Bio-functionalization of Electrospun Nanofibre Scaffolds for Cell Culture Applications

Ph.DDOCTOR OF PHILOSOPH

Screening therapeutic EMT blocking agents in a three-dimensional microenvironment

Epithelial–mesenchymal transition (EMT) plays a critical role in the early stages of dissemination of carcinoma leading to metastatic tumors, which are responsible for over 90% of all cancer-related deaths. Current therapeutic regimens, however, have been ineffective in the cure of metastatic cancer, thus an urgent need exists to revisit existing protocols and to improve the efficacy of newly developed therapeutics. Strategies based on preventing EMT could potentially contribute to improving the outcome of advanced stage cancers. To achieve this goal new assays are needed to identify targeted drugs capable of interfering with EMT or to revert the mesenchymal-like phenotype of carcinoma to an epithelial-like state. Current assays are limited to examining the dispersion of carcinoma cells in isolation in conventional 2-dimensional (2D) microwell systems, an approach that fails to account for the 3-dimensional (3D) environment of the tumor or the essential interactions that occur with other nearby cell types in the tumor microenvironment. Here we present a microfluidic system that integrates tumor cell spheroids in a 3D hydrogel scaffold, in close co-culture with an endothelial monolayer. Drug candidates inhibiting receptor activation or signal transduction pathways implicated in EMT have been tested using dispersion of A549 lung adenocarcinoma cell spheroids as a metric of effectiveness. We demonstrate significant differences in response to drugs between 2D and 3D, and between monoculture and co-culture.Singapore. National Research Foundation (Singapore MIT Alliance for Research and Technology's BioSystems and Micromechanics Inter-Disciplinary Research programme)National University of Singapore (Cancer Science Institute)Singapore. Agency for Science, Technology and ResearchSingapore. Institute of Molecular and Cell Biology (IMCB core funding A*STAR

Screening therapeutic EMT blocking agents in a three-dimensional microenvironment

Epithelial–mesenchymal transition (EMT) plays a critical role in the early stages of dissemination of carcinoma leading to metastatic tumors, which are responsible for over 90% of all cancer-related deaths. Current therapeutic regimens, however, have been ineffective in the cure of metastatic cancer, thus an urgent need exists to revisit existing protocols and to improve the efficacy of newly developed therapeutics. Strategies based on preventing EMT could potentially contribute to improving the outcome of advanced stage cancers. To achieve this goal new assays are needed to identify targeted drugs capable of interfering with EMT or to revert the mesenchymal-like phenotype of carcinoma to an epithelial-like state. Current assays are limited to examining the dispersion of carcinoma cells in isolation in conventional 2-dimensional (2D) microwell systems, an approach that fails to account for the 3-dimensional (3D) environment of the tumor or the essential interactions that occur with other nearby cell types in the tumor microenvironment. Here we present a microfluidic system that integrates tumor cell spheroids in a 3D hydrogel scaffold, in close co-culture with an endothelial monolayer. Drug candidates inhibiting receptor activation or signal transduction pathways implicated in EMT have been tested using dispersion of A549 lung adenocarcinoma cell spheroids as a metric of effectiveness. We demonstrate significant differences in response to drugs between 2D and 3D, and between monoculture and co-culture.Singapore. National Research Foundation (Singapore MIT Alliance for Research and Technology's BioSystems and Micromechanics Inter-Disciplinary Research programme)National University of Singapore (Cancer Science Institute)Singapore. Agency for Science, Technology and ResearchSingapore. Institute of Molecular and Cell Biology (IMCB core funding A*STAR

A Cell-Based Small Molecule Screening Method for Identifying Inhibitors of Epithelial-Mesenchymal Transition in Carcinoma

Epithelial Mesenchymal Transition (EMT) is a crucial mechanism for carcinoma progression, as it provides routes for in situ carcinoma cells to dissociate and become motile, leading to localized invasion and metastatic spread. Targeting EMT therefore represents an important therapeutic strategy for cancer treatment. The discovery of oncogene addiction in sustaining tumor growth has led to the rapid development of targeted therapeutics. Whilst initially optimized as anti-proliferative agents, it is likely that some of these compounds may inhibit EMT initiation or sustenance, since EMT is also modulated by similar signaling pathways that these compounds were designed to target. We have developed a novel screening assay that can lead to the identification of compounds that can inhibit EMT initiated by growth factor signaling. This assay is designed as a high-content screening assay where both cell growth and cell migration can be analyzed simultaneously via time-course imaging in multi-well plates. Using this assay, we have validated several compounds as viable EMT inhibitors. In particular, we have identified compounds targeting ALK5, MEK, and SRC as potent inhibitors that can interfere with EGF, HGF, and IGF-1 induced EMT signaling. Overall, this EMT screening method provides a foundation for improving the therapeutic value of recently developed compounds in advanced stage carcinoma

Controlled release of heparin from poly(epsilon-caprolactone) electrospun fibers

Sustained delivery of heparin to the localized adventitial surface of grafted blood vessels has been shown to prevent the vascular smooth muscle cell (VSMC) proliferation that can lead to graft occlusion and failure. In this study heparin was incorporated into electrospun poly(epsilon-caprolactone) (PCL) fiber mats for assessment as a controlled delivery device. Fibers with smooth surfaces and no bead defects could be spun from polymer solutions with 8% w/v PCL in 7:3 dichloromethane: methanol. A significant decrease in fiber diameter was observed with increasing heparin concentration. Assessment of drug loading, and imaging of fluorescently labeled heparin showed homogenous distribution of heparin throughout the fiber mats. A total of approximately half of the encapsulated heparin was released by diffusional control from the heparin/PCL fibers after 14 days. The fibers did not induce an inflammatory response in macrophage cells in vitro and the released heparin was effective in preventing the proliferation of VSMCs in culture. These results suggest that electrospun PCL fibers are a promising candidate for delivery of heparin to the site of vascular injury. (C) 2005 Elsevier Ltd. All rights reserved

EMT inhibitory property of an EGFR inhibitor, Gefitinib.

<p>T2 plate image and CDR dose response profile of Gefitinib, against EGF- (A) HGF- (B) and IGF-1- (C) induced EMT.</p

Cell dispersion ratio (CDR) vs. cell count ratio (CCR) plots.

<p>The graphs illustrate the behavior of NBT-II cells treated with different test compounds and growth factors in the spot migration assay. CDR threshold was set at 50% CDR between positive control CDR and negative control CDR. CCR threshold was set at 1.5 growth rate. We assessed compounds that inhibit cell dispersion (i.e. less than CDR threshold) and do not severely inhibit cell growth (i.e. more than CCR threshold). To further refine our hits, the test compounds were run at a low and high concentration (1.67 and 6.67 µM, respectively). Hit compounds (crossed squares) were classified as test compounds that satisfy the CDR and CCR threshold criteria at both concentrations.</p

EMT inhibitory property of an ALK5 inhibitor, A83-01.

<p>T2 plate image and CDR dose response profile of A83-01, against EGF- (A) HGF- (B) and IGF-1- (C) induced EMT.</p

EMT spot migration screening assay overview.

<p>(A) Schematic of the spot migration screening assay to identify EMT inhibitory compounds. EMT can be initiated and maintained in epithelial cells via growth factor signaling. This assay measures the dispersion of cells in the presence of a test compound and an EMT inducer (EGF, HGF or IGF-1). The prevention of cell dispersion directly correlates to the propensity of a test compound to block an induced EMT signaling pathway. (B) Screening assay image acquisition workflow. Robot-assisted plating of H2B-mcherry transfected NBT-II cells into the well centers of 96-well plates. The initial plate image acquired at T1 served as the baseline reference for calculating the CCR and CDR values for each well. The cells were treated with test compounds overnight and further incubated for 24 h with a growth factor to induce EMT. (C) Final plate image acquired at T2 depicted the dispersion response of cells 24 h after addition of the compounds and growth factor treatment. In the example shown, columns 2–11 were treated with 80 different test compounds at 6.67 µM and EGF. Column-1 served as negative controls treated with 0.67% DMSO and EGF, while column-12 served as positive controls treated with 6.67 µM AG1478 and EGF. (D) Magnified images of selected wells acquired at T1 and T2. Wells C12, H03 and D02 are examples of cell colonies treated by compounds that inhibited EGF-initiated cell dispersion and did not inhibit cell growth. Well C01 is a cell colony undergoing EGF-induced EMT without any dispersion inhibition. Well E09 is a cell colony treated by a growth inhibitory or toxic compound.</p

Image processing procedure to determine the cell count and dispersion values of a well.

<p>(A) Colony nuclei image of each well was obtained by stitching four adjacent, non-overlapping fields together. The example here shows a primary cell colony surrounded by several cell outliers. (B) Nuclei segmentation, which consists of a wavelet transform and watershed algorithm steps, was applied to identify all nuclei in the well. (C) The nuclei segmentation mask was then dilated to generate merging region areas where distinct cell clusters could be isolated. In general, the largest region (yellow), representing the cell colony of interest, and other smaller regions (other colors), representing outlier cell clusters, were identified. (D) Nuclei within the colony of interest were kept for measurement. Cell count was determined by the total nuclei count within the colony. Cell dispersion was determined by applying the spreading coefficient formula. The blue arrow represents a vector centered on the colony center with distance equal to the spreading coefficient.</p