kinome analysis, and systems biology across multiple biomaterial environments. Our work suggests that different model systems are important for evaluating cell response to receptor tyrosine kinase inhibitors, whose efficacy depends on cell-ECM interactions.
Citation Format: Alyssa Schwartz, Lauren Barney, Lauren Jansen, Christopher Hall, Aaron Meyer, Shelly R. Peyton. Biomaterials and systems biology to guide breast cancer drug screening abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 40.
Primary amebic meningoencephalitis (PAM) is a rare and lethal infection caused by Naegleria fowleri. We report an epidemiological and environmental investigation relating to a case of PAM in a ...previously healthy boy age 8 years. An interview of the patient's family was conducted to determine the likely exposure site and to assess risk factors. Data from the United States Geological Survey site at Waterloo, NE, on the Elkhorn River were used to estimate water temperature and streamflow at the time and site of exposure. Data from the National Weather Service were used to estimate precipitation and ambient air temperature at the time and site of exposure. Despite conventional treatment, the patient died 2 days after hospital admission. The patient participated in recreational water activities in the Elkhorn River in northeastern Nebraska 5 days before symptom onset. In the week before exposure, water and ambient air high temperatures reached annual highs, averaging 32.4°C and 35.8°C, respectively. The day before infection, 2.2 cm of precipitation was reported. Streamflow was low (407 ft3/s). Infections in several northern states, including Nebraska, suggest an expanding geographic range of N. fowleri transmission, which may lead to increased incidence of PAM in the United States. Similar environmental investigations at suspected exposure sites of future cases will allow data aggregation, enabling investigators to correlate environmental factors with infection risk accurately.
Extraction of genomic DNA (gDNA) from fresh whole blood is the first step in multiple translational research and molecular diagnostics applications, such as next-generation sequencing (NGS), ...multiplex PCR, qPCR, and droplet digital PCR (ddPCR). For example, DNA from whole blood is used as a matched-control for solid tumor somatic mutation profiling and for the detection of clinically-relevant variants in hematological malignancies, such as leukemia and myelodysplastic syndrome. An increasing number of clinical studies demonstrate the value of detecting disease-specific biomarkers from blood for early detection, diagnosis, monitoring treatment efficacy, and cohort study recruitment. Scaling down fresh blood volumes while scaling up processing capabilities is desirable to maximize laboratory throughput. At present, most DNA extraction methods require high volumes of blood and are challenging to automate because centrifugation or vacuum equipment are necessary. Additionally, conventional column and magnetic-based workflows need larger volumes for wash and elution steps, which require the use of deep well plates and other specialized consumables. Larger volumes are also needed to avoid excessive viscosity of the lysate, which can interfere with magnetic bead separation. To circumvent sample and process-related challenges, Covaris has adapted the oneTUBE-10 Plate for streamlining high-throughput nucleic acid extractions from whole blood using Adaptive Focused Acoustics (AFA) technology. Here, we performed 12 DNA extractions from healthy donors to evaluate recoveries, sample quality, and purity. Our results show recoveries in the range between 376 to 809 ng with an average fragment size of >1 kb. We also show that the extracted and purified DNA is devoid of any detectable PCR inhibiting contaminants. Taken together, this less than 90-minute AFA-enabled workflow significantly improves blood cell lysis and reduces hands-on time. The workflow can be performed on a liquid handler using the integrated Covaris R230 Focused-ultrasonicator, or off-deck using the LE220-plus Focused-ultrasonicator.
The U.S. maternal mortality rate has doubled in the past 25 years and has risen despite improvements in health care and an overwhelming global trend in the other direction. Forty-five countries have ...lower maternal mortality rates than the United States (CIA World Factbook, 2018). For a country that spends more than any other country on health care and more on childbirth-related care than any other area of hospitalization, this is a shockingly poor return on investment. After prolonged attention during pregnancy and birth, there is relatively little attention to the mother's health and well-being in the postpartum period. Yet more than half of childbirth-related deaths occur during this time (Muza, 2017). To minimize complications leading to maternal mortality, childbirth educators need to teach mother and families to identify and respond promptly to warning signs of postpartum complications.
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