title: PLD-Specific Small-Molecule Inhibitors Decrease Tumor-Associated Macrophages and Neutrophils Infiltration in Breast Tumors and Lung and Liver Metastases.
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.
authors.affiliation: Wright State University Boonshoft School of Medicine, Department of Biochemistry and Molecular Biology, Dayton, Ohio 45435, United States of America.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Henkels
authors.affiliation: Wright State University Boonshoft School of Medicine, Department of Biochemistry and Molecular Biology, Dayton, Ohio 45435, United States of America.
authors.forename: Naveen Reddy
authors.initials: NR
authors.lastname: Muppani
authors.affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women Hospital and Harvard Medical School, Boston, Massachusetts 02115, United States of America.|Wright State University Boonshoft School of Medicine, Department of Biochemistry and Molecular Biology, Dayton, Ohio 45435, United States of America.
authors.forename: Julian
authors.identifier: http://orcid.org/0000-0002-7581-9227
authors.initials: J
authors.lastname: Gomez-Cambronero
mesh_terms: D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism; D001119:Arginase / Q000378:metabolism; D001943:Breast Neoplasms / Q000473:pathology*; D063646:Carcinogenesis / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D016764:Cell Polarity / Q000187:drug effects; D060833:Cellular Microenvironment / Q000187:drug effects; D002633:Chemotaxis / Q000187:drug effects; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008113:Liver Neoplasms / Q000473:pathology / Q000556:secondary*; D008168:Lung / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008175:Lung Neoplasms / Q000473:pathology / Q000556:secondary*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D015674:Mammary Neoplasms, Animal / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D000950:Antigens, Ly; D016207:Cytokines; D004791:Enzyme Inhibitors; C480309:Ly6G antigen, mouse; D054852:Small Molecule Libraries; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D010739:Phospholipase D; C544448:Arg1 protein, mouse; D001119:Arginase