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.

Warburg metabolism in tumor-conditioned macrophages promotes metastasis in human pancreatic ductal adenocarcinoma

“…3 consisting of a central gel channel flanked by two fluidic channels was utilized. Similar microfluidic platforms have been used for investigating angiogenesis, 13,14 EMT, 15,16 cancer cell-macrophage interactions, 17 intravasation 18 and extravasation. 19,20 For the present work, GFPexpressing HUVECs were seeded into one of the fluidic channels to form a uniform monolayer mimicking a blood vessel wall (Fig.…”

Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

“…FIIN-2 and FIIN-3 also were evaluated using 3D dispersion assays in a microfluidic device. Compared with conventional 2D assays The 3D assay creates a 3D microenvironment that allows better evaluation of drugs that inhibit cell migration or epithelial-mesenchymal transition (66). SKOV-3 spheroids were suspended in the gel region of the microfluidic system and cultured in the presence or absence of FGF or EGF (20 ng/mL).…”

“…For 3D dispersion assays, SKOV-3 cells were allowed to grow in spheroids by resuspending cells at low density (2,000-4,000 cells∕mL) and were cultured for 10-14 d in ultra-low-attachment dishes (Corning Inc.). Spheroids of 40-to 70-μm diameter were selected with sieves as reported (66). Spheroids were seeded in the central region of a microfluidic device using standard soft lithography techniques (94,95).…”

Regulation of Survival by IKKe in Inflammatory Breast Cancer Involves EpCAM

“…These findings demonstrate that CYT387 treatment uniquely impairs not only cell viability in 2D culture but also growth factor-and cytokine-driven TNBC cell proliferation and dispersal in 3D culture. Next, we treated a panel of 15 breast cancer cell lines with CYT387 over a range of concentrations and found that TNBCs that exhibited high levels of IKBKE and pSTAT3 exhibited the greatest sensitivity, We further examined the effects of CYT387 treatment on MDA-MB-468 cells in a 3D culture tumor spheroid dispersal assay that captures features of the tumor microenvironment and also models aspects of the epithelial-mesenchymal transition (34). EGFinduced proliferation of MDA-MB-468 breast cancer cells in this assay was completely suppressed by CYT387 treatment at concentrations as low as 800 nM (Supplemental Figure 4A).…”