Ryan M. Phillips

Benchtop micromolding of polystyrene by soft lithography

Polystyrene (PS), a standard material for cell culture consumable labware, was molded into microstructures with high fidelity of replication by an elastomeric polydimethylsiloxane (PDMS) mold. The process was a simple, benchtop method based on soft lithography using readily available materials. The key to successful replica molding by this simple procedure relies on the use of a solvent, for example, gamma-butyrolactone, which dissolves PS without swelling the PDMS mold. PS solution was added to the PDMS mold, and evaporation of the solvent was accomplished by baking the mold on a hotplate. Microstructures with feature sizes as small as 3 μm and aspect ratios as large as 7 were readily molded. Prototypes of microfluidic chips made from PS were prepared by thermal bonding of a microchannel molded in PS with a flat PS substrate. The PS microfluidic chip displayed much lower adsorption and absorption of hydrophobic molecules (e.g. rhodamine B) compared to a comparable chip created from PDMS. The molded PS surface exhibited stable surface properties after plasma oxidation as assessed by contact angle measurement. The molded, oxidized PS surface remained an excellent surface for cell culture based on cell adhesion and proliferation. To demonstrate the application of this process for cell biology research, PS was micromolded into two different microarray formats, microwells and microposts, for segregation and tracking of non-adherent and adherent cells, respectively. The micromolded PS possessed properties that were ideal for biological and bioanalytical needs, thus making it an alternative material to PDMS and suitable for building lab-on-a-chip devices by soft lithography methods.

Measurement of protein tyrosine phosphatase activity in single cells by capillary electrophoresis.

A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn(2+) and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min(-1) mg(-1)) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min(-1) mg(-1)). Zn(2+), 1,2-naphthoquinone, and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 μM, and 100 nM, respectively. Dephosphorylation of the reporter, following loading into living single cells, occurred at rates of at least 2 pmol min(-1) mg(-1). When single cells were exposed to 1,2-naphthoquinone (50 μM), Zn(2+) (100 μM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens.

Chemoproteomic Discovery of AADACL1 as a Regulator of Human Platelet Activation

A comprehensive knowledge of the platelet proteome is necessary for understanding thrombosis and for envisioning antiplatelet therapies. To discover other biochemical pathways in human platelets, we screened platelets with a carbamate library designed to interrogate the serine hydrolase subproteome and used competitive activity-based protein profiling to map the targets of active carbamates. We identified an inhibitor that targets arylacetamide deacetylase-like 1 (AADACL1), a lipid deacetylase originally identified in invasive cancers. Using this compound, along with highly selective second-generation inhibitors of AADACL1, metabolomics, and RNA interference, we show that AADACL1 regulates platelet aggregation, thrombus growth, RAP1 and PKC activation, lipid metabolism, and fibrinogen binding to platelets and megakaryocytes. These data provide evidence that AADACL1 regulates platelet and megakaryocyte activation and highlight the value of this chemoproteomic strategy for target discovery in platelets.

Ex Vivo Chemical Cytometric Analysis of Protein Tyrosine Phosphatase Activity in Single Human Airway Epithelial Cells

We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min(-1) mg(-1)) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (76%, 69%, and 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min(-1) mg(-1) and was also effectively inhibited by preincubation of the cells with the inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min(-1) mg(-1) resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34 to 36 pmol min(-1) mg(-1) (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues.

Abstract 4128: A novel approach to the measurement of tyrosine phosphorylation dynamics in intact single cells using capillary electrophoresis.

Receptor tyrosine kinases (RTKs) are key components of physiological and pathological cell signaling, and are normally subject to tight regulation by protein tyrosine phosphatases (PTPs). Irregularities on either side of this equilibrium can lead to inflammation, metabolic disease, and cancer. While many established and emerging techniques have been used to characterize these signaling pathways, few approaches are well adapted to the analysis of single primary cells, and fewer still measure enzyme activity directly. We report a new method for measuring the balance between RTK and PTP activity using fluorescent peptide substrates in conjunction with capillary electrophoresis. Briefly, live cells are visualized on an inverted microscope, microinjected with a fluorescent peptide substrate which is acted upon by RTKs and/or PTPs. After the desired time interval, a single cell is lysed using a microsecond laser pulse and cell contents are aspirated into a fused-silica capillary where electrophoresis is performed and reporter species are separated and quantified based on phosphorylation status. We have applied this technique to the characterization of PTP inhibition in single A431 cells by several components of diesel exhaust particles, an important component of air pollution linked to cancer. We have also adapted this system to measure the effects of small-molecule epidermal growth factor receptor (EGFR) inhibitors on RTK activity in tumor cell lines as well as tumor xenograft models, also at the single cell level. This approach provides a unique set of advantages over alternative techniques and is a valuable tool for the study of cell signaling in cancer. The integrated microscopy permits selection of cells for analysis based on morphology, viability, or vital staining, an important consideration for analysis of heterogeneous samples such as tumor specimens, especially when total sample size is small. High-sensitivity detection allows chemical cytometry data from individual cells to be gathered and the presence of phenotypic subpopulations is not lost by observing only the average behavior of a large population. Additionally, peptide substrate reporters can be tuned for enzyme specificity, and the analytical separation allows multiple reporter pairs to be separated and detected in a single experiment, permitting simultaneous analysis of multiple enzymes. The introduction of reporter peptides by microinjection preserves cellular architecture, while being equally applicable to immortalized and primary cells since no genetic manipulation is necessary. This array of advantages makes our approach uniquely well suited to the study of RTK and PTP signaling in both cultured tumor cell lines and patient specimens and a valuable new tool for cancer research. Citation Format: Ryan M. Phillips, David S. Lawrence, Christopher E. Sims, Nancy L. Allbritton. A novel approach to the measurement of tyrosine phosphorylation dynamics in intact single cells using capillary electrophoresis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4128. doi:10.1158/1538-7445.AM2013-4128

Measuring the Enzymatic Activity of Clinically Important Proteins in Single Cells

Molecularly targeted therapies are at the forefront of clinical science, and are expected to lead to personalization of medical treatments for each patient. Most such therapies are directed at inhibiting specific signal transduction enzymes or pathways, thus creating a critical need for assays capable of measuring the activities of these proteins in disease models and in patient samples. The ability to measure relevant enzyme activity in primary cell samples at baseline and/or after treatment would provide the ability to tailor patient therapy based on aberrant signal transduction, validate mechanisms of resistance in patients, and would offer an invaluable pharmacodynamic tool to assess whether resistance is associated with inadequate target inhibition. Here we report our most recent efforts to create the analytical and chemical tools needed to directly measure the enzymatic activities of therapeutic targets including protein kinases, lipid-modifying enzymes and the proteasome. Fluorescent reagents are under development that report the activity of these various enzymes with the goal of performing biochemical assays in primary cells. The basic design incorporates enzyme substrates that are modified to create compounds which can be loaded into cells where they are modified by the enzyme of interest. Work has included modification of peptides to confer membrane permeability and to achieve long intracellular lifetimes. Microelectrophoretic separations combined with low-level fluorescence detection enable the quantitative analysis of these compounds from single mammalian cells. This capability addresses three major issues currently faced in the biochemical analysis of clinical samples: the need for direct measurement of the enzymatic activity of target proteins; sample size requirements that are feasible for clinical implementation; and sample heterogeneity that can mask pertinent aspects related to therapeutic response.