Mindy I. Davis Ph.D. National Institute of Health

This literature review describes a study where researchers used 3D high-content screening to identify compounds that target cells in inner MCTS core regions.

Cells within a tumor experience very different microenvironments depending on whether they are on the periphery or at the core of the tumor. This is due in part to the differing distance from the blood vessels that supply oxygen and nutrients to the tumors. There is a gradient of oxygen, nutrients, and metabolites in tumors. Cells that are deep in the core of a tumor often become dormant, which then also makes them resistant to many chemotherapeutics that target rapidly proliferating cells. These dormant cells could contribute to relapse so there is an interest in developing drugs that would be effective at killing these core cells.

Recapitulating the tumor microenvironment in the lab has led to the increase in assays that use spheroids rather than two-dimensional (2D)-plated cells. The authors* here sought to discover compounds that targeted the cells in the inner core of the spheroid while not affecting the outer shell of the spheroid or cells in 2D culture.

T47D cells were seeded into agarose-containing plates, and spheroids of ∼400 μM were formed by day 4 (Figure). Using high-content imaging with Hoechst and Sytox Green staining, the authors were able to differentiate all cells (Hoechst) from dead cells (Sytox Green). Initially, cytostatic agents, such as cisplatin and paclitaxel, were tested, and only the outer regions of the spheroid were affected and the inner core remained viable.

From a screen of U.S. Food and Drug Administration (FDA)-approved drugs and known bioactive compounds, they discovered nine compounds that specifically targeted the inner core of the spheroid while not affecting either the cells in the outer layer or cells plated in 2D culture. Because two of the hits were known to impact the respiratory chain, the authors tested oxygen consumption and lactate generation and found that all of the hits impacted both aspects of the respiratory chain. Additionally, they determined that the inner core had activated levels of caspase 3/7 prior to cell death indicating that apoptosis was initiated.

They hypothesized that cells at the exterior of the spheroid would have easier access to enough glucose to switch to glycolysis without encountering significant stress (i.e., the Warburg effect). By varying the glucose levels in the media, the authors could alter the extent of the observed phenotype, with lower glucose leading to an increase in the differential killing of the core region and higher glucose leading to a decrease in the killing of the core region. Lastly, the authors showed that sequential dosing of a drug that targets proliferating cells with one of the compounds that targets the dormant cells at the center of the spheroid was additive.

It will be interesting to see from screening additional libraries whether there are other pathways that are more sensitized in the inner core of a spheroid.

Figure. High-content screening (HCS) on multicellular tumor spheroids (MCTS). (A) HCS-workflow for T47D MCTS generated on 384-well agarose-coated multiplates. Development of custom image analysis routines allowed automated compound evaluation to specifically identify induction of cell death in MCTS core regions. Used drug libraries consisted of 1,120 compounds with known bioactivity (KBA) and a set of FDA-approved drugs. Staurosporine = general cytotoxicity control. Scale bar, 100 µm. (B) Representative images of a dilution series of the screening hit diphenyleneiodonium. Scale bar, 100 µm. (C) Comparison of dose response curve from screening hit diphenyleneiodonium on 3D spheroids vs. 2D cell culture. Similar results were obtained with all screening hits (see the article’s Supplementary Fig. S3C). 2D, two-dimensional; 3D, three-dimensional.

*Abstract from Experimental Cell Research 2014, Vol. 323: 131–143

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors.

With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected.

We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Experimental Cell Research titled "3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions". Authors of the paper are Wenzel C, Riefke B, Gründemann S, Krebs A, Christian S, Prinz F, Osterland M, Golfier S, Räse S, Ansari N, Esner M, Bickle M, Pampaloni F, Mattheyer C, Stelzer EH, Parczyk K, Prechtl S, and Steigemann P.