Acute Bacterial Infection Facilitates Lung Cancer Metastasis Through Toll-Like Receptor 2 and 4 Activation

INTRODUCTION: Lung cancer is the leading cause of cancer death, with low five-year survival rates and high percentage of local or metastatic recurrence. Surgical resection is integral for cure, however this procedure is associated with increased systemic inflammation and circulating tumour cells. This is further compounded by infectious postoperative complications, the most common of which is pneumonia, caused by both gram-positive and gram-negative bacteria. There is emerging clinical data to suggest that bacterial complications after lung cancer surgery may decrease survival, however the mechanism for this finding is not clear. Interactions between bacteria and host cells are mediated by specialized pathogen receptor proteins termed, Toll like receptors. Cancer cells have recently been shown to express Toll like receptors, but their function in these malignant cells is unknown. The aim of this study is to investigate the mechanisms of bacterial infection facilitated lung cancer metastasis with a particular focus on the roles of Toll like receptors.;METHODS: Murine H-59 Lewis lung carcinoma or Human A549 lung cancer cells were employed in a series of in vitro and in vivo experiments. Cancer cells were activated either directly with exposure to heat-inactivated S.pneumonia, E.coli or purified bacterial antigens, or indirectly with exposure to culture media from BEAS-2B bronchial epithelial cell incubation with heat-inactivated bacteria (S.pneumonia or E.coli) or bacterial antigens. In vitro assays include adhesion to extracellular matrix components fibronectin, collagen I and collagen IV, and migration/chemotaxis through a filter towards the BEAS-2B supernatant. In vivo assays include intravital microscopy to quantify in vivo cancer cell migration within the liver sinusoids, and intra-splenic hepatic metastasis assay. In some animals, a highly clinically relevant model of infection, cecal ligation and puncture (CLP) or sham control, was employed. To assess the role of Toll like receptors, we employed transgenic TLR4 knockout mice, monoclonal blocking antibodies, or small molecule inhibitors.;RESULTS: Direct and indirect incubation of both H-59 and A549 cells with S.pneumonia and E.coli increased adhesion to extracellular matrix components approximately 2-8 fold and 2-4 fold, respectively. Presence of BEAS-2B culture media after infection with S.pneumonia and E.coli increased in vivo migration of H-59 cells approximately 2-4 fold over negative control, and into the bottom chamber approximately 4-5 fold. Direct and indirect incubation of H-59 cells with S.pneumonia and E.coli increased adhesion to liver sinusoids 2-4 fold. In vivo infection with CLP increased adhesion of H-59 cells to liver sinusoids 3-fold and increased number of liver metastases at 2 weeks post-injection approximately 12 fold. These effects were attenuated with TLR2 and TLR4 inhibition.;CONCLUSION: Activation of murine and human lung cancer cells, either directly by S.pneumonia and E.coli, indirectly via activated bronchial epithelial cells, or via an in vivo infection, increases cancer cell adhesion, migration and metastasis, and this effect is mediated by toll like receptors.