ABSTRACT
Nanoparticle-based therapy represents a novel and promising approach to treat glioblastoma, the most common and lethal malignant brain cancer. Although similar therapies have achieved significant cytotoxicity in cultured glioblastoma or glioblastoma stem cells (GSCs), the lack of an appropriate approach to monitor interactions between cells and nanoparticle-based therapies impedes their further clinical application in human patients. To address this critical issue, we first obtained NOTCH1 positive GSCs from patient-derived primary cultures. We then developed a new imaging approach to directly observe the dynamic nature of nanoparticles at the molecular level using in situ transmission electron microscopy (TEM). Utilizing these tools we were able to visualize real-time movements of nanoparticles interacting with GSCs for the first time. Overall, we show strong proof-of-concept results that real-time visualization of nanoparticles in single cells can be achieved at the nanoscale using TEM, thereby providing a powerful platform for the development of nanotherapeutics.
Subject(s)
Glioblastoma/ultrastructure , Lab-On-A-Chip Devices , Microscopy, Electron, Transmission/instrumentation , Molecular Imaging/instrumentation , Nanoparticles/ultrastructure , Neoplastic Stem Cells/chemistry , Cell Line, Tumor , Computer Systems , Equipment Design , Equipment Failure Analysis , Glioblastoma/chemistry , Humans , Image Enhancement/instrumentation , Nanoparticles/chemistry , Neoplastic Stem Cells/ultrastructureABSTRACT
The precise manner in which physical changes to the breast cancer susceptibility protein (BRCA1) affect its role in DNA repair events remain unclear. Indeed, cancer cells harboring mutations in BRCA1 suffer from genomic instability and increased DNA lesions. Here, we used a combination of molecular imaging and biochemical tools to study the properties of the BRCA1 in human cancer cells. Our results reveal new information for the manner in which full-length BRCA1 engages its binding partner, the BRCA1-associated Ring Domain protein (BARD1) under oxidative stress conditions. We also show how physical differences between wild type and mutated BRCA15382insC impact the cell's response to oxidative damage. Overall, we demonstrate how clinically relevant changes to BRCA1 affect its structure-function relationship in hereditary breast cancer.
Subject(s)
BRCA1 Protein/chemistry , DNA Repair , Gene Expression Regulation, Neoplastic , Tumor Suppressor Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Binding Sites , Cell Line, Tumor , DNA Damage , Female , Guanine/analogs & derivatives , Guanine/metabolism , Humans , Hydrogen Peroxide/pharmacology , Mammary Glands, Human/drug effects , Mammary Glands, Human/metabolism , Mammary Glands, Human/pathology , Models, Molecular , Molecular Imaging , Mutation , Oxidative Stress , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Structural Homology, Protein , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Understanding the properties of protein-based therapeutics is a common goal of biologists and physicians. Technical barriers in the direct observation of small proteins or therapeutic agents can limit our knowledge of how they function in solution and in the body. Electron microscopy (EM) imaging performed in a liquid environment permits us to peer into the active world of cells and molecules at the nanoscale. Here, we employ liquid cell EM to directly visualize a protein-based therapeutic in its native conformation and aggregate state in a time-resolved manner. In combination with quantitative analyses, information from this work contributes new molecular insights toward understanding the behaviours of immunotherapies in a solution state that mimics the human body.
Subject(s)
Microscopy, Electron/methods , Protein Aggregates , Drug Compounding , Interferon-alpha/chemistry , Interferon-alpha/therapeutic use , Polyethylene Glycols/chemistry , Protein Conformation , Time FactorsABSTRACT
We present a new molecular toolkit to investigate protein assemblies natively formed in the context of human disease. The system employs tunable microchips that can be decorated with switchable adaptor molecules to select for target proteins of interest and analyze them using molecular microscopy. Implementing our new streamlined microchip approach, we could directly visualize BRCA1 gene regulatory complexes from patient-derived cancer cells for the first time.