Figure 1.
Multiple eicosanoids are increased in tumor bearing mice.
LLC-Luc cells or matrigel (control) were injected into left lung lobes of WT C57Bl/6 mice and tumor-bearing and control lungs were harvested 2 or 3 weeks after injection. Eicosanoid levels were analyzed by LC/MS/MS and normalized to the protein content of the sample. Levels on graphs are expressed as fold over control. A. Early eicosanoids – induced >3 fold at 2 weeks, and further increasing at 3 weeks. B. Late eicosanoids – not upregulated (<2-fold) at 2 weeks, but increased at 3 weeks. C. Unchanged eicosanoids.
Table 1.
Increase in eicosanoid levels during lung tumor progression in orthotopic lung cancer model in mice.
Figure 2.
Deletion of cPLA2 in the TME differentially affects eicosanoid production.
LLC-Luc cells or matrigel (control) were injected into left lung lobes of WT or cPLA2-KO mice. Injections were done on 3 separate days with control vs. tumor and WT vs. cPLA2-KO groups equally represented on each day. Left lobes harboring tumors and control lungs were harvested 2 or 3 weeks after injection, eicosanoids were analyzed by LC/MS/MS and normalized to protein content of the sample. Each point represents a single mouse. Bars represent medians. A. Weights of tumor-bearing and control lungs B. Select eicosanoids
Table 2.
Eicosanoid levels in tumor-bearing lungs of WT and cPLA2-KO mice.
Figure 3.
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.
Figure 4.
Expression of enzymes in the eicosanoid pathway in inflammatory cells recovered from tumor-bearing lungs.
Relative mRNA expression in cells recovered from tumor-bearing lungs by flow cytometry. Expression was assessed by qRT-PCR, normalized to the geometric average of β-actin, GAPDH, Ubiquitin C, and 18s, and expressed as % Maximum among the analyzed cell groups. Experiments were performed on 2 separately injected and analyzed pools of mice (4 mice per WT or cPLA2-KO group per pool). Filled circles – pool #1, open circles – pool #2, bar – mean of the pools.
Figure 5.
Eicosanoid production by inflammatory cells recovered by flow cytometry.
Eicosanoid production in cells recovered from tumor-bearing lungs by flow cytometry. Recovered cells were stimulated with ionophore A23187 (0.5 µM) for 12 minutes. Extracted eicosanoids were analyzed by LC/MS/MS. Experiments were performed on 2 separately injected and analyzed pools of mice (4 mice per WT or cPLA2-KO group per pool). Filled circles – pool #1, open circles – pool #2, bar – mean of the pools.