ABSTRACT
To determine the role for mutations of MECP2 in Rett syndrome, we generated isogenic lines of human induced pluripotent stem cells, neural progenitor cells, and neurons from patient fibroblasts with and without MECP2 expression in an attempt to recapitulate disease phenotypes in vitro. Molecular profiling uncovered neuronal-specific gene expression changes, including induction of a senescence-associated secretory phenotype (SASP) program. Patient-derived neurons made without MECP2 showed signs of stress, including induction of P53, and senescence. The induction of P53 appeared to affect dendritic branching in Rett neurons, as P53 inhibition restored dendritic complexity. The induction of P53 targets was also detectable in analyses of human Rett patient brain, suggesting that this disease-in-a-dish model can provide relevant insights into the human disorder.
Subject(s)
Cellular Senescence , Methyl-CpG-Binding Protein 2/deficiency , Neurons/metabolism , Neurons/pathology , Tumor Suppressor Protein p53/metabolism , Brain/metabolism , DNA Damage , Dendrites/metabolism , Gene Expression Regulation , Humans , Methyl-CpG-Binding Protein 2/metabolism , Models, Biological , Rett Syndrome/pathology , Transcriptome/geneticsABSTRACT
Intratumoral heterogeneity contributes to cancer drug resistance, but the underlying mechanisms are not understood. Single-cell analyses of patient-derived models and clinical samples from glioblastoma patients treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) demonstrate that tumor cells reversibly up-regulate or suppress mutant EGFR expression, conferring distinct cellular phenotypes to reach an optimal equilibrium for growth. Resistance to EGFR TKIs is shown to occur by elimination of mutant EGFR from extrachromosomal DNA. After drug withdrawal, reemergence of clonal EGFR mutations on extrachromosomal DNA follows. These results indicate a highly specific, dynamic, and adaptive route by which cancers can evade therapies that target oncogenes maintained on extrachromosomal DNA.
Subject(s)
Antineoplastic Agents/therapeutic use , Central Nervous System Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Glioblastoma/drug therapy , Molecular Targeted Therapy , Protein Kinase Inhibitors/therapeutic use , Animals , Central Nervous System Neoplasms/genetics , DNA/genetics , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride , Glioblastoma/genetics , Humans , Mice , Mutation , Neoplasm Transplantation , Quinazolines/therapeutic use , Single-Cell Analysis , Tumor Cells, Cultured , Withholding TreatmentABSTRACT
Nanoparticles are regarded as promising transfection reagents for effective and safe delivery of nucleic acids into a specific type of cells or tissues providing an alternative manipulation/therapy strategy to viral gene delivery. However, the current process of searching novel delivery materials is limited due to conventional low-throughput and time-consuming multistep synthetic approaches. Additionally, conventional approaches are frequently accompanied with unpredictability and continual optimization refinements, impeding flexible generation of material diversity creating a major obstacle to achieving high transfection performance. Here we have demonstrated a rapid developmental pathway toward highly efficient gene delivery systems by leveraging the powers of a supramolecular synthetic approach and a custom-designed digital microreactor. Using the digital microreactor, broad structural/functional diversity can be programmed into a library of DNA-encapsulated supramolecular nanoparticles (DNAâSNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNAâSNPs library identified the DNAâSNPs with the highest gene transfection efficiency, which can be attributed to cooperative effects of structures and surface chemistry of DNAâSNPs. We envision such a rapid developmental pathway can be adopted for generating nanoparticle-based vectors for delivery of a variety of loads.
Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Animals , Cell Line, Tumor , DNA/metabolism , Drug Delivery Systems , Gene Library , Humans , Light , Mice , Nanoparticles/chemistry , Scattering, Radiation , Surface Properties , TransfectionABSTRACT
The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform-microfluidic image cytometry (MIC)-capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine.
Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , Microfluidic Analytical Techniques/methods , Brain Neoplasms/metabolism , Cell Line, Tumor , ErbB Receptors/metabolism , Glioblastoma/metabolism , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/metabolism , Microfluidic Analytical Techniques/instrumentation , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Reproducibility of Results , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction , TOR Serine-Threonine KinasesABSTRACT
Microfluidic image cytometry (MIC) has been developed to study phenotypes of various hPSC lines by screening several chemically defined serum/feeder-free conditions. A chemically defined hPSC culture was established using 20 ng mL(-1) of bFGF on 20 microg mL(-1) of Matrigel to grow hPSCs over a week in an undifferentiated state. Following hPSC culture, we conducted quantitative MIC to perform a single cell profiling of simultaneously detected protein expression (OCT4 and SSEA1). Using clustering analysis, we were able to systematically compare the characteristics of various hPSC lines in different conditions.
Subject(s)
Cell Culture Techniques/instrumentation , Cell Separation/instrumentation , Flow Cytometry/instrumentation , Gene Expression Profiling/instrumentation , Microfluidic Analytical Techniques/instrumentation , Microscopy/instrumentation , Pluripotent Stem Cells/cytology , Cells, Cultured , Equipment Design , Equipment Failure Analysis , Humans , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
We demonstrated a convenient, flexible and modular synthetic approach for preparation of a small library of DNA-encapsulated supramolecular nanoparticles SNPs superset DNA and RGD-SNPs superset DNA with different sizes and RGD target ligand coverage for targeted gene delivery.
Subject(s)
DNA/chemistry , Gene Transfer Techniques , Nanoparticles/chemistry , Animals , Cell Line , DNA/metabolism , Humans , Mice , Nanoparticles/ultrastructure , Oligopeptides/chemistry , Oligopeptides/metabolism , Polyethylene Glycols/chemistryABSTRACT
Many biological and biomedical laboratory assays require the use of antibodies and antibody fragments that strongly bind to their cell surface targets. Conventional binding assays, such as the enzyme-linked immunosorbent assay (ELISA) and flow cytometry, have many challenges, including capital equipment requirements, labor intensiveness, and large reagent and sample consumption. Although these techniques are successful in mainstream biology, there is an unmet need for a tool to quickly ascertain the relative binding capabilities of antibodies/antibody fragments to cell surface targets on the benchtop at low cost. We describe a novel cell capture assay that enables several candidate antibodies to be evaluated quickly as to their relative binding efficacies to their cell surface targets. We used chimeric rituximab and murine anti-CD20 monoclonal antibodies as cell capture agents on a functionalized microscope slide surface to assess their relative binding affinities based on how well they capture CD20-expressing mammalian cells. We found that these antibodies' concentration-dependent cell capture profiles correlate with their relative binding affinities. A key observation of this assay involved understanding how differences in capture surfaces affect the assay results. This approach can find utility when an antibody or antibody fragment against a known cell line needs to be selected for targeting studies.