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

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

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

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

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

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

Discovering Molecules That Regulate Efferocytosis Using Primary Human Macrophages and High Content Imaging

<div><p>Defective clearance of apoptotic cells can result in sustained inflammation and subsequent autoimmunity. Macrophages, the “professional phagocyte” of the body, are responsible for efficient, non-phlogistic, apoptotic cell clearance. Controlling phagocytosis of apoptotic cells by macrophages is an attractive therapeutic opportunity to ameliorate inflammation. Using high content imaging, we have developed a system for evaluating the effects of antibody treatment on apoptotic cell uptake in primary human macrophages by comparing the Phagocytic Index (PI) for each antibody. Herein we demonstrate the feasibility of evaluating a panel of antibodies of unknown specificities obtained by immunization of mice with primary human macrophages and show that they can be distinguished based on individual PI measurements. In this study ~50% of antibodies obtained enhance phagocytosis of apoptotic cells while approximately 5% of the antibodies in the panel exhibit some inhibition. Though the specificities of the majority of antibodies are unknown, two of the antibodies that improved apoptotic cell uptake recognize recombinant MerTK; a receptor known to function in this capacity <i>in vivo</i>. The agonistic impact of these antibodies on efferocytosis could be demonstrated without addition of either of the MerTK ligands, Gas6 or ProS. These results validate applying the mechanism of this fundamental biological process as a means for identification of modulators that could potentially serve as therapeutics. This strategy for interrogating macrophages to discover molecules regulating apoptotic cell uptake is not limited by access to purified protein thereby increasing the possibility of finding novel apoptotic cell uptake pathways.</p></div

The majority of antibodies enhance apoptotic cell phagocytosis through recognition of unidentified surface molecules.

<p>Antibodies having a PI ≥ average PI + 1 SD (standard deviations) for cultures with no antibody treatment are considered as enhancing. Those having a PI≤ average PI—1 SD are considered inhibitory. All other antibodies having PI within 1 SD of average calculated for untreated MΦ’s are considered as having no effect. Frequency of each category of antibody is calculated as percent of total number of antibodies in the panel (left). Binding to known molecules could be detected in a small number of clones (right). Antibodies were evaluated by ELISA and readings that were ≥ 5-fold over background were considered positive.</p

Monoclonal antibodies capable of modulating efferocytosis are identified using high content image-based screening.

<p>Purified hybridoma supernatants were tested in a 384-well format using day 7 MΦ’s co-cultured with apoptotic Jurkat cells then analyzed on the Operetta High Content Imager. Results are expressed as PI (z-axis) and % positive cells (y-axis) for each sample (A) with controls represented as follows: cytochalasin D treated MΦ’s with apoptotic cells negative control (green squares); live cell, baseline phagocytosis (dark red squares); apoptotic cells with no antibody (blue squares); dexamethasone treated MΦ’s with apoptotic cells positive control (orange squares). Samples from each plate are grouped and represented by colored squares. The antibody panel contains molecules that exhibit positive and negative effects on MΦ efferocytosis as revealed by comparison of PI of treated cells to controls (B). Each diamond represents the PI data from an individual hybridoma and each is compared to the PI of MΦ’s incubated with apoptotic cells in absence of antibody (red line) which is geometric mean of 4 replicates 9.995 ± 7.54. Data shown are from testing antibodies on macrophages from a single donor, a second assay using cells from a different donor was also performed yielding similar results.</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