Engineering a scaffold-free 3D tumor model for in vitro drug penetration studies.

Three-dimensional (3D) in vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in vivo conditions. In cancer research, the multi-cellular tumor spheroid (MCTS) model is an established 3D cancer model that exhibits microenvironmental heterogeneity close to that of tumors in vivo. However, the established process of MCTS formation is time-consuming and often uncontrolled. Here, we report a method for engineering MCTS using a transient inter-cellular linker which facilitates cell-cell interaction. Using C3A cells (a hepatocellular carcinoma cell line) as a model, we formed linker-engineered spheroids which grew to a diameter of 250 microm in 7 days, as compared to 16 days using conventional non-adherent culture. Seven-day old linker-engineered spheroids exhibited characteristics of mature MCTS, including spheroid morphology, gene expression profile, cell-cell interaction, extracellular matrix secretion, proliferation and oxygen concentration gradients, and cellular functions. Linker-engineered spheroids also displayed a resistance to drug penetration similar to mature MCTS, with dose-dependent extracellular accumulation of the drug. The linker-engineered spheroids thus provide a reliable accelerated 3D in vitro tumor model for drug penetration studies.

[1]  M. Bissell,et al.  Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. , 2006, Annual review of cell and developmental biology.

[2]  James P. Freyer,et al.  The Use of 3-D Cultures for High-Throughput Screening: The Multicellular Spheroid Model , 2004, Journal of biomolecular screening.

[3]  Sophie Lelièvre,et al.  beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. , 2002, Cancer cell.

[4]  W. Mueller‐Klieser.  Tumor biology and experimental therapeutics. , 2000, Critical reviews in oncology/hematology.

[5]  Smadar Cohen,et al.  Enhancing the drug metabolism activities of C3A--a human hepatocyte cell line--by tissue engineering within alginate scaffolds. , 2006, Tissue engineering.

[6]  F. Pampaloni,et al.  The third dimension bridges the gap between cell culture and live tissue , 2007, Nature Reviews Molecular Cell Biology.

[7]  D. Gewirtz,et al.  Interference by doxorubicin with DNA unwinding in MCF-7 breast tumor cells. , 1994, Molecular pharmacology.

[8]  J. Jardillier,et al.  Multicellular resistance: a paradigm for clinical resistance? , 2000, Critical reviews in oncology/hematology.

[9]  M. Christian,et al.  Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials , 2001, British Journal of Cancer.

[10]  T. Lee,et al.  Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines , 2001, Cancer Chemotherapy and Pharmacology.

[11]  P. Indovina,et al.  Apoptosis, cell adhesion and the extracellular matrix in the three-dimensional growth of multicellular tumor spheroids. , 2000, Critical reviews in oncology/hematology.

[12]  Christopher S. Chen,et al.  Engineering cellular microenvironments to improve cell-based drug testing. , 2002, Drug discovery today.

[13]  I. Tannock,et al.  Drug penetration in solid tumours , 2006, Nature Reviews Cancer.

[14]  Juergen Friedrich,et al.  Experimental anti-tumor therapy in 3-D: Spheroids – old hat or new challenge? , 2007, International journal of radiation biology.

[15]  Y. Marcel,et al.  The HepG2 Extracellular Matrix Contains Separate Heparinase- and Lipid-releasable Pools of ApoE , 1998, The Journal of Biological Chemistry.

[16]  Hanry Yu,et al.  Transient inter-cellular polymeric linker. , 2007, Biomaterials.

[17]  Jayanta Debnath,et al.  Modelling glandular epithelial cancers in three-dimensional cultures , 2005, Nature Reviews Cancer.

[18]  M. Toner,et al.  Cellular Micropatterns on Biocompatible Materials , 1998, Biotechnology progress.

[19]  L. Kunz-Schughart,et al.  Multicellular tumor spheroids: intermediates between monolayer culture and in vivo tumor , 1999, Cell biology international.

[20]  Alok R Ray,et al.  Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen. , 2005, Biomaterials.

[21]  Mina J Bissell,et al.  Modeling tissue-specific signaling and organ function in three dimensions , 2003, Journal of Cell Science.

[22]  A. Ivascu,et al.  Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis , 2006, Journal of biomolecular screening.

[23]  L. Griffith,et al.  Capturing complex 3D tissue physiology in vitro , 2006, Nature Reviews Molecular Cell Biology.

[24]  Robert L Sah,et al.  Probing the role of multicellular organization in three-dimensional microenvironments , 2006, Nature Methods.

[25]  T. Boland,et al.  Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels. , 2003, The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology.

[26]  R E Durand,et al.  Resistance of tumor cells to chemo- and radiotherapy modulated by the three-dimensional architecture of solid tumors and spheroids. , 2001, Methods in cell biology.

[27]  A. Minchinton,et al.  Direct Assessment of Drug Penetration into Tissue Using a Novel Application of Three-Dimensional Cell Culture , 2004, Cancer Research.

[28]  R C Bates,et al.  Spheroids and cell survival. , 2000, Critical reviews in oncology/hematology.

[29]  Ian F Tannock,et al.  Limited penetration of anticancer drugs through tumor tissue: a potential cause of resistance of solid tumors to chemotherapy. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[30]  S. Siegel,et al.  Effect of adriamycin on DNA, RNA, and protein synthesis in cell-free systems and intact cells. , 1976, Cancer research.

[31]  Martin Fussenegger,et al.  Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. , 2003, Biotechnology and bioengineering.

[32]  Y. Toh,et al.  Integrating sensitive quantification of hepatic metabolic functions by capillary electrophoresis with laser-induced fluorescence detection. , 2008, The Analyst.

[33]  Tobias Schmelzle,et al.  Engineering tumors with 3D scaffolds , 2007, Nature Methods.

[34]  R. Sutherland.  Cell and environment interactions in tumor microregions: the multicell spheroid model. , 1988, Science.

[35]  Y. Maehara,et al.  Comparison of in vitro anticancer chemosensitivity between human squamous cell carcinoma and adenocarcinoma. , 1992, Oncology.

[36]  Ying Zhang,et al.  Development of an in Vitro Multicellular Tumor Spheroid Model Using Microencapsulation and Its Application in Anticancer Drug Screening and Testing , 2008, Biotechnology progress.

[37]  V. Mironov,et al.  Engineering biological structures of prescribed shape using self-assembling multicellular systems. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Maria Teresa Santini,et al.  Three-Dimensional Spheroid Model in Tumor Biology , 1999, Pathobiology.

[39]  Chin-Tarng Lin,et al.  Hepatocellular carcinoma cell-specific peptide ligand for targeted drug delivery , 2008, Molecular Cancer Therapeutics.

[40]  A. Abbott.  Cell culture: Biology's new dimension , 2003, Nature.