Analysis of anemia and iron supplementation among glioblastoma patients reveals sex-biased association between anemia and survival

The association between anemia and outcomes in glioblastoma patients is unclear. We analyzed data from 1346 histologically confirmed adult glioblastoma patients in the TriNetX Research Network. Median hemoglobin and hematocrit levels were quantified for 6 months following diagnosis and used to classify patients as anemic or non-anemic. Associations of anemia and iron supplementation of anemic patients with median overall survival (median-OS) were then studied. Among 1346 glioblastoma patients, 35.9% of male and 40.5% of female patients were classified as anemic using hemoglobin-based WHO guidelines. Among males, anemia was associated with reduced median-OS compared to matched non-anemic males using hemoglobin (HR 1.24; 95% CI 1.00-1.53) or hematocrit-based cutoffs (HR 1.28; 95% CI 1.03-1.59). Among females, anemia was not associated with median-OS using hemoglobin (HR 1.00; 95% CI 0.78-1.27) or hematocrit-based cutoffs (HR: 1.10; 95% CI 0.85-1.41). Iron supplementation of anemic females trended toward increased median-OS (HR 0.61; 95% CI 0.32-1.19) although failing to reach statistical significance whereas no significant association was found in anemic males (HR 0.85; 95% CI 0.41-1.75). Functional transferrin-binding assays confirmed sexually dimorphic binding in resected patient samples indicating underlying differences in iron biology. Anemia among glioblastoma patients exhibits a sex-specific association with survival

Liposomal delivery of ferritin heavy chain 1 (FTH1) siRNA in patient xenograft derived glioblastoma initiating cells suggests different sensitivities to radiation and distinct survival mechanisms.

Elevated expression of the iron regulatory protein, ferritin heavy chain 1 (FTH1), is increasingly being associated with high tumor grade and poor survival outcomes in glioblastoma. Glioma initiating cells (GICs), a small population of stem-like cells implicated in therapeutic resistance and glioblastoma recurrence, have recently been shown to exhibit increased FTH1 expression. We previously demonstrated that FTH1 knockdown enhanced therapeutic sensitivity in an astrocytoma cell line. Therefore, in this study we developed a liposomal formulation to enable the in vitro delivery of FTH1 siRNA in patient xenograft derived GICs from glioblastomas with pro-neural and mesenchymal transcriptional signatures to interrogate the effect of FTH1 downregulation on their radiation sensitivity. Transfection with siRNA decreased FTH1 expression significantly in both GICs. However, there were inherent differences in transfectability between pro-neural and mesenchymal tumor derived GICs, leading us to modify siRNA: liposome ratios for comparable transfection. Moreover, loss of FTH1 expression resulted in increased extracellular lactate dehydrogenase activity, executioner caspase 3/7 induction, substantial mitochondrial damage, diminished mitochondrial mass and reduced cell viability. However, only GICs from pro-neural glioblastoma showed marked increase in radiosensitivity upon FTH1 downregulation demonstrated by decreased cell viability, impaired DNA repair and reduced colony formation subsequent to radiation. In addition, the stemness marker Nestin was downregulated upon FTH1 silencing only in GICs of pro-neural but not mesenchymal origin. Using liposomes as a siRNA delivery system, we established FTH1 as a critical factor for survival in both GIC subtypes as well as a regulator of radioresistance and stemness in pro-neural tumor derived GICs. Our study provides further evidence to support the role of FTH1 as a promising target in glioblastoma

IL-13Rα2 is a Glioma-Restricted Receptor for Interleukin-13

We have found that binding sites for interleukin-13. ( IL13) are overexpressed in a vast majority of high-grade astrocytomas. (HGAs). These binding sites for IL-13 are distinct from the physiological receptor in that it does not bind IL-4. We also demonstrated that IL-13 receptor alpha 2 protein chain. (IL-13Rα2), an IL-4-independent receptor for IL-13, is abundant among HGAs, but not in normal organs. To examine if IL-13Rα2 is the tumorassociated site for IL-13, we stably transfected normal Chinese hamster ovary. (CHO) cells and glioma G-26 cells to express either human. (h) or murine. (m) IL13Rα2. CHO-hlL-13Rα2(+) cells and G-26-hlmlL13Rα2(+) cells, not CHO and G-26 parental or mock -transfected cells, specifically bound IL-13 in an IL-4-independent manner. The IL-13Rα2(+) cells also became highly susceptible to the killing by an IL-13-based cytotoxic fusion protein. In loss of function studies, a HGA cell line, SNB-19, was transfected with antisense. (as) hIL-13Rα2, as-SNB-19-hIL-13Rα2(+) cells lost their natural affinity towards IL-13 and became resistant to IL-13-based cytotoxins. The fact, that IL13Rα2-positive cells bind IL-13 independent of IL-4, become susceptible to IL-13 cytotoxins, cells deprived of IL-13Rα2 receptor lose these features, demonstrates that IL-13Rα2 is the brain tumor-associated receptor for IL-13

Tim-2 is the receptor for H-ferritin on oligodendrocytes.

Oligodendrocytes stain more strongly for iron than any other cell in the CNS, and they require iron for the production of myelin. For most cell types transferrin is the major iron delivery protein, yet neither transferrin receptor protein nor mRNA are detectable in mature oligodendrocytes. Thus an alternative iron delivery mechanism must exist. Given the significant long term consequences of developmental iron deficiency and the iron requirements for normal myelination, identification of the iron delivery mechanism for oligodendrocytes is important. Previously we have reported that oligodendrocytes bind H-ferritin and that H-ferritin binds to white matter tracts in vivo. Recently, T cell immunoglobulin and mucin domain-containing protein-2 (Tim-2) was shown to bind and internalize H-ferritin. In the present study we show that Tim-2 is expressed on oligodendrocytes both in vivo and in vitro. Further, the onset of saturable H-ferritin binding in CG4 oligodendrocyte cell line is accompanied by Tim-2 expression. Application of a blocking antibody to the extracellular domain of Tim-2 significantly reduces H-ferritin binding to the differentiated CG4 cells and primary oligodendrocytes. Tim-2 expression on CG4 cells is responsive to iron; decreasing with iron loading and increasing with iron chelation. Taken together, these data provide compelling evidence that Tim-2 is the H-ferritin receptor on oligodendrocytes suggesting it is the primary mechanism for iron acquisition by these cells

Characterization of a novel anti-cancer compound for astrocytomas.

The standard chemotherapy for brain tumors is temozolomide (TMZ), however, as many as 50% of brain tumors are reportedly TMZ resistant leaving patients without a chemotherapeutic option. We performed serial screening of TMZ resistant astrocytoma cell lines, and identified compounds that are cytotoxic to these cells. The most cytotoxic compound was an analog of thiobarbituric acid that we refer to as CC-I. There is a dose-dependent cytotoxic effect of CC-I in TMZ resistant astrocytoma cells. Cell death appears to occur via apoptosis. Following CC-I exposure, there was an increase in astrocytoma cells in the S and G2/M phases. In in vivo athymic (nu/nu) nude mice subcutaneous and intracranial tumor models, CC-I completely inhibited tumor growth without liver or kidney toxicity. Molecular modeling and enzyme activity assays indicate that CC-I selectively inhibits topoisomerase IIα similar to other drugs in its class, but its cytotoxic effects on astrocytoma cells are stronger than these compounds. The cytotoxic effect of CC-I is stronger in cells expressing unmethylated O6-methylguanine methyltransferase (MGMT) but is still toxic to cells with methylated MGMT. CC-I can also enhance the toxic effect of TMZ on astrocytoma when the two compounds are combined. In conclusion, we have identified a compound that is effective against astrocytomas including TMZ resistant astrocytomas in both cell culture and in vivo brain tumor models. The enhanced cytotoxicity of CC-I and the safety profile of this family of drugs could provide an interesting tool for broader evaluation against brain tumors

Cytotoxicity of CC-I, merbarone, and combination of CC-I and TMZ on the astrocytoma cells.

<p>(A) TMZ-resistant human CCF-STTG1 and T98G cell lines were cultured for 3 days with CC-I and other similar structure topoisomerase II inhibitor (merbarone) followed by cytotoxicity measurement by SRB assay. CC-I showed greater toxicity than merbarone on the astrocytomas. The symbols indicate a significant difference between the merbarone treated and CC-I treated groups (**p<0.01; ***p<0.001). (B) The MGMT methylated (T98G, CCF-STTG1) or un-methylated (LN-18) astrocytoma cell lines were cultured for 3 days with CC-I and determined cytotoxicity by SRB assay. T98G cells have methylated MGMT promoter, but show weak MGMT expression. CC-I is more cytotoxic to LN-18 cells which has un-methylated MGMT promoter and MGMT expression. The symbol (***) indicates the most difference between the cells (p<0.001).</p

The anti-tumor effect of CC-I in a subcutaneous mouse tumor model.

<p>(A) Mice were implanted with ten million cells with the SW1088 or CCF-STTG1 cells. The starting tumor size for the CCF-STTG1 cells ranged from 80–100 mm³. The SW1088 cells grew more slowly so CC-I treatment was started when the tumors reached 30 mm³. CC-I was injected intraperitoneally at a concentration of 25 mg/kg body weight once a week for 7 weeks (n = 7∼10). The control group was given PBS in the same volume and regimen (n = 3–8). The tumor slowly reoccurred in the TMZ-sensitive SW1088 astrocytoma injected nude mice but did not reoccur in the TMZ resistant CCF-STTG1 injected nude mice when CC-I was discontinued (beyond 7 weeks). CC-I inhibited the tumor growth and was not lethal in any of the treatment groups. Some error bars are too small to be visible. (B) Mean body weight of mice is presented in grams. Some error bars are too small to be visible.</p

CC-I-induced cell cycle arrest in CCF-STTG1 cells.

<p>The CCF-STTG1 cells were treated with 18 or 36 µM of CC-I for 24 or 48 hours. The cells were stained with propidium iodide and then analyzed for cell cycle distribution using a FACScan analyzer. CC-I treatment significantly increased the S and G2/M cell population, but decreased in G0/G1 phase. The symbols indicate a significant difference compared to the control. (*p<0.05; **p<0.01; ***p<0.001).</p