In glioblastoma, a fraction of malignant cells consists of therapy-resistant glioblastoma stem cells (GSCs) residing in protective niches that recapitulate hematopoietic stem cell (HSC) niches in bone marrow. We have previously shown that HSC niche proteins stromal cell-derived factor-1α (SDF-1α), C-X-C chemokine receptor type 4 (CXCR4), osteopontin (OPN), and cathepsin K (CatK) are expressed in hypoxic GSC niches around arterioles in five human glioblastoma samples. In HSC niches, HSCs are retained by binding of SDF-1α and OPN to their receptors CXCR4 and CD44, respectively. Protease CatK cleaves SDF-1α to release HSCs out of niches. The aim of the present study was to reproduce the immunohistochemical localization of these GSC markers in 16 human glioblastoma samples with the addition of three novel markers. Furthermore, we assessed the type of blood vessels associated with GSC niches. In total, we found seven GSC niches containing CD133-positive and nestin-positive GSCs as a single-cell layer exclusively around the tunica adventitia of 2% of the CD31-positive and SMA-positive arterioles and not around capillaries and venules. Niches expressed SDF-1α, CXCR4, CatK, OPN, CD44, hypoxia-inducible factor-1α, and vascular endothelial growth factor. In conclusion, we show that GSC niches are present around arterioles and express bone marrow HSC niche proteins.

Isocitrate dehydrogenase (IDH1)-1 is mutated in various types of human cancer, and the presence of this mutation is associated with improved responses to irradiation and chemotherapy in solid tumor cells. Mutated IDH1 (IDH1MUT) enzymes consume NADPH to produce d-2-hydroxyglutarate (d-2HG) resulting in the decreased reducing power needed for detoxification of reactive oxygen species (ROS), for example. The objective of the current study was to investigate the mechanism behind the chemosensitivity of the widely used anticancer agent cisplatin in IDH1MUT cancer cells. Oxidative stress, DNA damage, and mitochondrial dysfunction caused by cisplatin treatment were monitored in IDH1MUT HCT116 colorectal cancer cells and U251 glioma cells. We found that exposure to cisplatin induced higher levels of ROS, DNA double-strand breaks (DSBs), and cell death in IDH1MUT cancer cells, as compared with IDH1 wild-type (IDH1WT) cells. Mechanistic investigations revealed that cisplatin treatment dose dependently reduced oxidative respiration in IDH1MUT cells, which was accompanied by disturbed mitochondrial proteostasis, indicative of impaired mitochondrial activity. These effects were abolished by the IDH1MUT inhibitor AGI-5198 and were restored by treatment with d-2HG. Thus, our study shows that altered oxidative stress responses and a vulnerable oxidative metabolism underlie the sensitivity of IDH1MUT cancer cells to cisplatin.—Khurshed, M., Aarnoudse, N., Hulsbos, R., Hira, V. V. V., van Laarhoven, H. W. M., Wilmink, J. W., Molenaar, R. J., van Noorden, C. J. F. IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity.

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IntroductionIsocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer and the presence of a mutation is associated with improved responses to irradiation and chemotherapy in solid tumor cells. Mutated IDH1 (IDH1MUT) enzymes consume NADPH to produce D‐2‐hydroxyglutarate (D‐2HG) resulting in a decreased reducing power needed for detoxification of e.g. reactive oxygen species (ROS). The objective of the current study was to investigate the mechanism behind the chemosensitivity of the widely‐used anti‐cancer agent cisplatin in IDH1MUT cancer cells.MethodsOxidative stress, DNA damage and mitochondrial dysfunction caused by cisplatin treatment were monitored in IDH1MUT HCT116 cells and U251 glioma cells.ResultsBy performing histo‐ and cytochemical experiments, we found that exposure to cisplatin induced higher levels of ROS, DNA double‐strand breaks (DSBs), and cell death in IDH1MUT cancer cells as compared to IDH1 wild‐type (IDH1WT) cells. Mechanistic investigations as in situ histochemical enzyme activity assays revealed that cisplatin treatment dose‐dependently reduced oxidative respiration in lDH1MUT cells, which was accompanied by disturbed mitochondrial proteostasis, indicative for impaired mitochondrial activity. These effects were abolished by the IDH1MUT inhibitor AGI‐5198 and were recapitulated by treatment with D‐2HG.ConclusionsOur study shows that altered oxidative stress responses and a vulnerable oxidative metabolism underlie the sensitivity of IDH1MUT cancer cells to cisplatin. Our study may have clinical implications and imply that administration of IDH1MUT inhibitors to patients with IDH1MUT cancer abolishes the therapeutic effect of cisplatin. Our in vitro results suggest that concomitant administration of IDH1MUT inhibitors and cisplatin may result in an unfavorable clinical outcome.Support or Funding InformationThis work was supported by Dutch Cancer Society Grants KWF‐UVA 2014‐6839 (to M.K., V.V.V.H., R.J.M., and C.J.F.V.N.) and AMC2016.1‐10460 (to M.K., R.J.M., J.W.W., and C.J.F.V.N.). A) Proliferation assay using HCT116 cells after cisplatin exposure and in the presence or absence of ROS‐scavenging NAC.B) Colony‐forming assay after cisplatin exposure with HCT116 cells in the presence or absence of AGI‐5198 or of D‐2HG.C) Representative photomicrographs and plots of cells in the presence or absence of AGI‐5198, and cisplatin treatment. Y‐H2AX was stained immunocytochemically (red) to demonstrate DNA DSBs and with DAPI (blue) to demonstrate DNA nucleus content.imageA) Proliferation assay using HCT116 cells after cisplatin exposure and in the presence or absence of ROS‐scavenging NAC.B) Colony‐forming assay after cisplatin exposure with HCT116 cells in the presence or absence of AGI‐5198 or of D‐2HG.C) Representative photomicrographs and plots of cells in the presence or absence of AGI‐5198, and cisplatin treatment. Y‐H2AX was stained immunocytochemically (red) to demonstrate DNA DSBs and with DAPI (blue) to demonstrate DNA nucleus content. D) The basal oxygen consumption rate of HCT116 cells shown in the presence or absence of cisplatin. E) Western blot and mitonuclear protein imbalance of MTCO1 and nuclear DNA–encoded SDHA expression in HCT116 cells after exposure to cisplatin or carboplatin. F) Representative monochromatic light photomicrographs and quantification of histochemical enzyme activity of HCT116 cells treated with cisplatin or left untreated after staining for SDH activity in the presence or absence of sodium azide.imageD) The basal oxygen consumption rate of HCT116 cells shown in the presence or absence of cisplatin. E) Western blot and mitonuclear protein imbalance of MTCO1 and nuclear DNA–encoded SDHA expression in HCT116 cells after exposure to cisplatin or carboplatin. F) Representative monochromatic light photomicrographs and quantification of histochemical enzyme activity of HCT116 cells treated with cisplatin or left untreated after staining for SDH activity in the presence or absence of sodium azide.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.