A Phenotypic Screening Approach to Identify Anticancer Compounds Derived from Marine Fungi

Abstract

This study covers the isolation, testing, and identification of natural products with anticancer properties. Secondary metabolites were isolated from fungal strains originating from a variety of marine habitats. Strain culture protocols were optimized with respect to growth media composition and fermentation conditions. From these producers, isolated compounds were screened for their effect on the viability and proliferation of a subset of the NCI60 panel of cancer cell lines. Active compounds of interest were identified and selected for detailed assessments and structural elucidation using nuclear magnetic resonance. This revealed the majority of fungal-derived compounds represented known anticancer chemotypes, confirming the integrity of the process and the ability to identify suitable compounds. Examination of effects of selected compounds on cancer-associated cell signaling pathways used phospho flow cytometry in combination with 3D fluorescent cell barcoding. In parallel, the study addressed the logistical aspects of maintaining multiple cancer cell lines in culture simultaneously. A potential solution involving microbead-based cell culture was investigated (BioLevitator, Hamilton). Selected cell lines were cultured in microbead and 2D methods and cell viability tests showed comparable compound inhibition in both methods (R2=0.95). In a further technology assessment, an image-based assay system was investigated for its utility as a possible complement to ATP-based detection for quantifying cell growth and viability in a label-free manner.

Establishing the Secondary Metabolite Profile of the Marine Fungus: Tolypocladium geodes sp. MF458 and Subsequent Optimisation of Bioactive Secondary Metabolite Production

“…The detailed procedure was reported elsewhere [19] and showed a promising activity profile for a number of fractions from MF458. Purified compounds were analysed using a panel of cell lines from the NCI60 panel, consisting of cell lines from 8 different tissues and a number of different cancer stages and genetic characteristics.…”

“…Extracts from fermentations of fungi isolated from Mediterranean sponges, Indonesian corals and Chilean macroalgae were screened against tumour cell lines [19]. …”

Mass Cytometry: A Highly Multiplexed Single-Cell Technology for Advancing Drug Development

“…Typically, cells are stained with two DNA stains; the first DNA stain is a viability stain, whereas the second DNA stain (added at the end of the staining protocol) is used to distinguish cellular events from debris and for discriminating single events from doublets or cell aggregates. The cells can be stained with either a rhodium-based (Darzynkiewicz, 2010;Darzynkiewicz et al, 2010;Ornatsky et al, 2008b) or Ir-based DNA intercalator (Ornatsky et al, 2008b;Tinhofer et al, 2012), or a platinum-containing compound such as cisplatin (Ellinger et al, 2014). The Rh-and Ir-DNA intercalators stain by reaching pseudoequilibrium, and neither reach saturation nor are fixable and hence tend to leach out of the cells after staining.…”

“…The Rh-and Ir-DNA intercalators stain by reaching pseudoequilibrium, and neither reach saturation nor are fixable and hence tend to leach out of the cells after staining. In contrast, cisplatin is a fast DNA-labeling stain and is fixable (Ellinger et al, 2014).…”

“…For example, detailed phenotyping combined with identification of potential signaling nodes that constitute the intended and unintended targets of drugs have been successfully employed in drug screens (Zhang et al, 2009;Ellinger et al, 2014) and clinical studies with signaling inhibitors such as anticancer drugs (Guerrouahen et al, 2009;Tinhofer et al, 2012). As new drugs are developed for targeted therapy of patients stratified using a precision medicine approach, advances in multiplexed single-cell assay technology will be instrumental during the discovery and development phases of drug development.…”