Mars oxygen production system design

The design and construction phase is summarized of the Mars oxygen demonstration project. The basic hardware required to produce oxygen from simulated Mars atmosphere was assembled and tested. Some design problems still remain with the sample collection and storage system. In addition, design and development of computer compatible data acquisition and control instrumentation is ongoing

Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness <i>In Vitro</i> – Implication for Drug Development

<div><p>Three-dimensional (3D) cell culture is gaining acceptance in response to the need for cellular models that better mimic physiologic tissues. Spheroids are one such 3D model where clusters of cells will undergo self-assembly to form viable, 3D tumor-like structures. However, to date little is known about how spheroid biology compares to that of the more traditional and widely utilized 2D monolayer cultures. Therefore, the goal of this study was to characterize the phenotypic and functional differences between lung tumor cells grown as 2D monolayer cultures, versus cells grown as 3D spheroids. Eight lung tumor cell lines, displaying varying levels of epidermal growth factor receptor (EGFR) and cMET protein expression, were used to develop a 3D spheroid cell culture model using low attachment U-bottom plates. The 3D spheroids were compared with cells grown in monolayer for 1) EGFR and cMET receptor expression, as determined by flow cytometry, 2) EGFR and cMET phosphorylation by MSD assay, and 3) cell proliferation in response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In addition, drug responsiveness to EGFR and cMET inhibitors (Erlotinib, Crizotinib, Cetuximab [Erbitux] and Onartuzumab [MetMab]) was evaluated by measuring the extent of cell proliferation and migration. Data showed that EGFR and cMET expression is reduced at day four of untreated spheroid culture compared to monolayer. Basal phosphorylation of EGFR and cMET was higher in spheroids compared to monolayer cultures. Spheroids showed reduced EGFR and cMET phosphorylation when stimulated with ligand compared to 2D cultures. Spheroids showed an altered cell proliferation response to HGF, as well as to EGFR and cMET inhibitors, compared to monolayer cultures. Finally, spheroid cultures showed exceptional utility in a cell migration assay. Overall, the 3D spheroid culture changed the cellular response to drugs and growth factors and may more accurately mimic the natural tumor microenvironment.</p></div

cMET but not EGFR inhibitors reduced cell migration in HGF stimulated lung tumor spheroids.

<p>A) Day 4 lung spheroids generated from H292, H1650, H1975 and A549 were treated with Erlotinib, Cetuximab, Crizotinib and MetMab in a dilution series in the presence of 20 ng/ml of HGF for 48 hours to stimulate cell migration. Total area (μm²) of migrating and spheroid were determined by using bright field images in a fully automated Operetta high content imaging system (Perkin Elmer). Cell migration (total area) was normalized to media only control to create a percentage cell migration to control. Data are means <u>+</u> SEM with two to five replicates and is representative of two independent experiments. B) Representative bright field images showing drug response after 48 hours in 3D spheroids in cell migration at the highest concentration. Magnification: 2x objective, scan bar 1 mm.</p

EGFR (A) and cMET (B) receptor density was reduced in 3D spheroid culture compared to 2D monolayer.

<p>Day four 2D monolayer cultures and 3D spheroid from seven lung tumor cell lines were measured for EGFR (anti-human EGFR-PE) and cMET (anti-human HGF R-PE) receptor density by flow cytometry. 1×10⁴ total cell events were collected for each sample. Receptor density was determined by using QuantiBRITE PE beads and is representative of two independent experiments.</p

Generation of highly reproducible 3D lung tumor spheroids in culture.

<p>A) Eight cell lines were monitored over six days for formation and growth of tumor spheroids. Bright field images were taken daily. Magnification: 2x objective, scan bar: 1mm. B) Total area (μm²) of the tumor spheroids at day three were measured using the Operetta imaging system. N is equal to two to five replicates. C) Cell viability was determined at day one and three in six of the eight cell lines by CellTiter-Glo Assay (Promega). N is equal to fourteen replicates.</p

Summary of differences between lung tumor cells grown in a 2D monolayer or 3D spheroid culture.

<p>Summary of differences between lung tumor cells grown in a 2D monolayer or 3D spheroid culture.</p

Characteristics of Lung tumor cell lines compared in 3D and 2D culture systems [47].

<p>Characteristics of Lung tumor cell lines compared in 3D and 2D culture systems <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092248#pone.0092248-Thomson1" target="_blank">[47]</a>.</p

EC50 for EGFR/cMET compounds inhibiting cell migration and cell viability from a 3D lung tumor spheroid.

<p>Total area (μm²) of migration pattern and spheroid were determined by using bright field images in a fully automated Operetta high content imaging system (Perkin Elmer). Cell viability (RLU) was determined after cell migration by CellTiter Glo.</p

Proliferation response to EGF and HGF is altered in 3D compared to 2D.

<p>The plot displays the expected growth surface (i.e. the predicted growth response from the model that was estimated from the data) across varying concentrations of EGF and HGF using the baseline growth estimated for Plate 1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092248#pone.0092248.s003" target="_blank">Figure S3</a>). H1975 cells were plated as a monolayer on a flat bottom plate (2D) or a spheroid in a ULA round bottom plate (3D) and were stimulated with HGF and EGF (250 ng/ml with six serial 1∶2 dilutions) at various combinations at day 1 for 48 hours. These concentrations were dosed in either flat bottom or ULA round bottom plate. Growth (RLU values) was measured in each well by Cell Titer-Glo Assay and was normalized in terms of percent control. The expected growth surface was generated from the linear growth model that included a linear and quadratic terms for EGF and HGF, as well as an interaction term between EGF and HGF.</p

Compounds have an anti-proliferative effect in cell migration assay in HGF stimulated lung tumor spheroids.

<p>Day 4 lung spheroids generated from H292, H1650, H1975 and A549 were treated with Erlotinib, Cetuximab, Crizotinib and MetMab in a dilution series in the presence of 20/ml of HGF for 48 hours to stimulate cell migration. Cell proliferation was determined by CellTiter-Glo Assay (Promega). Cell proliferation (RLU) was normalized to untreated control. Data are means <u>+</u> SEM with two to five replicates and is representative of two independent experiments.</p