Impact of Hypoxia on Carbon Ion Therapy in Glioblastoma Cells: Modulation by LET and Hypoxia-Dependent Genes.

Tumor hypoxia is known to limit the efficacy of ionizing radiations, a concept called oxygen enhancement ratio (OER). OER depends on physical factors such as pO2 and linear energy transfer (LET). Biological pathways, such as the hypoxia-inducible transcription factors (HIF), might also modulate the influence of LET on OER. Glioblastoma (GB) is resistant to low-LET radiation (X-rays), due in part to the hypoxic environment in this brain tumor. Here, we aim to evaluate in vitro whether high-LET particles, especially carbon ion radiotherapy (CIRT), can overcome the contribution of hypoxia to radioresistance, and whether HIF-dependent genes, such as erythropoietin (EPO), influence GB sensitivity to CIRT. Hypoxia-induced radioresistance was studied in two human GB cells (U251, GL15) exposed to X-rays or to carbon ion beams with various LET (28, 50, 100 keV/µm), and in genetically-modified GB cells with downregulated EPO signaling. Cell survival, radiobiological parameters, cell cycle, and ERK activation were assessed under those conditions. The results demonstrate that, although CIRT is more efficient than X-rays in GB cells, hypoxia can limit CIRT efficacy in a cell-type manner that may involve differences in ERK activation. Using high-LET carbon beams, or targeting hypoxia-dependent genes such as EPO might reduce the effects of hypoxia.

M2 macrophages are more resistant than M1 macrophages following radiation therapy in the context of glioblastoma.

In some highly inflammatory tumors, such as glioblastoma (GB), macrophages (MΦ) represent the most abundant population of reactive cells. MΦ, initially denoted as M0 MΦ, can be polarized into two further phenotypes: the antitumor M1 MΦ, and the protumor M2 MΦ. The three phenotypes can reside simultaneously in the tumor mass and various external factors may influence MΦ polarization. Radiotherapy is a common modality of cancer treatment aiming to target tumor cells. However, the specific effects of X-ray radiation on the inflammatory cells are, so far, controversial and not fully understood. In the present investigation, we have first analyzed, in vivo, the effect of X-ray radiation on MΦ present in GB tumors. We have observed a decrease in MΦ number paralleled by an increase in the proportion of M2 MΦ. To understand this phenomenon, we then evaluated, in vitro, the effects of X-rays on the MΦ phenotypes and survival. We have found that X-ray radiation failed to modify the phenotype of the different MΦ. However, M1 MΦ were more sensitive to ionizing radiation than M2 MΦ, both in normoxia and in hypoxia, which could explain the in vivo observations. To conclude, M2 MΦ are more radioresistant than M0 and M1 MΦ and the present study allows us to propose that X-ray radiotherapy could contribute, along with other phenomena, to the increased density in the protumor M2 MΦ in GB.

Carbogen-induced increases in tumor oxygenation depend on the vascular status of the tumor: A multiparametric MRI study in two rat glioblastoma models.

The alleviation of hypoxia in glioblastoma with carbogen to improve treatment has met with limited success. Our hypothesis is that the eventual benefits of carbogen depend on the capacity for vasodilation. We examined, with MRI, changes in fractional cerebral blood volume, blood oxygen saturation, and blood oxygenation level dependent signals in response to carbogen. The analyses were performed in two xenograft models of glioma (U87 and U251) recognized to have different vascular patterns. Carbogen increased fractional cerebral blood volume, blood oxygen saturation, and blood oxygenation level dependent signals in contralateral tissues. In the tumor core and peritumoral regions, changes were dependent on the capacity to vasodilate rather than on resting fractional cerebral blood volume. In the highly vascularised U87 tumor, carbogen induced a greater increase in fractional cerebral blood volume and blood oxygen saturation in comparison to the less vascularized U251 tumor. The blood oxygenation level dependent signal revealed a delayed response in U251 tumors relative to the contralateral tissue. Additionally, we highlight the considerable heterogeneity of fractional cerebral blood volume, blood oxygen saturation, and blood oxygenation level dependent within U251 tumor in which multiple compartments co-exist (tumor core, rim and peritumoral regions). Finally, our study underlines the complexity of the flow/metabolism interactions in different models of glioblastoma. These irregularities should be taken into account in order to palliate intratumoral hypoxia in clinical trials.

Hypoxia induces macrophage polarization and re-education toward an M2 phenotype in U87 and U251 glioblastoma models.

Hypoxia is a common feature of solid tumors, particularly in glioblastoma (GBM), and known to be a poor prognosis factor in GBM patients. The growth of GBM is also associated with a marked inflammation partially characterized by an accumulation of macrophage (MΦ) of the M2 phenotype. However, the transition between M1 MΦ (antitumoral) and M2 MΦ (protumoral) phenotypes is a dynamic process. We made the assumption that oxygen (O2) availability could be a major regulator of this transition and that the intratumoral O2 gradient is of importance. We evaluated, in vivo, the impact of hypoxia on MΦ tropism and polarization in two models of human GBM, well differentiated by their degree of hypoxia. MΦ migration in the tumor was more pronounced in the more hypoxic tumor of the two GBM models. In the more hypoxic of the models, we have shown that MΦ migrated at the tumor site only when hypoxia takes place. We also demonstrated that the acquisition of the M2 phenotype was clearly an evolving phenomenon with hypoxia as the major trigger for this transition. In support of these in vivo finding, M0 but also M1 MΦ cultured in moderate or severe hypoxia displayed a phenotype close to that of M2 MΦ whose phenotype was further reinforced by severe hypoxia. These results highlight the role of hypoxia in the aggressiveness of GBM, in part, by transforming MΦ such that a protumoral activity is expressed.

Multimodal imaging based on MRI and PET reveals [(18)F]FLT PET as a specific and early indicator of treatment efficacy in a preclinical model of recurrent glioblastoma.

PURPOSE: The primary objective of this study was to compare the ability of PET and MRI biomarkers to predict treatment efficacy in a preclinical model of recurrent glioblastoma multiforme. METHODS: MRI (anatomical, diffusion, vasculature and oxygenation) and PET ([(18)F]FDG and [(18)F]FLT) parameters were obtained 3 days after the end of treatment and compared with late tumour growth and survival. RESULTS: Early after tumour recurrence, no effect of treatment with temozolomide combined with bevacizumab was observed on tumour volume as assessed by T2-W MRI. At later times, the treatment decreased tumour volume and increased survival. Interestingly, at the earlier time, temozolomide + bevacizumab decreased [(18)F]FLT uptake, cerebral blood volume and oedema. [(18)F]FLT uptake, oedema and cerebral blood volume were correlated with overall survival but [(18)F]FLT uptake had the highest specificity and sensitivity for the early prediction of treatment efficacy. CONCLUSION: The present investigation in a preclinical model of glioblastoma recurrence underscores the importance of multimodal imaging in the assessment of oedema, tumour vascular status and cell proliferation. Finally, [(18)F]FLT holds the greatest promise for the early assessment of treatment efficacy. These findings may translate clinically in that individualized treatment for recurrent glioma could be prescribed for patients selected after PET/MRI examinations.

Silencing erythropoietin receptor on glioma cells reinforces efficacy of temozolomide and X-rays through senescence and mitotic catastrophe.

Hypoxia-inducible genes may contribute to therapy resistance in glioblastoma (GBM), the most aggressive and hypoxic brain tumours. It has been recently reported that erythropoietin (EPO) and its receptor (EPOR) are involved in glioma growth. We now investigated whether EPOR signalling may modulate the efficacy of the GBM current treatment based on chemotherapy (temozolomide, TMZ) and radiotherapy (X-rays). Using RNA interference, we showed on glioma cell lines (U87 and U251) that EPOR silencing induces a G2/M cell cycle arrest, consistent with the slowdown of glioma growth induced by EPOR knock-down. In vivo, we also reported that EPOR silencing combined with TMZ treatment is more efficient to delay tumour recurrence and to prolong animal survival compared to TMZ alone. In vitro, we showed that EPOR silencing not only increases the sensitivity of glioma cells to TMZ as well as X-rays but also counteracts the hypoxia-induced chemo- and radioresistance. Silencing EPOR on glioma cells exposed to conventional treatments enhances senescence and induces a robust genomic instability that leads to caspase-dependent mitotic death by increasing the number of polyploid cells and cyclin B1 expression. Overall these data suggest that EPOR could be an attractive target to overcome therapeutic resistance toward ionising radiation or temozolomide.