Selection of efficient drugs for the therapy of gastrointestinal cancers using the multicellular spheroid model

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Advanced gastrointestinal tumors have a poor prognosis and are largely unresponsive to currently available chemotherapeutic drugs. The development of more effective therapies would be aided by better preclinical models. An in vitro multicellular cancer spheroid model was established using a modified liquid overlay technique and compared to the corresponding monolayers and xenografts in immunodeficient mice. Morphological and functional differentiation and expression of cell adhesion molecules were evaluated by histology, immunohistochemistry and flow cytometry. Cell cycle analysis was investigated by PI intercalation and the therapeutic efficacy of new drugs was tested with MTT and TUNEL assay. 77% of the gastrointestinal cancer cell lines reflected growth characteristics of their parental gastric carcinomas in three-dimensional culture. Thus, cell lines derived from peritoneal and pleural carcinomatosis grew as single cells and cell aggregates. Cell lines originated from solid primary and metastatic tumors formed partly or fully compact multicellular spheroids recapitulating the tumor architecture of the respective original tumor. The differentiated phenotype was lost after subcutaneous implantation of the in vitro spheroids in mice. The degree of morphological differentiation was reflected by the levels of mucin and constitutive E-cadherin expression. Heterogeneous changes of various molecules involved in tumor progression (e.g. EGF-R, HER2/neu, EpCAM, CD44s) were observed in 2D and 3D culture. Cell cycle analysis revealed most of the spheroid cells in G0/G1, which is contrary to the corresponding monolayers. These tumorbiological characteristics induced in 3D culture had a profound influence on testing of new drugs. Efficacy, side effects, penetration of a drug into the microtumor as well as various aspects of the pharmacodynamics were determined in the spheroid model. Recapitulating some complexity of their in vivo counterparts, multicellular cancer spheroids represent a physiologically valid model for studying the biology of gastrointestinal tumors, including the potential to test new therapeutic strategies.