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Eicosanoid profiling in an orthotopic model of lung cancer progression by mass spectrometry demonstrates selective production of leukotrienes by inflammatory cells of the microenvironment.

Poczobutt JM, Gijon M, Amin J, Hanson D, Li H, Walker D, Weiser-Evans M, Lu X, Murphy RC, Nemenoff RA - PLoS ONE (2013)

Bottom Line: AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer.Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS.The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Colorado Denver, Aurora, Colorado, United States of America.

ABSTRACT
Eicosanoids are bioactive lipid mediators derived from arachidonic acid(1) (AA), which is released by cytosolic phospholipase A2 (cPLA2). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA2 knockout vs wild-type mice, we demonstrated that prostaglandins (PGE2, PGD2 and PGF2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB4, LTC4, LTD4, LTE4) production required cPLA2 expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.

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Recovery of inflammatory cells from TME by flow cytometry.LLC-Luc cells were injected into left lung lobes of WT or cPLA2-KO mice and tumor-bearing lungs were harvested 2.5 weeks later. Tissues from 4 mice were pooled, and inflammatory cells were recovered by flow cytometry. A. Sequential flow cytometry gating strategy used to recover inflammatory cells: Neu: neutrophils (CD11b+/Ly6G+), MacA macrophages (SigF+/CD11c+/Ly6G-) and MacB macrophages (F480+/CD11b+/Ly6G-/SigF-). B. Numbers of cells recovered by flow cytometry from uninjected or tumor-bearing left lung lobes of WT and cPLA2-KO mice. Data show average from 3 separate experiments, with each sorting performed on a pool of 4 mice. Error bars show S.E.M.
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pone-0079633-g003: Recovery of inflammatory cells from TME by flow cytometry.LLC-Luc cells were injected into left lung lobes of WT or cPLA2-KO mice and tumor-bearing lungs were harvested 2.5 weeks later. Tissues from 4 mice were pooled, and inflammatory cells were recovered by flow cytometry. A. Sequential flow cytometry gating strategy used to recover inflammatory cells: Neu: neutrophils (CD11b+/Ly6G+), MacA macrophages (SigF+/CD11c+/Ly6G-) and MacB macrophages (F480+/CD11b+/Ly6G-/SigF-). B. Numbers of cells recovered by flow cytometry from uninjected or tumor-bearing left lung lobes of WT and cPLA2-KO mice. Data show average from 3 separate experiments, with each sorting performed on a pool of 4 mice. Error bars show S.E.M.

Mentions: The TME is composed of multiple cell types, including inflammatory cells, fibroblasts and adaptive immune cells. In order to begin to define which of these cell populations are producing leukotrienes in the setting of tumor progression, we used flow cytometry to isolate inflammatory cells from lungs of tumor-bearing mice. Using markers previously employed to characterize inflammatory cells in the lung, we defined neutrophils (Neu, CD11b+/Ly6G+), as well as two populations of macrophages, designated as MacA (SigF+/CD11c+/F480+/CD11b low) and MacB (F480+/CD11b+/Ly6G-/SigF-), (Fig. 3A). Based on previous studies [26], [27], MacA represent resident alveolar macrophages [27], whereas MacB is a heterogeneous population mainly composed of infiltrating monocytes and macrophages, and also contains interstitial macrophages, and CD11b+ dendritic cells [27], [28]. In the setting of tumors, the number of MacB cells was markedly increased, whereas the MacA population was not significantly altered compared to control mice not injected with cancer cells (Fig. 3B).


Eicosanoid profiling in an orthotopic model of lung cancer progression by mass spectrometry demonstrates selective production of leukotrienes by inflammatory cells of the microenvironment.

Poczobutt JM, Gijon M, Amin J, Hanson D, Li H, Walker D, Weiser-Evans M, Lu X, Murphy RC, Nemenoff RA - PLoS ONE (2013)

Recovery of inflammatory cells from TME by flow cytometry.LLC-Luc cells were injected into left lung lobes of WT or cPLA2-KO mice and tumor-bearing lungs were harvested 2.5 weeks later. Tissues from 4 mice were pooled, and inflammatory cells were recovered by flow cytometry. A. Sequential flow cytometry gating strategy used to recover inflammatory cells: Neu: neutrophils (CD11b+/Ly6G+), MacA macrophages (SigF+/CD11c+/Ly6G-) and MacB macrophages (F480+/CD11b+/Ly6G-/SigF-). B. Numbers of cells recovered by flow cytometry from uninjected or tumor-bearing left lung lobes of WT and cPLA2-KO mice. Data show average from 3 separate experiments, with each sorting performed on a pool of 4 mice. Error bars show S.E.M.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823604&req=5

pone-0079633-g003: Recovery of inflammatory cells from TME by flow cytometry.LLC-Luc cells were injected into left lung lobes of WT or cPLA2-KO mice and tumor-bearing lungs were harvested 2.5 weeks later. Tissues from 4 mice were pooled, and inflammatory cells were recovered by flow cytometry. A. Sequential flow cytometry gating strategy used to recover inflammatory cells: Neu: neutrophils (CD11b+/Ly6G+), MacA macrophages (SigF+/CD11c+/Ly6G-) and MacB macrophages (F480+/CD11b+/Ly6G-/SigF-). B. Numbers of cells recovered by flow cytometry from uninjected or tumor-bearing left lung lobes of WT and cPLA2-KO mice. Data show average from 3 separate experiments, with each sorting performed on a pool of 4 mice. Error bars show S.E.M.
Mentions: The TME is composed of multiple cell types, including inflammatory cells, fibroblasts and adaptive immune cells. In order to begin to define which of these cell populations are producing leukotrienes in the setting of tumor progression, we used flow cytometry to isolate inflammatory cells from lungs of tumor-bearing mice. Using markers previously employed to characterize inflammatory cells in the lung, we defined neutrophils (Neu, CD11b+/Ly6G+), as well as two populations of macrophages, designated as MacA (SigF+/CD11c+/F480+/CD11b low) and MacB (F480+/CD11b+/Ly6G-/SigF-), (Fig. 3A). Based on previous studies [26], [27], MacA represent resident alveolar macrophages [27], whereas MacB is a heterogeneous population mainly composed of infiltrating monocytes and macrophages, and also contains interstitial macrophages, and CD11b+ dendritic cells [27], [28]. In the setting of tumors, the number of MacB cells was markedly increased, whereas the MacA population was not significantly altered compared to control mice not injected with cancer cells (Fig. 3B).

Bottom Line: AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer.Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS.The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Colorado Denver, Aurora, Colorado, United States of America.

ABSTRACT
Eicosanoids are bioactive lipid mediators derived from arachidonic acid(1) (AA), which is released by cytosolic phospholipase A2 (cPLA2). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA2 knockout vs wild-type mice, we demonstrated that prostaglandins (PGE2, PGD2 and PGF2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB4, LTC4, LTD4, LTE4) production required cPLA2 expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.

Show MeSH
Related in: MedlinePlus