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Introduction Surgery is essential for cure of early-stage non-small cell lung cancer (NSCLC). Rates of postoperative bacterial pneumonias, however, remain high, and clinical data suggests that post-operative infectious complications confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory response to infection by recognizing evolutionarily conserved bacterial structures at the surface of numerous pulmonary cell types; yet, little is known about how host TLR activation influences NSCLC metastasis. TLR4 recognizes gram-negative bacterium lipopolysaccharide activating the innate immune system. Methods C57BL/6 and TLR4 knockout murine airways were inoculated with Escherichia coli or lipopolysaccharide. Hepatic metastasis assays and intravital microscopy were performed. Bronchoepithelial conditioned media was generated through coincubation of bronchoepithelial cells with TLR4 activating Escherichia coli or lipopolysaccharide. Subsequently, H59 NSCLC were stimulated with conditioned media and subject to various adhesion assays. Results We demonstrate that gram-negative Escherichia coli pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through TLR4 activation. Additionally, infected C57BL/6 mice demonstrate increased H59 NSCLC in vivo hepatic sinusoidal adhesion compared with negative controls, a response that is significantly diminished in TLR4 knockout mice. Similarly, intratracheal injection of purified TLR4 activating lipopolysaccharide increases in vivo adhesion of H59 cells to murine hepatic sinusoids. Furthermore, H59 cells incubated with bronchoepithelial conditioned medium show increased cell adhesion to in vitro extracellular matrix proteins and in vivo hepatic sinusoids through a mechanism dependent on bronchoepithelial TLR4 activation and interleukin-6 secretion. Conclusion TLR4 is a viable therapeutic target for NSCLC metastasis augmented by gram-negative pneumonia.

Surgery is required for the curative treatment of lung cancer but is associated with high rates of postoperative pneumonias predominantly caused by gram negative bacteria. Recent evidence suggests that these severe infectious complications may decrease long term survival after hospital discharge via cancer recurrence, but the mechanism is unclear. Lung cancer cells have recently been demonstrated to express Toll-like receptors (TLR) that mediate pathogen recognition. We hypothesized that incubation of non-small cell lung cancer (NSCLC) cells with heat-inactivated Escherichia coli can augment cancer cell adhesion, migration and metastasis via TLR4 signaling. Incubation of murine and human NSCLC cells with E. coli increased in vitro cell adhesion to collagen I, collagen IV and fibronectin, and enhanced in vitro migration. Using hepatic intravital microscopy, we demonstrated that NSCLC cells have increased in vivo adhesion to hepatic sinusoids after coincubation with gram negative bacteria. These enhanced cell adhesion and migration phenotypes following incubation with E. coli were attenuated at three levels: inhibition of TLR4 (Eritoran), p38 MAPK (BIRB0796) and ERK1/2 phosphorylation (PD184352). Incubation of murine NSCLC cells in vitro with E. coli prior to intrasplenic injection significantly augmented formation of in vivo hepatic metastases 2 weeks later. This increase was abrogated by NSCLC TLR4 blockade using Eritoran. TLR4 represents a potential therapeutic target to help prevent severe postoperative infection driven cancer metastasis.

Surgical resection of early stage nonsmall cell lung cancer (NSCLC) is necessary for cure. However, rates of postoperative bacterial pneumonias remain high and may confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory cascade by recognizing microbial products at the surface of numerous cell types in the lung; however, little is known about how host TLRs influence NSCLC metastasis. TLR2 recognizes gram-positive bacterial cell wall components activating innate immunity. We demonstrate that lower respiratory tract infection with Streptococcus pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through host TLR2 activation. Infected mice demonstrate increased H59 and human A549 NSCLC adhesion to hepatic sinusoids in vivo compared with noninfected controls, a response that is significantly diminished in TLR2 knock-out mice. Intra-tracheal injection of purified TLR2 ligand lipoteichoic acid into mice similarly augments in vivo adhesion of H59 cells to hepatic sinusoids. Additionally, H59 and A549 NSCLC cells incubated with bronchoepithelial conditioned media show increased cell adhesion to extracellular matrix components in vitro and hepatic sinusoids in vivo in a manner that is dependent on bronchoepithelial TLR2 activation and interleukin-6 secretion. TLR2 is therefore a potential therapeutic target for gram-positive pneumonia-driven NSCLC metastasis.

Surgery is required for the curative treatment of lung cancer but is associated with high rates of postoperative pneumonias predominantly caused by gram negative bacteria. Recent evidence suggests that these severe infectious complications may decrease long term survival after hospital discharge via cancer recurrence, but the mechanism is unclear. Lung cancer cells have recently been demonstrated to express Toll-like receptors (TLR) that mediate pathogen recognition. We hypothesized that incubation of non-small cell lung cancer (NSCLC) cells with heat-inactivated Escherichia coli can augment cancer cell adhesion, migration and metastasis via TLR4 signaling. Incubation of murine and human NSCLC cells with E. coli increased in vitro cell adhesion to collagen I, collagen IV and fibronectin, and enhanced in vitro migration. Using hepatic intravital microscopy, we demonstrated that NSCLC cells have increased in vivo adhesion to hepatic sinusoids after coincubation with gram negative bacteria. These enhanced cell adhesion and migration phenotypes following incubation with E. coli were attenuated at three levels: inhibition of TLR4 (Eritoran), p38 MAPK (BIRB0796) and ERK1/2 phosphorylation (PD184352). Incubation of murine NSCLC cells in vitro with E. coli prior to intrasplenic injection significantly augmented formation of in vivo hepatic metastases 2 weeks later. This increase was abrogated by NSCLC TLR4 blockade using Eritoran. TLR4 represents a potential therapeutic target to help prevent severe postoperative infection driven cancer metastasis.

Lung cancer is the leading cause of cancer death. Surgery remains essential for cure. However, postoperative infections particularly pneumonia remain high and confer an increased risk for metastasis. Emerging evidence implicates Toll-like receptor (TLR) signalling in tumor progression after systemic infection. We hypothesize bacterial pneumonia increases lung cancer cell adhesion and metastasis and that these effects are mediated in part by TLR signalling. C57BL6 mice were intranasally inoculated with 10^5 CFU/mL of S. pneumonia and intrasplenically injected with H59 murine lung carcinoma 3 days later. Gross liver metastases were quantified at 2 weeks. Similarly, H59 cell adhesion to liver sinusoids was assessed by intravital microscopy (IVM) in wild type (WT) and TLR2 KO and TLR4 KO mice. WT, TLR2 KO, TLR4 KO, and MyD88 KO mice were intratracheally injected with lipoteichoic acid (LTA), lipopolysaccharide (LPS), or control (PBS). H59 cell adhesion was assessed 4 hours later with liver IVM. Using an in vitro pneumonia model, bronchoepithelial BEAS2B cells were stimulated with LPS, LTA, heat-inactivated E. coli or heat-inactivated S. pneumonia and supernatants collected. H59 cells were exposed to various supernatants and adhesion to extracellular matrix components was assessed. BEAS2B cells were also stimulated in the presence of anti-TLR2 antibodies or eritoran tetrasodium to inhibit TLR2 and TLR4 signalling respectively. Adhesion of supernatant treated cells to liver sinusoids was also assessed with IVM. Mice inoculated with S. pneumonia demonstrated a 2-3 fold increase in gross liver metastases compared to non-infected controls. Similarly WT and TLR4 KO mice infected with S. pneumonia demonstrated a 4-5 fold increase in H59 adhesion to liver sinusoids versus control. TLR2 KO mice infected with S. pneumonia demonstrated no significant increase in H59 cell adhesion to liver. WT mice intratracheally injected with LTA or LPS demonstrated a 3-4 fold increase in cancer cell adhesion to liver compared to control. The increased adhesion for LTA was negated in TLR2 KO and MyD88 KO mice. For LPS conditions this increase was absent in TLR4 KO and MyD88 KO mice. Incubation with TLR-activated BEAS2B supernatants increased H59 cell adhesion to collagen 1, 4 and fibronectin 2-3 , 3-6 and 4-6 fold respectively versus media control and adhesion to liver sinusoids in vivo increased 3-4 fold. These effects were abrogated by blocking TLR2 for LTA or S. pneumonia conditions and by blocking TLR4 for LPS or E. coli conditions. Bacterial pneumonia increases the metastatic potential of circulating lung cancer cells. These effects are facilitated in part by TLR2 or TLR4 activation and are mediated via interactions of the respiratory epithelium with the host systemically and lung cancer cells directly. TLR2 and TLR4 are potential therapeutic targets to decrease cancer recurrence in patients who suffer severe post-operative infections. Citation Format: Stephen D. Gowing, Simon C. Chow, Jonathan J. Cools-Lartigue, Crystal B. Chen, Betty Giannias, France Bourdeau, Simon Rousseau, Salman T. Qureshi, Lorenzo E. Ferri. Toll-like receptor activation in bacterial pneumonia increases lung cancer cell adhesion and metastasis formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1963. doi:10.1158/1538-7445.AM2014-1963