Snapshots on bacterial tumor colonization [Elektronische Ressource] / von Kathrin Westphal

Kathrin Bartelheim

Sujets

• Gesundheit

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2007
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 22 Mo

Extrait

Snapshots on bacterial tumor
colonization



Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades einer

Doktorin der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n












Von: Kathrin Westphal
Aus: Bochum













































1. Referent: Professor Dr. Jürgen Wehland
2. Referent: Professor Dr. Stefan Dübel
eingereicht am: 14.03.2007
mündliche Prüfung (Disputation) am: 10.07.2007

Druckjahr 2007 Vorveröffentlichungen der Dissertation
Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab
veröffentlicht:
Publikationen:
Westphal K, Loessner H, Leschner S, Weiss S. Pharmaceutical composition for tumor
treatment. EP07102516.7 patent application filed, Feb. 15th, 2007
Loessner H, Endmann A, Leschner S, Westphal K, Rohde M, Miloud T, Hammerling G,
Neuhaus K, Weiss S. Remote control of tumour-targeted Salmonella enterica serovar
Typhimurium by the use of l-arabinose as inducer of bacterial gene expression in vivo.
Cellular Microbiology (2007).
Tagungsbeiträge:
Westphal K, Loessner H, Weiss S. Bacteria as carriers for tumor therapy (Poster),
"Molecular Interactions during Infection", Summer School, Quedlinburg (2006).
Westphal K, Zelmer A, Weiss S. Shigella flexneri mediated gene transfer into established
tumors (Poster), "New Approaches to Vaccine Development-From the bench to the field“,
Vaccine Congress, Berlin (2005). Table of contents I
1 Introduction ...................................................................................................................... 1
1.1 Cancer ......................................................................................................................... 2
1.2 Tumor therapy............................................................................................................. 5
1.2.1 Conventional tumor therapy............................................................................... 5
1.2.1.1 Chemotherapy ................................................................................................ 5
1.2.1.2 Radiotherapy .................................................................................................. 6
1.2.2 Innovative tumor therapy ................................................................................... 8
1.2.2.1 Bacteria-mediated tumor therapy................................................................. 11
1.3 Suitable bacteria for tumor therapy........................................................................... 12
1.3.1 Obligate anaerobic bacteria.............................................................................. 15
1.3.2 Facultative anaerobic bacteria.......................................................................... 17
1.3.2.1 Escherichia coli............................................................................................ 17
1.3.2.2 Salmonella enterica serovar Typhimurium.................................................. 20
1.3.2.3 Shigella flexneri............................................................................................ 23
1.4 Bacteria-mediated tumor therapy.............................................................................. 28
1.4.1 Bacteria-mediated gene transfer....................................................................... 28
1.4.2 Bacteria-mediated RNA transfer...................................................................... 32
1.4.3 Bacteria-mediated delivery of therapeutic proteins.......................................... 32
1.5 Bacterial interactions with the immune system in tumor therapies .......................... 38
1.5.1 First line of defense .......................................................................................... 39
1.5.2 Neutrophilic granulocytes ................................................................................ 40
1.5.3 Macrophages and the stimulation of the specific immune system................... 43
1.6 Aim of this work ....................................................................................................... 44
2 Material and Methods.................................................................................................... 46
2.1 Material ..................................................................................................................... 46
2.1.1 Mouse strain ..................................................................................................... 46
2.1.2 Cell lines and culture conditions ...................................................................... 46
2.1.3 Antibodies ........................................................................................................ 46
2.2 Methods..................................................................................................................... 47
2.2.1 Molecular biological methods.......................................................................... 47
2.2.2 Tissue culture ................................................................................................... 54
2.2.3 Calcium phosphate transfection ....................................................................... 55
2.2.4 S. flexneri-mediated transfer of eukaryotic expression plasmids to mammalian
cells .................................................................................................................. 56
2.2.5 Infection of tumor-bearing BALB/c mice with S. flexneri, S. typhimurium and
E. coli ............................................................................................................... 56
2.2.6 Quantitation of luciferase expression............................................................... 56 Table of contents II
2.2.7 Immunological methods................................................................................... 57
2.2.8 Electron microscopy......................................................................................... 58
3 Results ............................................................................................................................. 59
3.1 Shigella flexneri-mediated gene transfer into solid tumors ...................................... 59
3.1.1 The CMV promoter directs protein synthesis in S. flexneri M90T Serotyp 5,
dap and S. flexneri Serotyp 2a, aroD........................................................... 60
3.1.2 Distinction between eukaryotic and bacterial gene expression........................ 60
3.1.3 In vitro gene transfer into different tumor cell lines ........................................ 63
3.1.4 In vivo gene transfer into solid CT26 tumors................................................... 64
3.2 Distribution of Shigella flexneri dap in solid CT26 tumors ................................... 66
3.3 Distribution of Salmonella typhimurium SL7207 and Escherichia coli TOP10 in
solid CT26 tumors..................................................................................................... 69
3.4 Dramatic influx of neutrophils to the site of infection.............................................. 73
3.5 Depletion of host neutrophils.................................................................................... 81
3.5.1 Stronger accumulation of bacteria inside the tumor after depletion of
neutrophils........................................................................................................ 82
3.5.2 Bacteria are found in vital tumor tissue of neutrophil-depleted mice .............. 84
3.5.3 Depletion of neutrophils leads to increase of necrosis..................................... 88
3.6 Kinetics of bacterial colonization of solid CT26 tumors .......................................... 95
3.7 Salmonella typhimurium SL7207 resides mainly extracellularly inside solid CT26
tumors...................................................................................................................... 101
3.8 Bacterial encapsulation and biofilm formation inside solid CT26 tumors ............. 102
4 Discussion...................................................................................................................... 109
4.1 Early events in bacterial tumor colonization........................................................... 110
4.2 Containment of tumor colonizing bacteria by host neutrophils.............................. 113
4.3 Biofilm formation and encapsulation inside solid tumors ...................................... 114
4.4 Bacterial gene transfer into solid tumors ................................................................ 116
4.5 Outlook.................................................................................................................... 11