Identification of Agents that Induce Apoptosis of Multicellular Tumour Spheroids: Enrichment for Mitotic Inhibitors with Hydrophobic Properties

Abstract

Cell-based anticancer drug screening generally utilizes rapidly proliferating tumour cells grown as monolayer cultures. Hit compounds from such screens are not necessarily effective on hypoxic and slowly proliferating cells in 3-D tumour tissue. The aim of this study was to examine the potential usefulness of 3-D cultured tumour cells for anticancer drug screening. We used colon carcinoma multicellular spheroids containing hypoxic and quiescent cells in core areas for this purpose. Three libraries (∼11 000 compounds) were screened using antiproliferative activity and/or apoptosis as end-points. Screening of monolayer and spheroid cultures was found to identify different sets of hit compounds. Spheroid screening enriched for hydrophobic compounds: median XLogP values of 4.3 and 4.4 were observed for the hits in two independent screening campaigns. Mechanistic analysis revealed that the majority of spheroid screening hits were microtubuli inhibitors. One of these inhibitors was examined in detail and found to be effective against non-dividing cells in the hypoxic centres of spheroids. Spheroid screening represents a conceptually new strategy for anticancer drug discovery. Our findings have implications for drug library design and hit selection in projects aimed to develop drugs for the treatment of solid tumours.

Massive induction of apoptosis of multicellular tumor spheroids by a novel compound with a calmodulin inhibitor-like mechanism

“…NSC647889 shows antiproliferative activity in the NCI60 cell panel (average GI50: 1.1 µM; strongest activity on acute lymphoblastic leukaemia, promyelocytic leukemia and chronic myelogenous leukemia cell lines (http://dtp.nci.nih.gov/)). We previously reported that compounds active on multicellular spheroids generally are hydrophobic [19] and the hydrophobicity (XLogP 7.3) of NSC647889 is consistent with this observation. The high degree of hydrophobicity complicated systematic in vivo assessments of antineoplastic effects.…”

“…Sectioned spheroids were incubated with Hypoxyprobe-1 (HPI Inc., Burlington, MA, USA) to detect pimonidazole adducts. Serial sectioning was not required since the hanging drop procedure generates spheroids of homogenous size [19].…”

“…Spheroids were prepared as described previously [19]. Briefly, a cell suspension containing 10,000 cells was added to each well of poly-HEMA-coated 96-well plates.…”

Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer

“…Moreover, if uniform and equal in size, MTCS can be used for drug activity comparisons (8,13). We have previously shown that compounds identified in 3D-model screens are distinct from those found using 2D models (14,15). Thus, tumor cells in 3D models are not necessarily always drug resistant, opening a possibility for identification of more active cancer drugs if large-scale 3D-based drug screening could be pursued.…”

“…There are numerous MCTS formation methods (14,(16)(17)(18)(19)(20) but most are not suited for high-throughput screening (HTS); they are either costly, difficult to handle in a large scale or the spheroids often vary in size and number. Thus, MCTS-based screening efforts performed so far have used small drug libraries, not exceeding a few hundred compounds (14,15,17,21).…”