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 MTXloaded 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 supraphysiological 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.

Changes in CD73, CD39 and CD26 expression on T-lymphocytes of ANCA-associated vasculitis patients suggest impairment in adenosine generation and turn-over

“…In these patients, depletion of CD4 + CD73 + T-cells coincides with increased activation markers on CD4 + and CD8 + cells 39 suggesting a role in persistent immune activation and inflammation. Nonetheless, we cannot exclude that CD39 and CD73 expression are influenced by immunosuppressive medication as has been demonstrated for methotrexate in the treatment of rheumatoid arthritis patients 40 , 41 or azathioprine in inflammatory bowel disease patients 42 , 43 . Also rituximab and cyclophosphamide have been shown to modify peripheral T-cell subset frequencies 44 , 45 , but their effect on the expression of ecto-nucleotidases still remains elusive.…”

The influence of chemotherapy on adenosine-producing B cells in patients with head and neck squamous cell carcinoma

“…In addition, great differences may exist in the micro-environment of different kinds of cancer [ 22 ]. Interestingly, MTX may even have a promoting effect on adenosine metabolism in cancer cells as shown in glioblastoma cell lines due to the upregulation of the ectonucleotidase CD73 [ 51 ].…”

Multifaceted Effects of Extracellular Adenosine Triphosphate and Adenosine in the Tumor–Host Interaction and Therapeutic Perspectives

“…Elevated CD73 expression has been reported in several types of human cancers such as glioma ( 229 – 231 ), head and neck ( 128 ), melanoma ( 232 ), thyroid ( 233 ), breast ( 234 – 238 ), pancreas ( 239 ), colon ( 219 , 240 ), bladder ( 241 , 242 ), ovarian ( 243 ), prostate ( 244 ), and leukemia ( 126 ), being positively correlated with poor prognosis. In addition to tumor-derived CD73, host CD73 also negatively regulates tumor immunity ( 245 ).…”