Three dimensional cell cultures and light sheet-based fluorescence microscopy represent promising tools in phenotypic drug screening and personalized medicine

Undesired cytotoxic effects of many cancer drug candidates are mostly recognized quite late during the clinical trials. Just one out of ten drug candidates entering phase I trials reaches the market. This low success rate drives the high development costs and the prices of successfully marketed drugs. New assays predicting the efficacy of drugs in the early, pre-clinical stage are required to increase the success rate in drug development. Modern assays are performed with three-dimensional cell cultures. Three-dimensional cultures essentially avoid hard and flat surfaces. Hence, they favor less constrained physiological sample dynamics and promote cells growth in tissue-like environments. Conventional two-dimensional (2D) cell monolayers represent the state-of-the-art of cell-based assays in drug screening and toxicology. However, 2D systems do not reproduce the complex three-dimensional architecture of tissues. Thus, 2D cellular assays have a limited physiological significance and low predictivity to toxic compounds or drugs. Reliable screening assays based on three-dimensional cell cultures are necessary to reduce the effort and the cost of drug development and reduce failure rate of clinical studies.

We develop three-dimensional cultures of tumor cell spheroids for phenotypic drug screening issues. The cultivated spheroids resemble their microenvironment and serve as a model for tumors in the living organism (Figure 1). Tumor spheroids are easy to produce and culture. They do not need an exogenous extracellular matrix. They are well suited for high-content imaging with fluorescence microscopy and for biochemical assays. We evaluate the therapeutic benefits as well as the toxicity of drug candidates in spheroids by means of advanced light microscopy and light sheet-based fluorescence microscopy (LSFM). We develop a fluorescence imaging workstation for three-dimensional cultures based on LSFM that allows dynamic long-term observations of living three-dimensional cultures (Figure 2). Furthermore, we provide new solution to handle huge 3D data sets that arise from a time-lapse experiment, including multiple view angles and hundreds of samples by adapted data analysis algorithms and work flows. Next to cancer research, we also expect to promote novel scientific approaches in modern 3D cell biology like tissue homeostasis and development, inflammation, membrane dynamics and immunology. All the methods we employ as well as the technology we validated fulfill the requirements of good-laboratory practice.

Figure 1: Heterotypic spheroid formation of breast cancer cells (T-47D) and Normal Human Dermal Fibroblasts (NHDF).

Figure 2: Time lapse imaging of 3D cell cultures by the LSFM. (A) Perfusion system for live cell imaging in the LSFM. (B) Temperature distribution and pH stability within the perfusion system. (C) Analysis of mitotic events during spheroid formation of HepaRG cells.

Involved group members
Nariman Ansari
Francesco Pampaloni
Sabine Fischer
Biena Mathew
Alexander Schmitz
Christian Mattheyer
Roli Richa
Berit Reinhardt
Sigrun Becker
Sven Plath

Collaborators
University Hospital Frankfurt am Main
Georg Speyer Haus – Institute for Tumor Biology
Bayer HealthCare
Max Planck Institute of Molecular Cell Biology and Genetics
Max Planck Institute for Brain Research
SpheroTec GmbH
InSphero AG
Merz Pharmaceuticals GmbH
Carl Zeiss Microscopy GmbH

Funding
BMBF
DFG

Literature
Bissell MJ, Hines WC. Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression. Nature Medicine 17, 320-329, March 2011.

Friedrich J, et al. Spheroid-based drug screen: considerations and practical approach. Nat Protoc, 2009. 4(3): p. 309.

Tostões RM, Leite SB, Serra M, Jensen J, Björquist P, Carrondo MJ, Brito C, Alves PM. Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing. Hepatology. 2012 Apr;55(4):1227-36.

Genetic Engineering & Biotechnology News, 2012, Vol 32, No. 21

Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 2007 Sep-Oct;3(5):452-60.

Voss V, Senft C, Lang V, Ronellenfitsch MW, Steinbach JP, Seifert V, Kögel D. The pan-Bcl-2 inhibitor (-)-gossypol triggers autophagic cell death in malignant glioma. Mol Cancer Res. 2010 Jul;8(7):1002-16. doi: 10.1158/1541-7786.MCR-09-0562. Epub 2010 Jun 29.
 
Publications
Pampaloni F, Richa R, Ansari N, Stelzer EH. Live spheroid formation recorded with light sheet-based fluorescence microscopy. Methods Mol Biol. 2015; 1251:43-57.

Ansari N, Hardung S, Hötte K, Rakel S, Antonietti P, Kögel D, Stelzer EH,Pampaloni F. Quantifying the autophagy-triggering effects of drugs in cell spheroids with live fluorescence microscopy. Methods Mol Biol. 2014;1165:19-29.

Hengl T, Krischok S, Riegel K, Ansari N, Stelzer E and Abts HF. Cell interaction and growth in a 3D heterotypic hair-follicle spheroid model mimicking diffuse hair-loss. Journal of Investigative Dermatology 2014 134, S40–S48.

Swoger, J, Pampaloni, F, Stelzer, EH. Imaging MDCK Cysts with a Single (Selective) Plane Illumination Microscope.  Cold Spring Harb Protoc.2014.

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, Steigemann P. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions. Exp Cell Res. 2014 Apr 15;323(1):131-43.

Bunse S, Garg S, Junek S, Vogel D, Ansari N, Stelzer EH, Schuman E. Role of N-cadherin cis and trans interfaces in the dynamics of adherens junctions in living cells. PLoS One. 2013 Dec 2;8(12).

Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Pampaloni F, Stelzer EHK...Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013 Aug; 87(8):1315-530.

Pampaloni F, Ansari N and Stelzer EHK. High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy. Cell Tissue Res. 2013 Apr; 352(1):161-77.

Ansari N, Müller S, Stelzer EHK and Pampaloni F. Quantitative 3D cell-based assay performed with cellular spheroids and fluorescence microscopy. Methods Cell Biol. 2013 Jan; 113:295-309.

Pampaloni, F; Stelzer, EH; Leicht, S; Marcello, M. Madin-Darby canine kidney cells are increased in aerobic glycolysis when cultured on flat and stiff collagen-coated surfaces rather than in physiological 3-D cultures. Proteomics, 2010 Oct.

Verveer, PJ, Swoger, J, Pampaloni, F, Greger, K, Marcello, M, Stelzer, EH. High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nat Methods, 2007 Apr.

Pampaloni, F, Reynaud, EG, Stelzer, EH. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol, 2007 Oct.

Links & Websites
http://www.physikalischebiologie.de
http://www.bmls-institute.de/index.php?id=physical_biology
http://3dcellculture.com/