High-throughput screening in multicellular spheroids for target discovery in the tumor microenvironment.

INTRODUCTION: Solid tumors are highly influenced by a complex tumor microenvironment (TME) that cannot be modeled with conventional two-dimensional (2D) cell culture. In addition, monolayer culture conditions tend to induce undesirable molecular and phenotypic cellular changes. The discrepancy between in vitro and in vivo is an important factor accounting for the high failure rate in drug development. Three-dimensional (3D) multicellular tumor spheroids (MTS) more closely resemble the in vivo situation in avascularized tumors. AREAS COVERED: This review describes the use of MTS for anti-cancer drug discovery, with an emphasis on high-throughput screening (HTS) compatible assays. In particular, we focus on how these assays can be used for target discovery in the context of the TME. EXPERT OPINION: Arrayed MTS in microtiter plates are HTS compatible but remain more expensive and time consuming than their 2D culture counterpart. It is therefore imperative to use assays with multiplexed readouts, in order to maximize the information that can be gained with the screen. In this context, high-content screening allowing to uncover microenvironmental dependencies is the true added value of MTS-based screening compared to 2D culture-based screening. Hit translation in animal models will, however, be key to allow a broader use of MTS-based screening in industry.

High-throughput investigation of endothelial-to-mesenchymal transformation (EndMT) with combinatorial cellular microarrays.

In the developing heart, a specific subset of endocardium undergoes an endothelial-to-mesenchymal transformation (EndMT) thus forming nascent valve leaflets. Extracellular matrix (ECM) proteins and growth factors (GFs) play important roles in regulating EndMT but the combinatorial effect of GFs with ECM proteins is less well understood. Here we use microscale engineering techniques to create single, binary, and tertiary component microenvironments to investigate the combinatorial effects of ECM proteins and GFs on the attachment and transformation of adult ovine mitral valve endothelial cells to a mesenchymal phenotype. With the combinatorial microenvironment microarrays, we utilized 60 different combinations of ECM proteins (Fibronectin, Collagen I, II, IV, Laminin) and GFs (TGF-ß1, bFGF, VEGF) and were able to identify new microenvironmental conditions capable of modulating EndMT in MVECs. Experimental results indicated that TGF-ß1 significantly upregulated the EndMT while either bFGF or VEGF downregulated EndMT process markedly. Also, ECM proteins could influence both the attachment of MVECs and the response of MVECs to GFs. In terms of attachment, fibronectin is significantly better for the adhesion of MVECs among the five tested proteins. Overall collagen IV and fibronectin appeared to play important roles in promoting EndMT process. Great consistency between macroscale and microarrayed experiments and present studies demonstrates that high-throughput cellular microarrays are a promising approach to study the regulation of EndMT in valvular endothelium. Biotechnol. Bioeng. 2016;113: 1403-1412. © 2015 Wiley Periodicals, Inc.

A high-throughput-compatible 3D microtissue co-culture system for phenotypic RNAi screening applications.

Cancer cells in vivo are coordinately influenced by an interactive 3D microenvironment. However, identification of drug targets and initial target validations are usually performed in 2D cell culture systems. The opportunity to design 3D co-culture models that reflect, at least in part, these heterotypic interactions, when coupled with RNA interference, would enable investigations on the phenotypic impact of gene function in a model that more closely resembles tumor growth in vivo. Here we describe a high-throughput-compatible method to discover cancer gene functions in a co-culture 3D tumor microtissue model system composed of human DLD1 colon cancer cells together with murine fibroblasts. Strikingly, DLD1 cells in this model failed to expand upon siRNA-mediated depletion of Kif11/Eg5, a member of the mitotic kinesin-like motor protein family. In contrast, these cancer cells proved to be more resistant to Kif11/Eg5 depletion when grown as a 2D monolayer. These results suggest that growth of certain cancer cells in 3D versus 2D can unveil differential dependencies on specific genes for their survival. Moreover, they denote that the high-throughput-compatible, hanging drop technology-based 3D co-culture model will enable the discovery, characterization, and validation of gene functions in key biological and pathological processes.

Spot identification and quality control in cell-based microarrays.

Cell-based microarrays are being increasingly used as a tool for combinatorial and high throughput screening of cellular microenvironments. Analysis of microarrays requires several steps, including microarray imaging, identification of cell spots, quality control, and data exploration. While high content image analysis, cell counting, and cell pattern recognition methods are established, there is a need for new postprocessing and quality control methods for cell-based microarrays used to investigate combinatorial microenvironments. Previously, microarrayed cell spot identification and quality control were performed manually, leading to excessive processing time and potentially resulting in human bias. This work introduces an automated approach to identify cell-based microarray spots and spot quality control. The approach was used to analyze the adhesion of murine cardiac side population cells on combinatorial arrays of extracellular matrix proteins. Microarrays were imaged by automated fluorescence microscopy and cells were identified using open-source image analysis software (CellProfiler). From these images, clusters of cells making up single cell spots were reliably identified by analyzing the distances between cells using a density-based clustering algorithm (OPTICS). Naïve Bayesian classifiers trained on manually scored training sets identified good and poor quality spots using spot size, number of cells per spot, and cell location as quality control criteria. Combined, the approach identified 78% of high quality spots and 87% of poor quality spots. Full factorial analysis of the resulting microarray data revealed that collagen IV exhibited the highest positive effect on cell attachment. This data processing approach allows for fast and unbiased analysis of cell-based microarray data.