In vitro and in vivo effects of CpG-Oligodeoxynucleotides (CpG-ODN) on murine transitional cell carcinoma and on the native murine urinary bladder wall.
BACKGROUND
Intravesical BCG instillation is established and efficient in the prophylaxis of recurrent transitional cell carcinoma. A Th-1 biased immune response is postulated. Recent work has proven the efficacy of synthetic CpG-Oligodeoxynucleotides (ODN) as inducers and adjuvants for a strong Th1-response and there is evidence for a direct and/or adjuvant anti-neoplastic effect. The purpose of this study was to examine the local effects of CpG-ODN on the murine bladder wall after intravesical instillation and the effects on cytokine expression in an orthotopic murine bladder cancer model.


METHODS
Histopathology, immunohistochemistry and fluorescence microscopy were performed after different instillation schedules of stimulatory, non-stimulatory biotinylized and FITC-labelled CpG-ODN into the murine bladder. MB-49 murine bladder cancer cells were tested for TLR-9 expression to exclude a potential direct responsiveness to CpG-ODN. Furthermore induction of apoptosis was tested by annexin V staining and FACS analysis of CpG-ODN stimulated tumor cells. In an orthotopic C57/Bl6 murine bladder cancer model, the expressions of IL-12, IFNgamma, IL-10 and TGF-beta were evaluated after repeated CpG-ODN treatment.


RESULTS
Single and repeated instillation of CpG-ODN induced subepithelial and urothelial lymphocytic infiltrations with consecutive apoptoses. PBS and non-stimulative ODN induced no visible reaction. Bladder submucosa stained positive for biotin. Controls showed no endogenic biotin staining. FITC-labelled ODN adhered to the bladder mucosa and penetration of the mucosal barrier was not detected. MB-49 TCC cells did not express TLR-9 and CpG-ODN did not induce apoptosis in these cells. Repeated intravesical instillations of CpG-ODN in orthotopic murine tumor bearing urinary bladders resulted in significant up-regulation of both Th-1 and Th-2 cytokines.


CONCLUSIONS
CpG-ODNs have promising anti-neoplastic potential. They exert a pronounced immunological response both in the native murine urinary bladder and in murine TCC. The mechanisms of action appear to be mediated immunologically, There was no direct effect of CpG-ODN on the tumor cells in this model.
Olbert
Peter Jochen
PJ
Department of Urology and Pediatric Urology, Medical School, Phillips University Marburg, 35043 Marburg, Germany. peter.olbert@med.uni-marburg.de
Schrader
Andres Jan
AJ
Simon
Corinna
C
Dalpke
Alexander
A
Barth
Peter
P
Hofmann
Rainer
R
Hegele
Axel
A
D000276:Adjuvants, Immunologic / Q000627:therapeutic use*; D000818:Animals; D017209:Apoptosis; D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D005260:Female; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism; D018664:Interleukin-12 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D009838:Oligodeoxyribonucleotides / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D001743:Urinary Bladder / Q000502:physiology*; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000628:therapy*
D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9; D016212:Transforming Growth Factor beta; D016753:Interleukin-10; D018664:Interleukin-12; D007371:Interferon-gamma