Chemical carcinogen-induced rat mammary carcinogenesis is a potential model of p21-activated kinase positive female breast cancer.

The p21-activated kinase 1 (PAK1) gene encodes a serine/threonine kinase that is overexpressed in a subset of human breast carcinomas with poor prognosis. The laboratory rat (Rattus norvegicus) orthologous gene is located at Mammary carcinoma susceptibility 3 (Mcs3) QTL on rat chromosome 1. We used quantitative PCR to determine effects of Mcs3 genotype and 7,12-dimethylbenz(a)anthracene (DMBA) exposure on Pak1 expression. There was no effect of Mcs3 genotype; however, there was a 3.5-fold higher Pak1 level in DMBA-exposed mammary glands (MGs) than in unexposed glands (P < 0.05). Sequence variants in Pak1 exons did not alter amino acid sequence between Mcs3-susceptible and -resistant strains. Protein expression of PAK1/Pak1 in human breast carcinomas and DMBA-exposed rat mammary glands was detected using immunohistochemistry (IHC). Rat mammary glands from 12-wk-old females unexposed to DMBA were negative for Pak1, whereas 24% of carcinogen-exposed mammary glands from age-matched females stained positive for Pak1. The positive mammary glands exposed to carcinogen had no pathological signs of disease. Human breast carcinomas, used as comparative controls, had a 22% positivity rats. This was consistent with other human breast cancer studies of PAK1 expression. Similar frequencies of human/rat PAK1/Pak1 expression in female breast carcinomas and carcinogen-induced rat mammary glands, showing no visible pathogenesis of disease, suggests aberrant PAK1 expression is an early event in development of some breast cancers. Laboratory rats will be a useful experimental organism for comparative studies of Pak1-mediated mechanisms of breast carcinogenesis. Future studies of PAK1 as a diagnostic marker of early breast disease are warranted.

A microwell array device capable of measuring single-cell oxygen consumption rates.

Due to interest in cell population heterogeneity, the development of new technology and methodologies for studying single cells has dramatically increased in recent years. The ideal single cell measurement system would be high throughput for statistical relevance, would measure the most important cellular parameters, and minimize disruption of normal cell function. We have developed a microwell array device capable of measuring single cell oxygen consumption rates (OCR). This OCR device is able to diffusionally isolate single cells and enables the quantitative measurement of oxygen consumed by a single cell with fmol/min resolution in a non-invasive and relatively high throughput manner. A glass microwell array format containing fixed luminescent sensors allows for future incorporation of additional cellular parameter sensing capabilities. To demonstrate the utility of the OCR device, we determined the oxygen consumption rates of a small group of single cells (12 to 18) for three different cells lines: murine macrophage cell line RAW264.7, human epithelial lung cancer cell line A549, and human Barrett's esophagus cell line CP-D.

Interface of an array of five capillaries with an array of one-nanoliter wells for high-resolution electrophoretic analysis as an approach to high-throughput chemical cytometry.

We report a system that allows the simultaneous aspiration of one or more cells into each of five capillaries for electrophoresis analysis. A glass wafer was etched to create an array of 1-nL wells. The glass was treated with poly(2-hydroxyethyl methacrylate) to control cell adherence. A suspension of formalin-fixed cells was placed on the surface, and cells were allowed to settle. The concentration of cells and the settling time were chosen so that there was, on average, one cell per well. Next, an array of five capillaries was placed so that the tip of each capillary was in contact with a single well. A pulse of vacuum was applied to the distal end of the capillaries to aspirate the content of each well into a capillary. Next, the tips of the capillaries were placed in running buffer and potential was applied. The cells lysed upon contact with the running buffer, and fluorescent components were detected at the distal end of the capillaries by laser-induced fluorescence. The electrophoretic separation efficiency was outstanding, generating over 750,000 theoretical plates (1,800,000 plates/m). In this example, AtT-20 cells were used that had been treated with TMR-G(M1). The cells were allowed to metabolize this substrate into a series of products before the cells were fixed. The number of cells found in each well was estimated visually under the microscope and was described by a Poisson distribution with mean of 0.98 cell/well. This system provides an approach to high-throughput chemical cytometry.

A cellular isolation system for real-time single-cell oxygen consumption monitoring.

The development of a cellular isolation system (CIS) that enables the monitoring of single-cell oxygen consumption rates in real time is presented. The CIS was developed through a multidisciplinary effort within the Microscale Life Sciences Center (MLSC) at the University of Washington. The system comprises arrays of microwells containing Pt-porphyrin-embedded polystyrene microspheres as the reporter chemistry, a lid actuator system and a gated intensified imaging camera, all mounted on a temperature-stabilized confocal microscope platform. Oxygen consumption determination experiments were performed on RAW264.7 mouse macrophage cells as proof of principle. Repeatable and consistent measurements indicate that the oxygen measurements did not adversely affect the physiological state of the cells measured. The observation of physiological rates in real time allows studies of cell-to-cell heterogeneity in oxygen consumption rate to be performed. Such studies have implications in understanding the role of mitochondrial function in the progression of inflammatory-based diseases, and in diagnosing and treating such diseases.

A New Approach for Measuring Single-Cell Oxygen Consumption Rates.

A novel system that has enabled the measurement of single-cell oxygen consumption rates is presented. The experimental apparatus includes a temperature controlled environmental chamber, an array of microwells etched in glass, and a lid actuator used to seal cells in the microwells. Each microwell contains an oxygen sensitive platinum phosphor sensor used to monitor the cellular metabolic rates. Custom automation software controls the digital image data collection for oxygen sensor measurements, which are analyzed using an image-processing program to yield the oxygen concentration within each microwell versus time. Two proof-of-concept experiments produced oxygen consumption rate measurements for A549 human epithelial lung cancer cells of 5.39 and 5.27 fmol/min/cell, closely matching published oxygen consumption rates for bulk A549 populations.

Phosphorescence lifetime based oxygen micro-sensing using a digital micromirror device.

A digital light modulation microscope (DLMM) that utilizes a digital micromirror device (DMD) on an epifluorescence microscope has been developed to modulate excitation light in spatial and temporal domains for phosphorescence lifetime detection. Local O2 concentration can be inferred through the detected lifetime around an O2-quenching phosphorescent porphyrin microsensor. Combined with microsensor arrays, the DLMM can sequentially address light to each microsensor element to construct a discrete lifetime image or O2 distribution. In contrast to conventional phosphorescence lifetime imaging, the new method eliminates the need for a pulsed light source and a time-gated camera. To demonstrate O2 sensing with lab-on-a-chip devices, an array of 150-mum-diameter micro-wells coated with phosphorescent porphyrin were observed. The locations of the sensor elements were automatically identified though image analysis. The goal of this platform is to measure the O2 consumption of individual cells trapped in the microwells.

Single cell methods for methane oxidation analysis.

Respiration is widely used for evaluation of the metabolic capabilities or physiological state of the microbial culture. This chapter describes novel approaches for characterization of respiration at a single cell level: (1) flow cytometry-based redox sensing (FCRS) of actively metabolizing microbes; (2) respiration response imaging (RRI) for real-time detection of substrate stimulated redox responses of individual cells; (3) respiration detection system: microobservation chamber (RDS: MC), a single cell analysis system for carrying out the physiological and genomic profiling of cells capable of respiring C(1) compounds. The techniques are suitable for description of physiological heterogeneity among cells in a single microbial population and could be used to characterize distribution of methylotrophic ability among microbial cells in the natural environmental samples.

Direct measurement of oxygen consumption rates from attached and unattached cells in a reversibly sealed, diffusionally isolated sample chamber.

Oxygen consumption is a fundamental component of metabolic networks, mitochondrial function, and global carbon cycling. To date there is no method available that allows for replicate measurements on attached and unattached biological samples without compensation for extraneous oxygen leaking into the system. Here we present the Respiratory Detection System, which is compatible with virtually any biological sample. The RDS can be used to measure oxygen uptake in microliter-scale volumes with a reversibly sealed sample chamber, which contains a porphyrin-based oxygen sensor. With the RDS, one can maintain a diffusional seal for up to three hours, allowing for the direct measurement of respiratory function of samples with fast or slow metabolic rates. The ability to easily measure oxygen uptake in small volumes with small populations or dilute samples has implications in cell biology, environmental biology, and clinical diagnostics.

FORMING SELF-ASSEMBLED CELL ARRAYS AND MEASURING THE OXYGEN CONSUMPTION RATE OF A SINGLE LIVE CELL.

We report a method for forming arrays of live single cells on a chip using polymer micro-traps made of SU8. We have studied the toxicity of the microfabricated structures and the associated environment for two cell lines. We also report a method for measuring the oxygen consumption rate of a single cell using optical interrogation of molecular oxygen sensors placed in micromachined micro-wells by temporarily sealing the cells in the micro-traps. The new techniques presented here add to the collection of tools available for performing "single-cell" biology. A single-cell self-assembly yield of 61% was achieved with oxygen draw down rates of 0.83, 0.82, and 0.71 fmol/minute on three isolated live A549 cells.