Abstract
BackgroundCpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology.MethodsThe expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system.ResultsWe found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis.ConclusionsHere we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology.
Highlights
CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer
Expression of TLR9 in malignant tumors To investigate the expression of TLR9 in human lung tumors and lung tumor cell lines we used the recently described HOPE-fixation method
To confirm the results obtained by in situ hybridization (ISH) we analyzed TLR9-transcripts in tumor cell lines by RT-PCR
Summary
CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. The Toll gene, the expression of one of it's relatives we are reporting here concerning human malignant tumors, originally was characterized for its role in specifying dorsoventral polarity of the Drosophila embryo[1]. Human TLR recognize pathogen-derived products, termed pathogen-associated molecular patterns (PAMP) [3]. These are bacterial lipoproteins (sBLP) [4], viral double stranded RNA/poly (I:C) [5], lipopolysaccharides (LPS) [6], flagellin [7] and bacterial DNA [8], which engage TLR2, TLR3, TLR4, TLR5 and TLR9, respectively. TLR9, in contrast to the other TLR, is not located at the cell surface, but intracellularily and, inhibition of endocytosis or endosome formation completely ablates the effects of CpG-ODN [10]
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