Minori Ohashi

Photothermal effects of supramolecularly assembled gold nanoparticles for the targeted treatment of cancer cells.

Noble-metal nanostructures with unique photophysical properties have been considered as prime candidate agents for the photothermal treatment of cancer.[1–4] Typically, the photothermal properties of these nanostructures can be controlled by manipulating their sizes and shapes.[4,5] Over the past decade, significant endeavors have been devoted to the production of a variety of gold nanostructures, such as nanoparticles,[6,7] nanoshells,[8–10] nanorods,[11,12] and nanocages,[5,13,14] which are able to overcome limitations of organic-dye-based photothermal agents,[7] such as low light absorption and undesired photobleaching. For sufficient energy to be harvested/generated to damage tumor cells, the size of these nanostructure-based agents are required in the range of tens to hundreds nm.[15] However, the relatively “large” size of the agents often leads to poor bioclearance (i.e., accumulation in the liver, spleen, and kidneys), which is a major obstacle to their in vivo application.[16–18] Alternatively, the photophysical properties of noble-metal nanostructures can be altered systematically by the formation of aggregates through self-assembly.[19–30] The antibody-assisted aggregation of Au nanoparticles on cell membranes or in intracellular environments led to the enhancement of photothermal performance[31] as a result of the collective effects[32,33] associated with the assembled structures. Therefore, the self-assembly of small noble-metal building blocks, that is, noble-metal colloids with diameters of less than 8 nm[16–18] (compatible with renal clearance) would be a promising approach toward a new class of noble-metal photothermal agents.

Targeted Therapy Resistance Mediated by Dynamic Regulation of Extrachromosomal Mutant EGFR DNA

Playing Hide and Seek Targeted cancer therapies have shown promising results in patients, but few of these drugs provide long-term benefits because tumor cells rapidly develop drug resistance. Nathanson et al. (p. 72, published online 5 December) show that glioblastoma cells can become resistant to erlotinib, an epidermal growth factor receptor (EGFR)–targeted drug, by eliminating extrachromosomal copies of the mutant EGFR gene. After a period of drug withdrawal, the mutant EGFR gene reappears on extrachromosomal DNA and the tumor cells become resensitized. The discovery that cancer cells can evade drug therapy by this “hide and seek” mechanism may help to optimize the dosing schedule of erlotinib in glioblastoma patients. Tumor cells become resistant to targeted therapies by eliminating the gene encoding the drug target from extrachromosomal DNA. 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.

A rapid pathway toward a superb gene delivery system: programming structural and functional diversity into a supramolecular nanoparticle library.

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 (DNA⊂SNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNA⊂SNPs library identified the DNA⊂SNPs with the highest gene transfection efficiency, which can be attributed to cooperative effects of structures and surface chemistry of DNA⊂SNPs. We envision such a rapid developmental pathway can be adopted for generating nanoparticle-based vectors for delivery of a variety of loads.

A Microfluidic Platform for Systems Pathology: Multiparameter Single-Cell Signaling Measurements of Clinical Brain Tumor Specimens

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.

Microfluidic image cytometry for quantitative single-cell profiling of human pluripotent stem cells in chemically defined conditions †

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.

A small library of DNA-encapsulated supramolecular nanoparticles for targeted gene delivery

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.

Loss of MECP2 Leads to Activation of P53 and Neuronal Senescence

Summary 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.

Loss of MECP2 leads to telomere dysfunction and neuronal stress

To determine the role for mutations of MECP2 in Rett Syndrome, we generated isogenic lines of human iPSCs (hiPSCs), neural progenitor cells (NPCs), 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 show 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. These disease-in-a-dish data suggest that loss of MECP2 can lead to dendritic defects due to an increase in aspects of neuronal aging.