Validation of bone marrow derived dendritic cells as an appropriate model to study tumor-mediated suppression of DC maturation through STAT3 hyperactivation.
OBJECTIVE
Tumors can escape immune eradication by harnessing dendritic cell (DC) maturation. However, DC types used as in vitro models to study tumor-mediated immunosuppression possess fundamental variability that could influence research outcomes. Therefore, we assessed the behavior of two distinct murine DC models upon exposure to tumor-conditioned medium of B16.F10 melanoma (B16-CM).


METHODS
Using primary bone-marrow derived dendritic cells (BMDCs) or immortalized DC2.4 cell line, we evaluated the level of signal transducer and activator of transcription 3 (STAT3) phosphorylation by Western blot as a molecular parameter. We also examined the surface expression of co-stimulatory molecules on DCs by flow cytometry as a phenotypic parameter.


RESULTS
Our results revealed critical discrepancies between the two models in response to tumor-conditioned medium. While conditioned medium was able to induce STAT3 phosphorylation in BMDCs, it did not significantly induce STAT3 phosphorylation in DC2.4 cell line. Moreover, only in BMDCs, the expression of CD86 and CD40 was remarkably downregulated by B16-CM and was not totally recovered after LPS stimulation. In contrast, DC2.4 cells did not show any signs of harnessed maturation upon exposure to B16-CM.


CONCLUSIONS
In order to study the effect of tumor-mediated immunosuppression on DC maturation in vitro via tumor-induction of STAT3 activation, primary BMDCs are more reliable as a model than DC2.4.
Alshamsan
Aws
A
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada.
Hamdy
Samar
S
Das
Saswati
S
Lavasanifar
Afsaneh
A
Samuel
John
J
El-Kadi
Ayman O S
AO
D000818:Animals; D051940:B7-2 Antigen / Q000235:genetics; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism; D019013:CD40 Antigens / Q000235:genetics; D002460:Cell Line; D003713:Dendritic Cells / Q000276:immunology*; D015536:Down-Regulation; D005434:Flow Cytometry; D007108:Immune Tolerance; D008546:Melanoma, Experimental / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D010766:Phosphorylation; D050796:STAT3 Transcription Factor / Q000276:immunology*
D051940:B7-2 Antigen; D019013:CD40 Antigens; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse