Multiple Irradiation Affects Cellular and Extracellular Components of the Mouse Brain Tissue and Adhesion and Proliferation of Glioblastoma Cells in Experimental System In Vivo

Intensive adjuvant radiotherapy (RT) is a standard treatment for glioblastoma multiforme (GBM) patients; however, its effect on the normal brain tissue remains unclear. Here, we investigated the short-term effects of multiple irradiation on the cellular and extracellular glycosylated components of normal brain tissue and their functional significance. Triple irradiation (7 Gy*3 days) of C57Bl/6 mouse brain inhibited the viability, proliferation and biosynthetic activity of normal glial cells, resulting in a fast brain-zone-dependent deregulation of the expression of proteoglycans (PGs) (decorin, biglycan, versican, brevican and CD44). Complex time-point-specific (24–72 h) changes in decorin and brevican protein and chondroitin sulfate (CS) and heparan sulfate (HS) content suggested deterioration of the PGs glycosylation in irradiated brain tissue, while the transcriptional activity of HS-biosynthetic system remained unchanged. The primary glial cultures and organotypic slices from triple-irradiated brain tissue were more susceptible to GBM U87 cells’ adhesion and proliferation in co-culture systems in vitro and ex vivo. In summary, multiple irradiation affects glycosylated components of normal brain extracellular matrix (ECM) through inhibition of the functional activity of normal glial cells. The changed content and pattern of PGs and GAGs in irradiated brain tissues are accompanied by the increased adhesion and proliferation of GBM cells, suggesting a novel molecular mechanism of negative side-effects of anti-GBM radiotherapy.

The Study of Cerebral Blood Flow Variations during Brain Metastasis Radiotherapy

Purpose: To investigate the cerebral blood flow (CBF) variations during brain metastases (BMs) radiotherapy (RT) applying with MR 3D-arterial spin labeling (ASL). Materials and Methods: A total of 26 BMs patients with 54 tumors were retrospectively enrolled. MR examinations were performed before and during RT (30-50 Gy) with a total dose of 36-60 Gy (12-30 fractions) including contrast-enhanced T1-weighted, T2 Flair and 3D-ASL images. The relationship between CBF changes and the largest cross-sectional area changes in BMs was investigated. And CBF changes in BMs, normal brain tissue, and peritumoral edema areas were analyzed under different dose gradients that was divided into 10 Gy intervals. Results: The largest cross-sectional areas and CBF of 54 BMs decreased by 26.46% and 29.64% respectively during RT (P<0.05), but there was no correlation between the two changes (P>0.05). The rates of CBF decrease in BMs were 33.75%, 24.61% and 27.55% at 30-40, 40-50 and >50 Gy, respectively (P<0.05). In normal brain tissue with dose gradients of 0-10, 10-20, 20-30, 30-40, 40-50 and > 50 Gy, the CBF decreased by 7.65%, 11.12%, 18.42%, 20.23%, 19.79% and 17.89%, respectively (P <0.05). The CBF decreases reached a maximum at 30-40 Gy in normal brain tissue as well as BMs. In contrast, the CBF decreases of peritumoral edema areas increased as the dose gradients increased. Moreover, the CBF changes of BMs were more notable than those in normal brain tissue and peritumoral edema areas. Conclusion: CBF changes can be feasibly assessed in different brain regions during RT based on 3D-ASL. The changes should be considered as a critical factor to determine the personal radiation dose for BMs, normal brain tissue and peritumoral edema areas.

EXTH-38. TARGETING KIF11 TO RADIOSENSITIZE GLIOBLASTOMA

Glioblastoma (GBM) is the most lethal primary brain tumor, with a 5 year survival rate of only 5%. The standard of care for GBM is maximal surgical resection of the tumor, followed by irradiation and chemotherapy. Despite treatment, tumors recur in almost 100% of patients. There are subpopulations of cells in GBM that are radioresistant and chemoresistant, and new treatments will need to inclusively target these cells. KIF11 is a mitotic protein that drives bipolar spindle formation and is crucial for successful completion of mitosis. We previously reported that KIF11 is overexpressed in GBM over normal brain tissue, and that inhibiting KIF11 in a patient-derived xenograft (PDX) GBM mouse model increased survival. However, in this model, tumors recurred after treatment was stopped, indicating that treatment may have had a cytostatic effect, rather than cytotoxic. Importantly, it has been reported that cells are most vulnerable to irradiation when they are in mitosis. Because using a KIF11 inhibitor arrests cells in mitosis, we hypothesized that combining irradiation and a KIF11 inhibitor would radiosensitize GBM cells, and lead to greater tumor cell death. In this study, we found that combination therapy increased cell death over vehicle or either treatment used alone in patient-derived GBM cells in vitro. Additionally, we found that inhibiting KIF11 combined with radiotherapy increased survival over vehicle or monotherapy in orthotopic PDX models. Our results demonstrate that combining KIF11 inhibitors with radiotherapy is a promising potential therapy for GBM.

BIOM-33. EV-ASSOCIATED IL13Ra2 AS A LIQUID BIOPSY BIOMARKER FOR GBM

Glioblastoma (GBM) is both the most common and deadly malignant primary brain tumor with a 1-year survival of 37.2% and a 5-year survival of just 5.1%. Despite advances in imaging technology, distinguishing between pseudoprogression and pseudoresponse remains problematic and there is a dire need for additional diagnostic methods. Tumor-derived extracellular vesicles are found in a variety of biofluids and are good candidates for a liquid biopsy. A unique characteristic of GBM is the overexpression of the α2 variant of IL-13 receptor (IL13Rα2) in 78-96% of patients while virtually undetectable in normal brain tissue. We hypothesize that biofluids of patients with GBM contains sufficient IL13Rα2-positive extracellular vesicles (EV) to inform the clinical picture regarding disease status such as tumor recurrence and response to therapy. We have measured the levels of IL13Rα2 in the EV fraction of plasma from GBM patients and correlated these levels to patient survival, and patient clinical characteristics (n=35). We have demonstrated that the EV fraction of some patients with GBM is enriched for the IL13Rα2 receptor. The plasma level of IL13Rα2 could also predict the level of IL13Rα2 in the GBM tumor (n=9). Further, IL13Rα2 positive EVs from patient-derived GBM cell lines could be immunoprecipitated for further interrogation of genomic and proteomic molecular markers. These exciting findings lay the groundwork for a GBM liquid biopsy based on tumor-specific EVs in patient biofluids.

Shugosin 2 is a biomarker for pathological grading and survival prediction in patients with gliomas

Glioblastomas are the most common type of adult primary brain neoplasms. Clinically, it is helpful to identify biomarkers to predict the survival of patients with gliomas due to its poor outcome. Shugoshin 2 (SGO2) is critical in cell division and cell cycle progression in eukaryotes. However, the association of SGO2 with pathological grading and survival in patients with gliomas remains unclear. We analyzed the association between SGO2 expression and clinical outcomes from Gene Expression Omnibus (GEO) dataset profiles, The Cancer Genome Atlas (TCGA), and Chinese Glioma Genome Atlas (CGGA). SGO2 mRNA and protein expression in normal brain tissue and glioma cell lines were investigated via quantitative RT-PCR, Western blot, and IHC staining. The roles of SGO2 in proliferation, migration, and apoptosis of GBM cells were studied with wound-healing assay, BrdU assay, cell cycle analysis, and JC-1 assay. The protein–protein interaction (PPI) was analyzed via Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). SGO2 mRNA expression predicted higher grade gliomas than non-tumor brain tissues. Kaplan–Meier survival analysis showed that patients with high-grade gliomas with a higher SGO2 expression had worse survival outcomes. SGO2 mRNA and protein expression were upper regulated in gliomas than in normal brain tissue. Inhibition of SGO2 suppressed cell proliferation and migration. Also, PPI result showed SGO2 to be a potential hub protein, which was related to the expression of AURKB and FOXM1. SGO2 expression positively correlates with WHO pathological grading and patient survival, suggesting that SGO2 is a biomarker that is predictive of disease progression in patients with gliomas.

miR-190a-3p Promotes Proliferation and Migration in Glioma Cells via YOD1

Introduction. To investigate the function of miR-190a-3p on the proliferation and migration of glioma. Methods. Twenty glioma samples and 6 normal brain tissue samples were collected. Normal human glial cell line HEB and glioma cell lines were used for the experiments. We then used TargetScan to predict the target genes of miR-190a-3p. Dual-luciferase reporter assay was also used to validate. Results. Combined with dual-luciferase reporter experiment, we finally verified that YOD1 was the aim, and it was low-expressed in glioma. Besides, a series of mechanism experiments then proved that miR-190a-3p negatively regulates YOD1 expression. Conclusions. Our research was the first to demonstrate the promoting function of miR-190a-3p in the proliferation and migration of glioma and provided new views for the treatment of glioma. miR-190a-3p was expected to be a new target for molecular therapy of glioma.

Yes-associated protein (YAP) promotes progression of glioma and poor prognosis of human

Background: Yes-associated protein(YAP) plays an important role in signal transduction and gene transcription regulation in 1 normal cells, with elevated and over-expressed YAP levels observed in various malignant tumors. The aim of this study was 2 to investigate the expression of YAP in malignant glioma, and to study the possible relationship of YAP expression with the 3 occurrence and development of malignant glioma. 4 Methods: Immunohistochemical staining was used to assess the expression of YAP and phosphor-YAP in malignant glioma 5 tissue and normal brain tissue, and their protein and mRNA levels were evaluated through Western blotting and reverse 6 transcription-polymerase chain reaction (RT-PCR), respectively. Normal brain tissue obtained from the functional lesion of 7 the epilepsy patients. After transfection of YAPsiRNA oligonucleotides or pcDNA3.1-hYAP plasmid, their effects on glioma 8 cells were investigated using western blot, cell proliferation, cycle, apoptosis and invasion, respectively. We conducted the 9 2 co-Immunoprecipitation to verify the combination of YAP and PPARγ, explore the mechanism of action. 10 Results: YAP-positive expression was found in 9 cases of normal brain and 60 cases of glioma. A significantly higher 11 expression of YAP in glioma tissue as compared with normal brain tissue at both protein and mRNA levels, and YAP proteins 12 mainly expressed and located in the nucleus and only a small percentage in the cytoplasm of glioma tissue. Phosphor-YAP 13 protein expression showed high staining of the cytoplasm, but no staining of the nuclear. While, with the enhancement of 14 the malignant degree, the cytoplasm YAP(p-YAP) expression is lower gradually than normal brain tissues. Further study in 15 glioma cell lines in which YAP was either overexpressed or depleted confirmed that YAP markedly promoted the cell 16 proliferation, cycle, invasion and inhibited the cell apoptosis. Moreover, YAP in company with PPARγ regulates the cell 17 proliferatin and effects the gliomagenesis. 18 Conclusion: These results indicate that YAP plays an important role in glioma and might be a useful therapeutic target of 19 glioma. 20

Maximum Lesion-To-Contralateral Normal Brain Tissue Ratio of 11C-Methionine PET as a Prognostic Imaging Biomarker for Newly Diagnosed and Untreated Astrocytic Glioma

Purpose: This study aimed whether the uptake of amino tracer positron emissiontomography (PET) can be used as an additional imaging biomarker to estimatethe prognosis of glioma.Methods: Participants comprised 56 adult patients with newly diagnosed and untreated World Health Organization (WHO) gradeⅡ-Ⅳ astrocytic glioma who underwent surgical excision and were evaluated by 11C-methionine PET prior to the surgical excision at Osaka City University Hospital from July 2011 to March 2018. Clinical and imaging studies were retrospectively reviewed based on medical records at our institution.Results: Preoperative Karnofsky Performance Status (KPS) only influenced progression-free survival (PFS) (hazard ratio [HR] 0.20; 95% confidence interval [CI] 0.10-0.41, p<0.0001), whereas histology (anaplastic astrocytoma: HR 5.30, 95%CI 1.23-22.8, p=0.025; glioblastoma: HR 11.52, 95%CI 2.27-58.47, p=0.0032), preoperative KPS≥80 (HR 0.23, 95%CI 0.09-0.62, p=0.004), maximum lesion-to-contralateral normal brain tissue (LN max)≥4.03 (HR 0.24, 95%CI 0.08-0.71, p=0.01), and isocitrate dehydrogenase (IDH) status (HR 14.06, 95%CI 1.81-109.2, p=0.011) were factors influencing overall survival (OS) in multivariate Cox regression. OS was shorter in patients with LN max≥ 4.03 (29.3 months) than in patients with LN max<4.03 (not reached; p=0.03). OS differed significantly between patients with IDH mutant/LN max<4.03 and patients with IDH mutant/LN max ≥4.03.Conclusions: LN max using 11C-methionine PET may be used in prognostic markers for newly identified and untreated WHO gradeⅡ-Ⅳ astrocytic glioma.

Longitudinal Grouping of Target Volumes for Volumetric-Modulated Arc Therapy of Multiple Brain Metastases

PurposeTreatment of multiple brain metastases with single-isocenter volumetric modulated arc therapy causes unnecessary exposure to normal brain tissue. In this study, a longitudinal grouping method was developed to reduce such unnecessary exposure.Materials and MethodsThis method has two main aspects: grouping brain lesions longitudinally according to their longitudinal projection positions in beam’s eye view, and rotating the collimator to 90° to make the multiple leaf collimator leaves conform to the targets longitudinally group by group. For 11 patients with multiple (5–30) brain metastases, two single-isocenter volumetric modulated arc therapy plans were generated using a longitudinal grouping strategy (LGS) and the conventional strategy (CVS). The prescription dose was 52 Gy for 13 fractions. Dose normalization to 100% of the prescription dose in 95% of the planning target volume was adopted. For plan quality comparison, Paddick conformity and the gradient index of the planning target volume, and the mean dose, the V100%, V50%, V25%, and V10% volumes of normal brain tissue were calculated.ResultsThere were no significant differences between the LGS and CVS plans in Paddick conformity (p = 0.374) and the gradient index (p = 0.182) of the combined planning target volumes or for V100% (p = 0.266) and V50% (p = 0.155) of the normal brain. However, the V25% and V10% of the normal brain which represented the low-dose region were significantly reduced in the LGS plans (p = 0.004 and p = 0.003, respectively). Consistently, the mean dose of the entire normal brain was 12.04 and 11.17 Gy in the CVS and LGS plans, respectively, a significant reduction in the LGS plans (p = 0.003).ConclusionsThe longitudinal grouping method can decrease unnecessary exposure and reduces the low-dose range in normal brain tissue.

Maximum Lesion-to-contralateral Normal Brain Tissue Ration of 11C-methionine PET as a Prognostic Imaging Biomarker for Newly Diagnosed and Untreated Astrocytic Glioma

Purpose: This study aimed whether the uptake of amino tracer positron emission tomography (PET) can be used as an additional imaging biomarker to estimate the prognosis of glioma.Methods: Participants comprised 56 adult patients with newly diagnosed and untreated World Health Organization (WHO) gradeⅡ-Ⅳ astrocytic glioma who underwent surgical excision and were evaluated by 11C-methionine PET prior to the surgical excision at Osaka City University Hospital from July 2011 to March 2018. Clinical and imaging studies were retrospectively reviewed based on medical records at our institution.Results: Preoperative Karnofsky Performance Status (KPS) only influenced progression-free survival (PFS) (hazard ratio [HR] 0.20; 95% confidence interval [CI] 0.10-0.41, p<0.0001), whereas histology (anaplastic astrocytoma: HR 5.30, 95%CI 1.23-22.8, p=0.025; glioblastoma: HR 11.52, 95%CI 2.27-58.47, p=0.0032), preoperative KPS≥80 (HR 0.23, 95%CI 0.09-0.62, p=0.004), maximum lesion-to-contralateral normal brain tissue (LN max)≥4.03 (HR 0.24, 95%CI 0.08-0.71, p=0.01), and isocitrate dehydrogenase (IDH) status (HR 14.06, 95%CI 1.81-109.2, p=0.011) were factors influencing overall survival (OS) in multivariate Cox regression. OS was shorter in patients with LN max≥ 4.03 (29.3 months) than in patients with LN max<4.03 (not reached; p=0.03). OS differed significantly between patients with IDH mutant/LN max<4.03 and patients with IDH mutant/LN max ≥4.03.Conclusions: LN max using 11C-methionine PET may be used in prognostic markers for newly identified and untreated WHO gradeⅡ-Ⅳ astrocytic glioma.