The CMT167 cell line derived from a spontaneous alveolar lung carcinoma of a C57BL/6 female mouse(6
) was stably transfected with firefly luciferase (CMT167/luc) to allow bioluminescent imaging to be performed. CMT167/luc cells suspended in Matrigel were injected through the rib cage into the left lobe of the lung of C57/BL6 mice as previously described(7
), and tumor formation analyzed as a function of time by bioluminescent imaging (). At 3 weeks, mice consistently developed large primary tumors at the sight of injection (). Lungs were inflated with India ink to visualize and quantify additional large (>0.4 mm) and micro (<0.3 mm) secondary tumors. Wild-type mice exhibited multiple secondary tumors throughout the left and right lung lobes (). By four weeks post-injection, all mice showed clinical features of mediastinal lymph node metastasis ().
Immune-competent lung cancer model
To assess the role of cPLA2 in the TME, equal numbers of CMT167/luc cells were injected into WT or cPLA2-KO mice, and lungs harvested after four weeks. Quantification of tumor metastases was limited to large secondary tumors visible under a dissecting microscope. Growth of the primary tumor was not significantly different between the two groups of mice (). CMT167 cells injected into WT mice metastasized to other lung lobes and into mediastinal lymph nodes. However, CMT167 cells growing in cPLA2-KO mice showed fewer metastases and little lymph involvement; 5/5 WT mice, but only 1/5 KO mice, exhibited lung metastases and lymph involvement by macroscopic morphological examination (). To extend the generality of these findings we performed similar experiments with Lewis lung carcinoma cells, another lung cancer cell line derived from C57BL/6 mice. These cells are more aggressive as reflected by more rapid proliferation of the primary tumor and increased numbers of secondary tumors. However, as with CMT167 cells, we observed no statistical difference in the size of primary tumors (not shown), but a marked decrease in secondary tumor number () in cPLA2-KO mice compared to WT. CMT167 cells were used for all subsequent studies.
cPLA2 in the tumor microenvironment is essential for lung cancer progression
While numerous cell types comprise the TME, attention has focused on tumor-associated macrophages (TAMs) as mediators of tumor progression and metastasis(9
). Macrophages express high levels of cPLA2
, and produce pro-angiogenic cytokines. We demonstrated that macrophage recruitment to the lung is impaired in cPLA2
-KO mice following an inflammatory stimulus(10
). TAMs surrounding tumors were assessed by F4/80 staining. Tumors grown in WT mice showed abundant accumulation of TAMs surrounding developing tumors. In contrast, fewer TAMs were detected in tumors of KO mice ().
Recruitment of bone marrow-derived circulating monocytes/macrophages is associated with more aggressive, malignant tumors. To assess the role of cPLA2 in bone marrow-derived cells on tumor progression, wild-type mice received bone marrow transplants from either WT or cPLA2-KO mice. Animals were allowed to recover for 5 weeks, and then injected with CMT167/luc cells. Mice transplanted with cPLA2-KO bone marrow had a marked survival advantage over mice transplanted with WT bone marrow (). In separate studies, bone marrow derived from either WT or cPLA2-KO mice was transplanted into a limited number of either WT or cPLA2-KO recipients to examine recruitment of bone marrow-derived macrophages to the tumor. These studies used UBI-EGFP/B6 transgenic mice, which express EGFP in all cells, as WT bone marrow donors. Lungs were harvested 4 weeks after CMT167/luc injection and analyzed histologically. Mice receiving cPLA2-KO bone marrow, independent of genotype, exhibited less inflammatory infiltrate surrounding the primary tumor compared to mice receiving WT bone marrow (). Mice receiving wild-type bone marrow were examined for EGFP and stained for F4/80. Both groups of mice receiving WT bone marrow showed large numbers of EGFP+;F4/80+ cells surrounding the tumor () indicating recruitment of bone marrow-derived macrophages. In both groups of mice receiving cPLA2-KO bone marrow, fewer F4/80 positive cells were detected, consistent with fewer macrophages. These data suggest that the stromal protective effects of cPLA2 depletion are mediated in large part through bone marrow-derived cells, and support a model in which cPLA2 expression in bone marrow-derived macrophages is critical for recruitment of these cells to the site of the tumor.
Increased survival and alterations in TAMs surrounding tumors grown in WT and cPLA2-KO mice transplanted with cPLA2 KO bone marrow
Production of IL-6 by both tumor and stromal cells contributes to tumor progression as well as angiogenesis(11
). To determine if cPLA2
plays a role in IL-6 production in vivo, sections from tumors grown in WT or cPLA2
-KO mice were stained for IL-6. Tumors grown in cPLA2
-KO mice had markedly lower levels of IL-6 both within and surrounding the tumor, compared to tumors grown in WT mice (). To examine IL-6 production in vitro, bone marrow-derived cells isolated from WT or cPLA2
-KO mice were cultured in the presence of M-CSF to promote macrophage maturation as previously described(4
). These cells have the morphology of macrophages, and are >95% F4/80 positive. Macrophages derived from WT mice produced twice the levels of IL-6 compared to macrophages derived from cPLA2
-KO mice (). Since interactions between cancer cells and macrophages impact cytokine production(12
), IL-6 production was also assessed in co-cultures of bone marrow-derived macrophages with CMT167 cells using Transwells, which allows diffusible mediators to act on each cell type. Co-culture of macrophages with CMT167 cells increased IL-6 production in both WT and cPLA2
-KO macrophages, with WT macrophages continuing to produce twice the levels compared to cPLA2
-KO macrophages (). CMT cells grown alone failed to produce detectable levels of IL-6. However, co-culture with macrophages resulted in significant IL-6 production. Importantly, IL-6 production by CMT167 co-cultured with cPLA2
-KO macrophages was only 40% of the levels seen with WT macrophage-CMT cell co-culture ().
cPLA2 deficiency attenuates production of IL-6 in co-cultures of CMT cells and bone marrow-derived macrophages
expression by NSCLC has been demonstrated to be important for transformed growth(2
), this is to our knowledge the first report indicating the importance of this enzyme in the TME. Our data indicate that two independent mouse lung cancer cell lines show a marked impairment in formation of secondary tumors when grown in cPLA2
-deficient mice. While the TME comprises many types of cells, we have focused on the role of bone marrow-derived cells, specifically macrophages. Specific deletion of cPLA2
in bone marrow-derived cells inhibits tumor progression and metastasis and promotes cancer survival. This protection is associated with decreased numbers of TAMs surrounding the tumors in cPLA2
-KO mice. In human lung cancer, increased numbers of macrophages surrounding the tumor has been associated with an unfavorable prognosis(13
). Our findings are consistent with these observations.
Macrophages play a complex role in cancer progression. While initially mediating cytotoxic effects on tumors, TAMs have been implicated in promoting tumor progression and metastasis. Production by TAMs of pro-angiogenic cytokines in cooperation with tumor cells stimulates tumor angiogenesis(14
). TAMs secrete factors with immunomodulatory activity, inhibiting T-cell function and other immune anti-tumorigenic effects. IL-6, a critical cytokine for tumor progression, promotes cancer progression through effects on tumor cells or stroma(15
). Our data indicate that tumors grown in cPLA2
-KO mice are exposed to lower levels of IL-6, associated with reduced tumor progression and metastasis. Consistent with this, in vitro data indicate that cPLA2
contributes to IL-6 production by macrophages, and is critical for the synergistic induction of IL-6 seen in co-cultures of cancer cells and macrophages.
The mechanism whereby cPLA2
regulates IL-6 production remains to be established. cPLA2
is critical for PGE2
production, the major prostaglandin produced by macrophages and NSCLC cells. PGE2
can induce IL-6 production in cholangiocarcinoma cells(16
), and modulates cytokine production by macrophages leading to promotion of M2-type macrophages (17
can act as an immune suppressor by inhibiting proliferation of T cells, effecting immunoglobulin production by B cells, and regulating dendritic cell maturation(18
). Further studies will be required to define the role of PGE2
in this model. Since cPLA2
is critical for production of other eicosanoids, including leukotrienes, other mediators may contribute to the effects of stromal cPLA2
. In addition, our studies also do not rule out a role for cPLA2
in other stromal cells, including fibroblasts and vascular cells. Recent studies have implicated production of PGE2
by myeloid-derived suppressor cells as a mediator of breast cancer progression(19
). Finally it should be noted that numerous studies have examined NSAIDs as therapeutic treatments for lung cancer. It appears that these agents may not only impact tumor growth, but may also directly impact metastasis through targeting prostaglandin production in macrophages and other stromal cells(20