Eicosanoids and exosomes : a link between macrophages and lung cancer

Chronic inflammation increases the risk of lung cancer. Macrophages (MO) are important players in inflammation, with regulatory and executive functions. Eicosanoids and exosomes can be both triggers and mediators of these functions. Cysteinyl leukotrienes (CysLTs) are the most potent mediators of broncho-constriction in the lungs, a function exerted via CysLT1 receptor. Their function in asthma is well described, but little is known about CysLTs and lung cancer. In the first study we investigated how the interaction between pulmonary epithelium and leukocytes affects CysLTs formation. Monocytic cells and eosinophils formed LTC4, which was exported and promptly converted to LTD4 by pulmonary epithelial cells in a transcellular manner. The lung cancer cell line A549 expressing γ-glutamyl transpeptidase 1 (GGT-1) showed a high activity. Exosomes released by A549 cells also contained GGT-1 and efficiently converted LTC4 to LTD4. On the other hand, healthy bronchial epithelial cells (PBEC) expressing GGT-5 formed LTD4 12 times more slowly. The results highlight an active role for epithelial cells and their exosomes in biosynthesis of LTD4, which may be of particular relevance in the lung, given that LTD4 is the most potent agonist of CysLT1. MOs can be differentiated from blood monocytes with GM-CSF and M-CSF, resulting in cells primed toward the inflammatory M1and resolving M2-states. A comprehensive analysis of eicosanoid formation in these two in vitro models is missing and our second study focused on this gap. By LC-MS analysis, we observed that both MO phenotypes released pro-resolving lipid mediators (PGE metabolite, LXA4) in resting conditions. When the same cells were incubated (30 min) with bacterial stimuli, there was a shift to pro-inflammatory eicosanoids: M-CSF MOs produced high amounts of LTC4, relevant for M2 functions in asthma. GM-CSF cells expressed the highest levels of cPLA2, 5-LO and FLAP; and in ionophore incubations these cells also produced the highest levels of 5-HETE. However, MCSF MO formed more products apparently due to a better response to bacterial stimuli, demonstrated by enhanced mobilization and activation of cPLA2 and 5-LO. In conclusion, GM-CSF and M-CSF can regulate specific pathways in MOs, and it appears that eicosanoid biosynthesis primarily reflect the cellular response and activation mechanisms, rather than the protein expression profile. In colon cancer a pro-tumorigenic effect of LTD4 but not LTC4 has been demonstrated. A pro-tumorigenic effect has been shown also for exosomes. To extend the findings of our first study, we used pleura exudates from lung cancer patients to isolate primary cancer cells and exosomes. Both cells and exosomes metabolized LTC4 to LTD4, and we also found that exosomes stimulated CysLTs formation in the cancer cells. Cancer cells from all patients expressed CysLT1, and exosomes promoted their migration and survival in a CysLT1 dependent manner, as demonstrated by the inhibition by montelukast (MK) treatment, a CysLT1 antagonist used to treat asthma. In cancer, interactions between the transformed cancer cells and other recruited cell types in the tumor are important. Tumor associated macrophages (TAMs) provide cancer cells with a suitable low-grade inflammation milieu including growth promoting factors. Taken together, the results in this thesis suggest a novel pro-tumorigenic mechanism based on this theme, driven by the exosomes/CysLT1 cascade: TAMs provide LTC4 that lung cancer cells and their exosomes convert to LTD4. Via CysLT1 receptor this promotes survival and migration of the cancer cells. A protective effect in lung cancer has been previously described for MK and our results suggest a possible mechanism for this, driven by the exosomes/ CysLT1 cascade, further encouraging the use of this drug in lung cancer treatment. LIST OF SCIENTIFIC PAPERS I. Lukic, A. , Ji, J., Idborg, H. , Samuelsson, B. , Palmberg, L. , Gabrielsson, S. , & Rådmark, O. (2016). Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4. Journal of lipid research, 57(9), 1659-1669 II. Lukic, A. , Larssen, P., Fauland, A. , Samuelsson, B. , Wheelock, C. E. , Gabrielsson, S. , & Radmark, O. (2017). GM-CSF–and M-CSF–primed macrophages present similar resolving but distinct inflammatory lipid mediator signatures. The FASEB Journal, 31(10), 4370-4381. III. Lukic, A. , Wahlund, C. , Gomez, C. , Brodin, D. , Samuelsson, B. , Wheelock, C. E. , Gabrielsson, S. , & Radmark, O. Exosomes and malignant cells from lung cancer pleura exudates form LTD4, promoting cell migration and survival in a CysLT1 dependent mechanism. Manuscript Publications not included in this thesis: I. Torregrosa Paredes, P., Esser, J., Admyre, C. , Nord, M. , Rahman, Q. K. , Lukic, A. , . & Scheynius, A. (2012). Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma. Allergy, 67(7), 911-919. II. Basavarajappa, D. , Wan, M. , Lukic, A. , Steinhilber, D. , Samuelsson, B. , & Rådmark, O. (2014). Roles of coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP) in cellular leukotriene biosynthesis. Proceedings of the National Academy of Sciences, 111(31), 11371-11376. III. Martinez-Bravo, M. J., Wahlund, C. J., Qazi, K. R. , Moulder, R. , Lukic, A. , Rådmark, O. , . & Gabrielsson, S. (2017). Pulmonary sarcoidosis is associated with exosomal vitamin D–binding protein and inflammatory molecules. Journal of Allergy and Clinical Immunology, 139(4), 1186-1194. LIST OF ABBREVIATIONS AA AERD BAL BEC BLT CD COPD COX cPLA2 CYP CysLT DHA DiHET DiHOME EDP EET EGFR EMT EP EPA EpOME ESCRT FLAP fMLP GGT GM-CSF Arachidonic acid Aspiring exacerbated respiratory disease Bronco-alveolar lavage Bronchial epithelial cells Leukotriene B4 receptor Cluster of differentiation Chronic obstructive pulmonary disease Cyclooxygenase Cytosolic phospholipase A2 Cytochrome P450 Cysteinyl leukotriene Docosahexaenois acid Dihydroxyeicosatrienoic acids Dihydroxyoctadecenoic acid Epoxydocosapentaenoic acid Epoxyeicosatrienoic acid Epithelial growth factor receptor Epithelial to mesothelial transition Prostaglandin E2 receptor Epoxydocosapentaenoic acid Epoxyoctadecenoic acid Endosomal complex required for transport Five lipoxygenase activating protein N-formylmethionyl-leucyl-phenylalanine Gamma-glutamyl transpeptidase Granulocyte macrophage-colony stimulating factor M-CSF GSH GTP HDoHE HETE HODE HpETE HPLC IL IFN-γ LA LC-MS LO LPS Macrophage-colony stimulating factor Glutathione Guanosine Triphosphate Hydroxydocosahexaenoic acid Hydroxyeicosatetraenoic acid Hydroxyoctadecadienoic acid Hydroperoxy eicosatetraenoic acid High-performance liquid chromatography Interleukin Interferon γ Linoleic acid Liquid chromatography-mass spectrometry Lipoxygenase Lipopolysaccharide LT LTC4s LTB4h LX MDSC MHC miRNA MK MM6 mPGES MSC MVB NSCLC PBEC PBMC Leukotriene LTC4 synthase LTB4 hydrolase Lipoxin Myeloid derived suppressor cells Major histocompatibility complex Micro RNA Montelukast Mono Mac 6 Microsomal prostaglandin E2 synthase Mesenchymal stem cells Multivesicular bodies Non-small cell lung cancer Primary BEC Peripheral blood mononuclear cells PE PG PGN PMN PTGIS PUFA RvE/RvD TLR SBC sEH SPM TAM Th TME TNFα Treg TXAS VEGF Pleural exudate Prostaglandin Peptidoglycan Polymorphonuclear neutrophils PGI2 synthase Polyunsaturated fatty acids Resolvins E/D series Toll like receptor Serine Borate Complex soluble Epoxide Hydrolase Specialized proresolving mediators Tumor associated macrophages T helper cell Tumor micro-environment Tumor necrosis factor alpha Regulatory T cell Thromboxane A synthase 1 Vascular endothelial growth factor