Screening therapeutic EMT blocking agents in a three-dimensional microenvironment

Abstract

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.

Fig. 1

Schematic and photograph of 3D co-culture microfluidic device. A. Schematic diagram of device layout depicts the inlets for injecting cells, filling collagen, and replenishing medium. B. Enlarged view of gel region and HUVEC-lined channel. Cytokines in conditioned medium from HUVEC monolayer diffuse into the gel region triggering spheroids to undergo EMT. C. Photograph of the PDMS-molded device bonded on a glass cover-slip. D. A 3D co-culture image combining phase contrast and fluorescence with enlarged HUVECs monolayer structure and 3D cancer spheroid dispersion. Blue: Hoechst; green: VE-Cadherin; red: nuclei mCherry.

Fig. 2

Dispersion measurements of A549 spheroids in 2D conditions in a 96-well plate. A. Spheroids were seeded in wells in control medium and allow to spread. B. Treatment with 10 μM CI-1033, an EGFR inhibitor, reduced the dissociation of cancer cells from spheroids. C. 50% inhibition concentration of CI-1033 was achieved at 5,200 nM.

Fig. 3

Fluorescent images in time-series showing A549 cell dissemination in the 3D collagen gel. A. Control condition in the absence of a HUVEC monolayer, i.e., 3D monoculture. B. Control condition in the presence of a HUVEC monolayer in the side channel, i.e. 3D co-culture. C. EGF-targeted drug (300nM of CI-1033) applied in the presence of HUVEC monolayer. Red: nuclei of A549 cells; green: HUVEC. Triangles show the PDMS posts on the edge of collagen gel.

Fig. 4

Immunostaining of EMT markers on A549 spheroids at 0 h and 36 h in co-culture with HUVECs. A–D. Expression of vimentin in spheroids at 0 h and 36 h. Blue: nuclei; green: vimentin. E–H. Expression of E-cadherin in spheroids at 0 h and 36 h. Blue: nuclei; green: E-cadherin.

Fig. 5

Analysis of cell dispersion and proliferation in the presence or absence of drug treatment. A. Normalized dispersion for twelve drugs. B. Representative normalized dispersion measured over time for three concentrations of AZD 0530. C. Normalized proliferation for twelve drugs. D. Representative normalized proliferation measured over time for three concentrations of AZD 0530. The values for the effective dose for full inhibition are given in Table 1.

Fig. 6

Normalized cell dispersion and corresponding cell number at 36 h. Four quadrants were defined to characterize the dose response for each drug. The \upper right quadrant indicates cases with high rates of proliferation and dispersion, where we find the control conditions. Most drug-treated data lie in the lower left quadrant with low proliferation and low dispersion activity. Therefore, it appears that for the conditions tested, drugs reduced cell dispersion and cell proliferation simultaneously.