BAFF, APRIL, TWEAK, BCMA, TACI and Fn14 proteins are related to human glioma tumor grade: immunohistochemistry and public microarray data meta-analysis

Abstract

Gliomas are common and lethal tumors of the central nervous system (CNS). Genetic alterations, inflammatory and angiogenic processes have been identified throughout tumor progression; however, treatment still remains palliative for most cases. Biological research on parameters influencing cell survival, invasion and tumor heterogeneity identified several cytokines interfering in CNS inflammation, oxidative stress and malignant transformation, including TNF-superfamily (TNFSF) members. In this report we performed a meta-analysis of public gene-array data on the expression of a group of TNFSF ligands (BAFF, APRIL, TWEAK) and their receptors (BAFF-R, TACI, BCMA, Fn14) in gliomas. In addition, we investigated by immunohistochemistry (IHC) the tumor cells' expression of these ligands and receptors in a series of 56 gliomas of different grade. We show that in IHC, BAFF and APRIL as well as their cognate receptors (BCMA, TACI) and Fn14 expression correlate with tumor grade. This result was not evidenced in micro-arrays meta-analysis. Finally, we detected for the first time Fn14, BAFF, BCMA and TACI in glioma-related vascular endothelium. Our data, combined with our previous report in glioma cell lines, suggest a role for these receptors and ligands in glioma biology and advance these molecules as potential markers for the classification of these tumors to the proliferative, angiogenic or stem-like molecular subtype.

Figure 1. Representative immunohistochemical images of APRIL, BAFF, TWEAK and their cognate receptor expression in human gliomas.

All pictures are taken under 20× magnification. APRIL immunoreactivity (A,B) increases from low grade to higher grade lesions. Note the intense staining in larger cells, while smaller ones are negative (A). BAFF immunoreactivity declines from lower grade (C, diffuse and intense), to higher grade gliomas (D). TACI displayed higher immunoreactivity in higher grade gliomas (F) than in low grade ones (E). Similar findings were found for BCMA as well (low grade G, high grade F). Low and high grade tumors displayed heterogeneous regarding percentage of positive cells and/or intensity of staining for TWEAK and Fn14. Here representative photos from Grade III gliomas for TWEAK (I, J) and Fn14 (K, L), respectively are shown.

Figure 2. Representative images of BAFF, TWEAK and their cognate receptor BCMA, TACI and Fn14 expression in vascular endothelial cells of human glioma specimens.

Upper row: vascular endothelium immunoreactivity for CD31 (A), Fn14 (B), and TWEAK (C). Lower row: vascular endothelium immunoreactivity for BAFF (D) and TACI (E). In panel F, vascular endothelial immunoreactivity for BCMA (direct immunofluorescence assay, with anti-BCMA-FITC antibody. Similar results were obtained by indirect immunostaining with non-fluorescent anti-BCMA antibody (not shown). Arrows show tumor vessels. APRIL was not detected in endothelial cells (not shown). All pictures are taken under 20× magnification. Bar = 200 µm. Inserts in each panel show a higher magnification of a representative part of the vessel.

Figure 3. Representative images of BAFF, APRIL, TWEAK and their cognate receptors as well as inflammatory markers CD3, CD20, CD68 expression in human glioma specimens.

Parallel immunostaining of BAFF, APRIL, TWEAK, BCMA, TACI, Fn14 and inflammatory markers CD3, CD20 and CD68 showing that the main source of these TNFSF members are reactive and tumoral glial cells per se in areas with minimal inflammation. Red arrows (black in 3B) indicate gemistocytes immunopositive for BAFF (A), APRIL (B), TWEAK (C), TACI (D), BCMA (E) and Fn14 (F). Gemistocytes negative for lymphocytic CD3 (G), CD20 (H) and panmacrophage CD68 (I) immunostaining. CD68 immunopositive macrophages/microglia and monocytes in I. All pictures are taken under 40× magnification.

Figure 4. Quantification of BAFF, APRIL, TWEAK and their receptors in gliomas, stratified by grade.

Data are expressed as mean±SEM and medians of the corresponding H-score of tumors, presented in Table S4 in A and bar graph (mean±SEM) in B.