Ferritin in glioblastoma

Abstract

Elevated levels of serum ferritin (SF) are observed in several types of cancer; however, little is known on the association between ferritin and glioma, the most frequent type of human primary brain tumour. Here we report that GBM patients show significantly increased pre-surgical SF levels (i.e. ferritinaemia) within the SF reference range and a marked ferritin immunoreactivity of resected tumour tissue. Our findings account for an indirect association between ferritin synthesis in glioma-tissue and altered SF levels, which limits the clinical value of SF as a tumour marker in glioma. Importantly, we show for the first time that GBM-derived glioma cells release ferritin in vitro, which exerts an apoptosis-stimulating activity. Albeit the pathophysiologic context of apoptosis induction by a tumour-derived ferritin remains to be defined, our findings account for a distinct growth-regulatory role of these ferritin species in tumour biology.

Fig. 1. Ferritin in serum and resected tumour tissue of GBM and meningioma patients.

a Based on SF quantification, patients were assigned to two cohorts: the RefR cohort (open boxes) showing SF levels inside the reference range (i.e. below the 95th percentile) of the TINA-Quant ferritin assay and the ExtR cohort (black boxes) with SF levels elevated above the 95th percentile. Dotted lines refer to the median reference levels reported for healthy male and female donors. Note that only a minority of GBM patients (ExtR cohort) showed very high SF levels (462–1355 ng/ml; median = 727 ng/mL), which is in line with existing data, with only 4 of a total of 57 glioma patients investigated so far, showing extremely high SF levels (Supplemental Table 1). Meningioma patients showed lower SF levels, which is significant (p > 0.05) for the RefR cohorts, and the highest SF value observed in meningioma patients (766 ng/mL) locates close to the median SF level of GBM–ExtR patients. b Immunohistochemistry of resected GBM and meningioma tissue evaluated by the labelling index (i.e. the percentage of immune-positive cells) demonstrates a significantly (p < 0.005) higher immunoreactivity for ferritin in GBM samples. In both types of tumour specimens, labelling for the FTL subunit was significantly (p < 0.05; p < 0.005) higher compared with FTH. c In GBM tissue, no differences of ferritin labelling were found between samples obtained from the RefR and ExtR cohort, the highest FTL labelling being observed in RefR cohort samples: 328 ng/mL (upper dot) and 206 ng/mL (lower dot). *p < 0.05; **p < 0.005 compared with the healthy reference population in (a) (Wilcoxon sign test for median difference based on gender-specific SF values) or as indicated (Mann–Whitney U exact test for independent samples for the comparison between the two patient cohorts in (a, c), and Wilcoxon signed-rank test for paired samples in b); ⁺p < 0.05; ⁺⁺p < 0.005 compared with meningioma patients (Mann–Whitney U exact test for independent samples). The dots in meningioma RefR cohort (a) and (b) highlight statistical outliers. N refers to the number of investigated patients (a) or tumour specimens (b).

Fig. 2. Cultured human glioma cells release an apoptosis-stimulating ferritin in vitro.

a Ferritin is detectable in culture supernatants (GBM-CM) collected from human glioma-derived cells after 24 h of serum-free culture (inset: dot blot of GBM-CM [1.5 µg of total protein] immunoreacted with monoclonal anti-ferritin Ab rH02). Upon 24 h of incubating primary rat hepatocytes under serum-free conditions in native GBM-CM [8.5 µg of total protein/mL], a significant (p < 0.05) shift of the percentage of apoptotic cells was observed (black bars), which was suppressed by anti-FTH antibody rH02 (hatched bar). b The ferritin was purified from GBM-CM according to a protocol described elsewhere (left inset: dot blot of purified ferritin [0.5 µg] immunoreacted with Ab rH02). Treatment of serum-free primary rat hepatocyte cultures for 48 h with 100 ng/ml of the purified GBM ferritin (GBM, closed bar) also had a significant (p < 0.05) apoptosis-stimulating effect that was suppressed by anti-FTH Ab rH02 (hatched bar). In contrast, a 48-h exposure of primary rat hepatocytes to 100 ng/mL of a ferritin purified from newborn mouse astrocyte culture supernatants had no effect (NBA). (b right inset: NBA culture supernatants [10 µg of total protein] immunoreacted with Ab rH02). Bars represent the mean ± SD of N ≥ 3 independent experiments. *P < 0.05 compared with the control or as indicated; Student’s double-sided t-test for independent samples.