title: Three-dimensional tumor spheroids for in vitro analysis of bacteria as gene delivery vectors in tumor therapy.
abstract: BACKGROUND
  Several studies in animal models demonstrated that obligate and facultative anaerobic bacteria of the genera Bifidobacterium, Salmonella, or Clostridium specifically colonize solid tumors. Consequently, these and other bacteria are discussed as live vectors to deliver therapeutic genes to inhibit tumor growth. Therapeutic approaches for cancer treatment using anaerobic bacteria have been investigated in different mouse models. In the present study, solid three-dimensional (3D) multicellular tumor spheroids (MCTS) of the colorectal adenocarcinoma cell line HT-29 were generated and tested for their potential to study prodrug-converting enzyme therapies using bacterial vectors in vitro.
  
  
  RESULTS
  HT-29 MCTS resembled solid tumors displaying all relevant features with an outer zone of proliferating cells and hypoxic and apoptotic regions in the core. Upon incubation with HT-29 MCTS, Bifidobacterium bifidum S17 and Salmonella typhimurium YB1 selectively localized, survived and replicated in hypoxic areas inside MCTS. Furthermore, spores of the obligate anaerobe Clostridium sporogenes germinated in these hypoxic areas. To further evaluate the potential of MCTS to investigate therapeutic approaches using bacteria as gene delivery vectors, recombinant bifidobacteria expressing prodrug-converting enzymes were used. Expression of a secreted cytosine deaminase in combination with 5-fluorocytosine had no effect on growth of MCTS due to an intrinsic resistance of HT-29 cells to 5-fluorouracil, i.e. the converted drug. However, a combination of the prodrug CB1954 and a strain expressing a secreted chromate reductase effectively inhibited MCTS growth.
  
  
  CONCLUSIONS
  Collectively, the presented results indicate that MCTS are a suitable and reliable model to investigate live bacteria as gene delivery vectors for cancer therapy in vitro.
authors.affiliation: Institute of Microbiology and Biotechnology, University of Ulm, 89069, Ulm, Germany. annika.osswald.ext@boehringer-ingelheim.com.|Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, 1121, Vienna, Austria. annika.osswald.ext@boehringer-ingelheim.com.
authors.forename: Annika
authors.initials: A
authors.lastname: Osswald
authors.affiliation: Institute of Microbiology and Biotechnology, University of Ulm, 89069, Ulm, Germany. szj008@126.com.|College of Life Sciences and Agriculture, Zhoukou Normal University, Chuanhui District, Zhoukou, 466001, People's Republic of China. szj008@126.com.
authors.forename: Zhongke
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Institute of Microbiology and Biotechnology, University of Ulm, 89069, Ulm, Germany. verena.grimm@uni-ulm.de.
authors.forename: Verena
authors.initials: V
authors.lastname: Grimm
authors.affiliation: Institute of Microbiology and Biotechnology, University of Ulm, 89069, Ulm, Germany. grace.ampem@uni-ulm.de.
authors.forename: Grace
authors.initials: G
authors.lastname: Ampem
authors.affiliation: Institute of Microbiology and Biotechnology, University of Ulm, 89069, Ulm, Germany. karin.riedel@uni-ulm.de.
authors.forename: Karin
authors.initials: K
authors.lastname: Riegel
authors.affiliation: Institute of Medical Microbiology, University Hospital Essen, University Duisburg-Essen, Essen, Germany. astrid.westendorf@uk-essen.de.
authors.forename: Astrid M
authors.initials: AM
authors.lastname: Westendorf
authors.affiliation: Department of Lead Discovery, Boehringer Ingelheim RCV GmbH & Co KG, 1121, Vienna, Austria. wolfgang.sommergruber@boehringer-ingelheim.com.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sommergruber
authors.affiliation: Institute of Applied Biotechnology, University of Applied Sciences Biberach, 88400, Biberach, Germany. otte@hochschule-bc.de.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Otte
authors.affiliation: Institute of Microbiology and Biotechnology, University of Ulm, 89069, Ulm, Germany. peter.duerre@uni-ulm.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Dürre
authors.affiliation: Institute of Microbiology and Biotechnology, University of Ulm, 89069, Ulm, Germany. christian.riedel@uni-ulm.de.
authors.forename: Christian U
authors.initials: CU
authors.lastname: Riedel
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods*; D051379:Mice; D018874:Spheroids, Cellular / Q000378:metabolism*