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Bladder cancer is among the most common malignancies worldwide. The majority (75%-85%) of the patients are diagnosed early, prior to muscle invasive stage. BCG immunotherapy, in addition to transurethral resection, is recommended in high risk non muscle invasive tumors. This is often the only therapy required.
The mechanism of action of BCG has not been fully elucidated. Toll-like receptors, modulators of innate immunity, play a major role in its therapeutic mechanisms. In a mouse model, the TLR-9 ligand CpG-ODN has proven more effective than BCG in treating early stage bladder cancer.
Little is known about the direct effect of TLRs on bladder cancer cells. This work studied the expression of TLR-4 and TLR-9 on various bladder cancer cell lines. On activation of these receptors outcomes measured were cytokine expression, cell viability and cell invasiveness.
The cell lines tested universally expressed TLR-9 mRNA, and most expressed TLR-4 mRNA. From those cell lines and based on their TLR mRNA expression four cell lines were chosen for further investigation. In these cell lines TLR expression was verified using Immuncytology (for TLR-4) and Western Blot (for TLR-9).
T24 cells in which high TLR-4 and BFTC cells in which no TLR-4 expression was detected, were subjected to the TLR-4 agonist LPS. In T24 cells this significantly induced IL-6 and TNFα. There was no effect on BFTC. No impact on cell viability in either cell line.
UMUC3 cells, in which high TLR-9 and RT112 cells, in which low TLR-9 expression was detected, were subjected to the TLR-9 agonists CpG-A and CpG-B. In this work it was demonstrated that INTERFERin® increased the uptake of CpG-ODN making it available to intracellular TLR-9.To some extend it enhanced CpG-ODN effects.
In UMUC3 CpG-A boosted with INTERFERin® significantly induced IL-8, TNFα, INFß, VEGF-A and PlGF. Whereas CpG-B boosted with INTERFERin® only induced IL-8 and INFß. In RT112 no induction was noted in the CpG-A group. However in the CpG-B group both IL-8 and TNFα were induced.
Cell viability of UMUC3 was reduced when exposed to CpG-B alone or in combination with INTERFERin®. However, CpG-A reduced cell viability only when boosted with INTERFERin®. In RT112, only CpG-B reduced viability. This effect was seen with and without INTERFERin®.
Cell invasion in both tested cell lines was increased significantly by CpG-ODN. CpG-B caused the highest increase.
Immunotherapeutics, in current use (BCG) or those being under investigation (CpG-ODN, mycobacterial cell wall-DNA complex) mediate their effect partially via TLR-4 and TLR-9. This means, that a direct effect on the tumor cells has to be taken into concern, because many tumors are expressing functionally active TLRs, too.
Findings in this work suggest these effects to be inhibitory regarding tumor growth and stimulatory regarding metastasis. Further research is needed to verify these effects and their therapeutic relevance in the hope that therapies with BCG and potential future immunotherapeutics become more predictable.