gms | German Medical Science

Objective: The ENPP (ectonucleotide pyrophosphatase/phosphodiesterase) ectoenzymes 1, 2 and 3 are type II transmembrane proteins with ATPase and ATP pyrophosphatase activities. ENPP3 (CD203c) is an activation marker for mast cells and basophils. In addition, it is regularly expressed in both uterus and prostate. First publications indicate a role of ENPP3 in glial tumors. The expression has been shown in both rat glial precursor cells and in human glioma cell lines. The exposure to N-ethyl-N-nitrosurea (EtNU) causes an overexpression of ENPP3 in neuroectodermal tumors originated from glial precursor cells in rats. A recent study demonstrated high ENPP3 expression in clear renal cell carcinoma and tumor growth inhibition was noticed via antibody-drug conjugates. The goal of this study was to evaluate whether ENPP3 is expressed on low- and high-grade gliomas. In order to assess the further value of targeting ENPP3 only patient tissue and no cell-line was used for this study.

Methods: Quantitative real-time PCR was performed with Cycler Rotor Gene Q and the Rotor-Gene SYBR Green using two primers with different target locations. SDHA was used as a housekeeping gene. Data analysis was performed with Graphpad Prism 7. Statistical tests used: Kruskal-Wallis for ANOVA and Mann-Whitney U for two-taled t-tests. Expression levels are displayed in mean values and standard error of the mean (SEM). In addition, immunostaining was performed with a primary antibody from Biolegend (ABIN2661579) and with Diaminobenzidine.

Results: Fresh frozen samples of 50 patients (10 control brain tissue, 10 grade II astrocytomas, 10 anaplastic astrocytomas, 10 secondary- and 10 primary-glioblastomas (GBM)). Both primers detected a significantly higher level of ENPP3 in astrocytomas compared to control brain tissue (Primer A: 0.51 ± 0.09 vs. 0.15 ± 0.04, p=0.0045; Primer B: 1.13 ± 0.32 vs. 0.37 ± 0.14, p=0.0216), whereas primary GBM did not show elevation in comparison to control brain tissue (Primer A: 0.144 ± 0.016 vs. 0.15 ± 0.04; Primer B: 0.476 ± 0.104 vs. 0.37 ± 0.14). The expression levels of primary and secondary GBMs did not differ significantly. Immunostaining mainly showed positive staining in astrocytomas, while GBM exhibited little positive staining only.

Conclusion: To the best of our knowledge, this is the first study to prove ENPP3 expression on glioma patient-samples. Low-grade gliomas have significantly higher ENPP3 expression than control brain tissue. Unlike most cell surface molecules, ENPP3 shows stronger expression in low-grade glioma compared to glioblastoma. This finding could play a role for immunotargeting ENPP3 in gliomas.