title: Methotrexate up-regulates ecto-5'-nucleotidase/CD73 and reduces the frequency of T lymphocytes in the glioblastoma microenvironment.
abstract: Glioblastoma multiforme (GBM) is a deadly cancer characterized by a pro-tumoral immune response. T-regulatory (Treg) lymphocytes suppress effector immune cells through cytokine secretion and the adenosinergic system. Ecto-5'-nucleotidase/CD73 plays a crucial role in Treg-mediated immunosuppression in the GBM microenvironment (GME). Methotrexate (MTX) is an immunosuppressive drug that can increase the extracellular concentration of adenosine. In this manuscript, C6 GBM cells were treated with 1.0 μM MTX, and ecto-5'-nucleotidase/CD73 expression and extracellular AMP metabolism were analyzed in vitro. For in vivo studies, rats with implanted GBM were treated for 10 days with MTX-loaded lipid-core nanocapsules (MTX-LNCs, 1 mg/kg/day). The activity of ectonucleotidase and the expression of NTPDase1/CD39 and ecto-5'-nucleotidase/CD73 were measured. The frequencies of T lymphocytes (CD3(+)CD4(+), CD3(+)CD8(+), and CD4(+)CD25(high)CD39(+)) were quantified. In vitro, treatment with MTX increased CD73 expression and activity in C6 cells, which is in agreement with higher levels of extracellular adenosine. In vivo, MTX-LNC treatment increased CD39 expression on CD3(+)CD8(+) lymphocytes. In addition, MTX-LNC treatment up-regulated CD73 expression in tissue isolated from GBM, a finding that is in agreement with the higher activity of this enzyme. More specifically, the treatment increased CD73 expression on CD3(+)CD4(+) and CD3(+)CD8(+) lymphocytes. Treatment with MTX-LNCs decreased the frequencies of T-cytotoxic, T-helper, and Treg lymphocytes in the GME. Although more studies are necessary to better understand the complex cross-talk mediated by supra-physiological concentrations of adenosine in the GME, these studies demonstrate that MTX treatment increases CD73 enzyme expression and AMP hydrolysis, leading to an increase in adenosine production and immunosuppressive capability.
authors.affiliation: Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Fabrício
authors.initials: F
authors.lastname: Figueiró
authors.affiliation: Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Catiúscia P
authors.initials: CP
authors.lastname: de Oliveira
authors.affiliation: Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Letícia S
authors.initials: LS
authors.lastname: Bergamin
authors.affiliation: Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Liliana
authors.initials: L
authors.lastname: Rockenbach
authors.affiliation: Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Franciane B
authors.initials: FB
authors.lastname: Mendes
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Av. Ramiro Barcelos, 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil.
authors.forename: Elisa Helena F
authors.initials: EH
authors.lastname: Jandrey
authors.affiliation: Programa de Pós-Graduação em Medicina: Ciências Médicas, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Cesar Eduardo J
authors.initials: CE
authors.lastname: Moritz
authors.affiliation: Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Letícia F
authors.initials: LF
authors.lastname: Pettenuzzo
authors.affiliation: Centre de Recherche du CHU de Québec, Université Laval, Québec, QC, Canada.|Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
authors.forename: Jean
authors.initials: J
authors.lastname: Sévigny
authors.affiliation: Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Silvia S
authors.initials: SS
authors.lastname: Guterres
authors.affiliation: Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.|Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Adriana R
authors.initials: AR
authors.lastname: Pohlmann
authors.affiliation: Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. abattastini@gmail.com.|Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Av. Ramiro Barcelos, 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil. abattastini@gmail.com.
authors.forename: Ana Maria O
authors.initials: AM
authors.lastname: Battastini
mesh_terms: D015720:5'-Nucleotidase / Q000096:biosynthesis*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D001932:Brain Neoplasms / Q000201:enzymology / Q000276:immunology*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004195:Disease Models, Animal; D005909:Glioblastoma / Q000201:enzymology / Q000276:immunology*; D007150:Immunohistochemistry; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008727:Methotrexate / Q000494:pharmacology*; D051381:Rats; D013601:T-Lymphocytes / Q000187:drug effects*; D019139:Tumor Escape / Q000276:immunology; D059016:Tumor Microenvironment / Q000187:drug effects / Q000276:immunology; D015854:Up-Regulation
chemical_list: D007166:Immunosuppressive Agents; D000249:Adenosine Monophosphate; D015720:5'-Nucleotidase; D008727:Methotrexate
keywords: Adenosine; Ecto-5′-nucleotidase/CD73; Glioblastoma; Methotrexate; NTPDase1/CD39; Regulatory T lymphocytes