Eicosanoid Profiling in an Orthotopic Model of Lung Cancer Progression by Mass Spectrometry Demonstrates Selective Production of Leukotrienes by Inflammatory Cells of the Microenvironment

Joanna M Poczobutt,Deandra Walker,Howard Li,Robert C Murphy,Mary Weiser-Evans,Xian Lu,Miguel Gijon,Raphael A Nemenoff,Jay Amin,Dwight Hanson,Anthony Peter Sampson

doi:10.1371/journal.pone.0079633

Abstract

Eicosanoids are bioactive lipid mediators derived from arachidonic acid1 (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.

Highlights

Eicosanoids represent a family of bioactive lipids produced through metabolism of arachidonic acid
We have demonstrated that tumors growing in mice globally deficient in cytosolic phospholipase A2 (cPLA2) have a marked impairment in progression and metastasis [21]
Multiple eicosanoids are increased in tumor bearing mice Using an orthotopic immunocompetent mouse model, in which tumor cells are injected into lungs of mice, we previously showed that loss of cPLA2 in the tumor microenvironment inhibited lung cancer metastasis[21]

Summary

Introduction

Eicosanoids represent a family of bioactive lipids produced through metabolism of arachidonic acid. The family of PLA2 enzymes hydrolyze membrane phospholipids to produce free fatty acids and lysophospholipids. Cyclooxygenases (COX1, 2) produce prostaglandins, including PGE2 and PGI2 as well as thromboxanes, lipoxygenases produce hydroxyeicosatetraenoic acids (HETEs) and leukotrienes, and cytochrome P450 epoxygenases produce epoxygenated fatty acids (EETs). Over 100 distinct eicosanoid species have been identified [3] The majority of these molecules are secreted from cells and act in an autocrine or paracrine fashion through a family of G-protein coupled receptors [4]. The repertoire of eicosanoids produced by a particular cell type will be governed by expression of enzymes in the pathway, including specific downstream synthases. PGE2 production will be regulated by expression of cyclooxygenase enzymes as well as prostaglandin E2 synthases, while specific leukotrienes, such as LTC4 require expression of 5lipoxygenase, as well as LTC4 synthase

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