Translation of a Tumor Microenvironment Mimicking 3D Tumor Growth Co-culture Assay Platform to High-Content Screening

Abstract

For drug discovery, cell-based assays are becoming increasingly complex to mimic more realistically the nature of biological processes and their diversifications in diseases. Multicellular co-cultures embedded in a three-dimensional (3D) matrix have been explored in oncology to more closely approximate the physiology of the human tumor microenvironment. High-content analysis is the ideal technology to characterize these complex biological systems, although running such complex assays at higher throughput is a major endeavor. Here, we report on adapting a 3D tumor co-culture growth assay to automated microscopy, and we compare various imaging platforms (confocal vs. nonconfocal) with correlating automated image analysis solutions to identify optimal conditions and settings for future larger scaled screening campaigns. The optimized protocol has been validated in repeated runs where established anticancer drugs have been evaluated for performance in this innovative assay.

Advanced Cell Culture Techniques for Cancer Drug Discovery

“…Krausz et al [72] recently published anchorage-dependent 3D cell culture assay methodology for prostate cancer, which is suitable for high-content screening (HCS) (96-well microtitre plate format). Initially, comparisons of various imaging platforms (MIAS-2 ® , IN Cell Analyzer 2000 ® and an Opera ® ) and their respective analysis programs along with their in house tool, Plate-based High-Content Analysis Evaluation and Dynamic Reliability Assurance (Phaedra) were performed.…”

“…Several of these programs consist of features which enable batch analysis of large data sets that are becoming readily accessible for most academic groups, and are relatively user friendly. In addition, various analysis protocols which focus on deconstructing elements of cells cultured in 3D, such as morphological profiles and biomarkers, designed for low-throughput and HCS experiments have been developed and published [72,81,82]. Technological advances such as these may not only allow a broader scope of models to be developed, but may further encourage the incorporation of 3D models in the search for anti-cancer drug candidates in the drug discovery pipeline.…”

“…3D cancer co-culture models have been developed in both low-throughput [89,90,91,92] and high-throughput [5,72,73] formats. However, at the present time, only cancer cells along with one other cell type (e.g., fibroblasts) have been incorporated into models suitable in screening new compounds in a high-throughput manner.…”

3-Dimensional Patient-Derived Lung Cancer Assays Reveal Resistance to Standards-of-Care Promoted by Stromal Cells but Sensitivity to Histone Deacetylase Inhibitors

“…This was achieved by development of the 3-Dimensional Tumor Growth Assay (3D-TGA; refs. 25,26), which utilizes a low stiffness laminin-rich extracellular matrix (ECM; ref. 27) to embed tumor cells and admixing with stromal cells to provide the paracrine signaling present in the TME of solid tumors (13,14).…”

High-throughput imaging: Focusing in on drug discovery in 3D

“…An interesting study by Krausz et al described an anchorage-dependent 3D co-culture model including tumor cells and stromal cells organized in a complex ECM microenvironment [46]. This model was used to assess tumor growth and focused on non-uniform 3D-colony structures as opposed to uniform single MCTS.…”