Abstract
Phospholipase D-2 (PLD2) has a key role in breast cancer formation and metastasis formation with PLD small inhibitors reducing primary tumor growth. This study aimed to evaluate the importance of targeting PLD on the tumor microenvironment. We provide evidence about the beneficial effect of PLD inhibitors [FIPI (dual PLD1/PLD2) or VU0155072-2 (PLD2 inhibitor)] on avoiding infiltration of tumor-helping macrophages and neutrophils. Tumor growth and metastasis within the primary tumors had low (<20% over controls) PLD enzyme activity. Unexpectedly, we found that the inhibitors also affected PLD2 gene expression and protein albeit at a lesser extent. The later could indicate that targeting both the actual PLD enzyme and its activity could be beneficial for potential cancer treatments in vivo. F4/80 and Ly6G staining of macrophages and neutrophils, respectively, and Arg1 staining data were consistent with M2 and N2 polarization. NOS2 staining increased in xenotransplants upon treatment with PLD2 inhibitors suggesting the novel observation that an increased recruitment of M1 macrophages occurred in primary tumors. PLD inhibitor-treated primary tumors had large, fragile, necrotic areas that were Arg1+ for M2 macrophages. The xenotransplants also caused the formation of large F4/80+ and Ly6G+ (>100 μm) clusters in lungs. However, PLD inhibitors, particularly FIPI, were able to diminish leukocyte presence. Ex vivo chemotaxis and PLD activity of peripheral blood neutrophils (PMN) and peritoneal macrophages was also determined. Whereas PMN had impaired functionality, macrophages did not. This significantly increased (“emboldened”) macrophage function was due to PLD inhibition. Since tumor-associated leukocytes in primary tumors and metastases were targeted via PLD inhibition, we posit that these inhibitors have a key role in cancer regression, while still affording an appropriate inflammatory response at least from off-site innate immunity macrophages.
Highlights
Macrophages and neutrophils have been implicated in many studies of human breast cancer with a growing emphasis currently being placed on the study of inflammatory breast cancer (IBC), whereby leukocytes isolated from the tumor microenvironment of such patients secrete cytokines involved in cell movement, which contributes to propagation and metastatic spreading of IBC cells [1,2]
We found that the fluoro-2-indolyl des-chlorohalopemide (FIPI) and VU0155072-2 phospholipase D (PLD) inhibitors inhibited leukocyte (TAM and tumorassociated neutrophils (TAN)) recruitment to the tumors, which suggests a shift in macrophage polarization occurred from M2 to that of the less invasive and anti-tumoral M1 macrophage in the PLD-specific inhibitor-treated samples
Our data shows a decreased involvement of F4/80, Arginine 1 (Arg1)- and Ly6G-mediated pathways in our orthotopic breast cancer xenograft model as a result of PLD-specific inhibitor treatment. Data from this current study indicates that primary breast tumors increased in both tumor volume and metastases as a function of time and that PLD-specific inhibitors decreased both of these factors, which strongly suggests phospholipase D2 (PLD2) has a role in breast tumor progression
Summary
Macrophages and neutrophils have been implicated in many studies of human breast cancer with a growing emphasis currently being placed on the study of inflammatory breast cancer (IBC), whereby leukocytes isolated from the tumor microenvironment of such patients secrete cytokines involved in cell movement, which contributes to propagation and metastatic spreading of IBC cells [1,2]. Both macrophages and neutrophils are associated with poor prognosis in breast cancer studies [3,4]. TAMs and TANs can be detected immunohistochemically using antibodies specific to many different macrophage- (F4/80 and arginase 1 (Arg1)) or neutrophil-specific (Ly6G) proteins [7,8,9,10]
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