title: CD73 on B16F10 melanoma cells in CD73-deficient mice promotes tumor growth, angiogenesis, neovascularization, macrophage infiltration and metastasis.
abstract: Ecto-5'-nucleotidase (CD73), an enzyme providing interstitial adenosine, mediates diverse physiological and pathological responses. In tumor progression, it has primarily an immunosuppressive role but is also thought to regulate neovascularization. However, the latter role is still in debate. When B16F10 melanoma was subcutaneously injected into CD73 knockout mice, changes in the tumor vasculature were not always observed. However, we demonstrated earlier that the growth and vascularization of B16F10 melanoma in CD73 knockout mice depend on the low presence of CD73 on tumor cells. To further analyze the role of CD73 on tumor growth and vascularization, we compared the changes in B16F10 melanoma subcutaneously injected into right flank of wild-type mice, CD73 knockout mice lacking host CD73 only, and CD73 knockout mice with tumor cell CD73 either inhibited with AOPCP (adenosine α,β-methylene 5'-diphosphate) or permanently knocked down through genetic modification. We report here that both inhibition and knockdown of tumor CD73 further inhibited tumor growth compared to host CD73 knockout alone. MAP-kinase signaling pathway activation also decreased more strongly in the stable knockdown. There was a significant reduction in the angiogenic activation of blood microvessels as observed by decreased anti-VEGFR2 staining. Stable CD73 knockdown also reduced endothelial cell proliferation as measured by anti-CD105 staining. However, only chemical inhibition with AOPCP significantly augmented the reduction in intratumoral microvessel density induced by host CD73 knockout. Such reduction was not observed when tumor CD73 was knocked down due to the much slower tumor growth and decreased oxygen demand as indicated by the low expression of Bad, a hypoxia marker. Decreased CD73 activity also led to the decreased expression of angiogenic factors, including VEGF and bFGF that was only partially reversed by hypoxia in tumors treated with AOPCP. Both inhibition and knockdown of tumor CD73 significantly decreased tumor macrophage infiltration and induced microenvironment changes, thereby influencing MI or MII macrophage polarization. Additionally, tumor cell CD73 is important in metastasis formation through adenosine-independent attachment to endothelium. We conclude that even low tumor cell CD73 expression has an undeniable role in melanoma progression, including the regulation of many aspects of angiogenesis. CD73 is thus a viable target in anti-angiogenic melanoma therapy.
authors.affiliation: Laboratory of Cell Biology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: pkosz@gumed.edu.pl.
authors.forename: Patrycja
authors.initials: P
authors.lastname: Koszałka
authors.affiliation: Laboratory of Cell Biology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: golunska.m@gmail.com.
authors.forename: Monika
authors.initials: M
authors.lastname: Gołuńska
authors.affiliation: Department of Histology, Faculty of Medicine, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: m.stanislawowski@gumed.edu.pl.
authors.forename: Marcin
authors.initials: M
authors.lastname: Stanisławowski
authors.affiliation: Laboratory of Cell Biology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: ola.urban90@gmail.com.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Urban
authors.affiliation: Laboratory of Cell Biology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: gstasilojc@gumed.edu.pl.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Stasiłojć
authors.affiliation: Laboratory of Cell Biology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: marcelmajewski@o2.pl.
authors.forename: Marceli
authors.initials: M
authors.lastname: Majewski
authors.affiliation: Department of Histology, Faculty of Medicine, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: pwierzb@gumed.edu.pl.
authors.forename: Piotr
authors.initials: P
authors.lastname: Wierzbicki
authors.affiliation: Laboratory of Molecular Enzymology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: acskla@gumed.edu.pl.
authors.forename: Andrzej C
authors.initials: AC
authors.lastname: Składanowski
authors.affiliation: Laboratory of Cell Biology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Electronic address: jjbgid@gumed.edu.pl.
authors.forename: Jacek
authors.initials: J
authors.lastname: Bigda
mesh_terms: D015720:5'-Nucleotidase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D008264:Macrophages / Q000502:physiology*; D008297:Male; D008546:Melanoma, Experimental / Q000098:blood supply / Q000378:metabolism* / Q000556:secondary; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic; D012878:Skin Neoplasms / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D047368:Tumor Burden
chemical_list: D015720:5'-Nucleotidase
keywords: B16F10 melanoma; Ecto-5′-nucleotidase; Metastasis; Tumor macrophage infiltration; Tumor neovascularization and angiogenesis