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BackgroundGlioblastoma remains highly lethal due to its inevitable recurrence. This recurrence is found locally in most cases, indicating that post-surgical tumor-initiating cells (TICs) accumulate at tumor edge. These edge TICs then generate recurrent tumors harboring new core lesions. Here, we investigated the clinical significance of the edge-to-core transition (ECT) signature causing glioblastoma recurrence and sought to identify central mediators for ECT.MethodsFirst, we examined the association of the ETC-related expression changes and patient outcome in matched primary and recurrent samples (n=37). Specifically, we tested whether the combined decrease of the edge TIC marker PROM1 (CD133) with the increase of the core TIC marker CD109 representing ECT during the primary-to-recurrence progression indicates poorer patient outcome. We then investigated the specific molecular mediators that trigger tumor recurrence driven by the ECT signature. Subsequently, the functional and translational significance of the identified molecule was validated within our patient-derived tumor edge-TIC modelsin vitroandin vivo.ResultsPatients exhibiting a CD133down/CD109upsignature during recurrence representing ECT displayed a strong association with poorer progression-free survival and overall survival among all tested patients. Differential gene expression identified that PLAGL1 was tightly correlated with the core TIC marker CD109 and was linked to a shorter survival of glioblastoma patients. Experimentally, forced PLAGL1 overexpression enhanced, while its knockdown reduced, the glioblastoma edge-derived tumor growthin vivoand subsequent mouse survival, suggesting its essential role in the ECT-mediated glioblastoma development.ConclusionsECT is likely an ongoing lethal process in primary glioblastoma contributing to its recurrence partly in a PLAGL1/CD109-mediated mechanism.Key PointsECT is a pathobiological process contributing to glioblastoma lethalityThe CD133down/CD109upsignature is a novel prognostic molecular biomarker in ECTPLAGL1 regulates growth of edge-located tumor-initiating cellsImportance of the StudyVery few studies have sought to longitudinally characterize the transition of molecular landscapes from primary to recurrent glioblastoma. Post-surgical edge-located TICs are presumably the predominant source of tumor recurrence, yet this cellular subpopulation in glioblastoma remains largely uncharacterized. This study evaluates the significance of glioblastoma edge-derived core transition (ECT) for tumor recurrence in the primary-recurrent paired sample set. We elucidate a prognostically-significant shift in molecular and cellular phenotypes associated with ECT in the CD133down/CD109upgroup. Moreover, our results provide clinical and experimental evidence that PLAGL1 is a mediator of glioblastoma ECT and its subsequent tumor development by the direct transcriptional regulation of the core TIC marker CD109.

The rise in population aging worldwide is causing an unparalleled increase in death from many cancers, including glioblastoma (GBM). In advanced countries, the number of elderly people living alone is increasing due to the rapid aging of the population and the socialization of nuclear families. Here, we explored the impact of aging and social isolation on GBM tumorigenesis. In normal brain tissue, aging promoted pathways related to cytokines and inflammation, which were further promoted by social isolation. In tumor tissues, the expression of neuron/synapse-related genes was significantly reduced in aged mice, and their expression was further reduced by social isolation. In addition, the survival period of aged mice was significantly shorter than that of young mice, and the survival period was further shortened by social isolation, which was characteristic of males. This phenomenon was the same in humans, and the survival period in the young group was significantly longer than that in the elderly group, and in the elderly group, the survival period was shortened in the male elderly group living alone. Our data indicate that social isolation contributes to the highly aggressive GBM by the shift to neuro-inflammation in the elderly brain.

PURPOSE Primary central nervous system lymphoma (PCNSL) has been immunohistochemically classified into two subtypes, germinal center (GC) B-cell and non-GC B-cell, but prognostic impact of these subtypes remains debated. We investigated clinical features and prognostic significance of newly defined immunohistochemical subtypes that were identified by expression patterns of three B-cell differentiation markers in PCNSL. In addition, we analyzed a factor related to responsiveness to high-dose methotrexate (HD-MTX) chemotherapy. METHODS Tumors from 32 PCNSL patients were immunohistochemically evaluated regarding expression of CD10, BCL-6, and MUM-1 and classified into subtypes according to the expression patterns of these markers. Clinical features and prognostic outcome of these subtypes were investigated. RESULTS Twenty-three patients were treated with HD-MTX-based chemotherapy followed by whole-brain radiation therapy (WBRT), and nine were treated with WBRT alone. Three immunohistochemical subtypes were identified, including A-type expressing CD10, BCL-6, and MUM-1 (12 patients), B-type expressing BCL-6 and MUM-1 (12 patients) and C-type expressing MUM-1 only (8 patients). Response rate in the HD-MTX therapy group was 57.1% (4/7) in A-type, 87.5% (7/8) in B-type, and 75% (6/8) in C-type. C-type with the lowest metabolic activity showed significantly longer overall survival than A-type with the higher uptake of methionine (71.6 versus 39.6 months). CONCLUSIONS Immunohistochemical identification of PCNSL based on the B-cell differentiation stage revealed three types of tumors, showing different metabolic activity and survival time. Refined immunohistochemical classification of PCNSL subtypes may become a useful tool for predicting more accurate prognosis and accessing the sensitivity to HD-MTX therapy.

Chloride intracellular channel protein 2 (CLIC2) belongs to the CLIC family of conserved metazoan proteins. However, CLIC2 is the least studied among its family members, and its function remains to be elucidated. Recently, we have shown that CLIC2 is correlated with the development and/or maintenance of tight junctions in blood vessel endothelial cells in human non-cancer tissues. CLIC2-expressing endothelial cells supposedly prevent hematogenous spread of cancer cells. In this study, we investigated CLIC2 expression in human brain tumor tissues and also addressed its function by employing human meningioma cells, rat glioma cells and rat malignant brain tumor model. Thirty-one meningioma cases, six SFT/HPC cases, twelve pituitary adenoma cases and twenty-three glioblastoma cases who underwent surgery at Ehime University Hospital were included. CLIC2 mRNA expression levels were investigated with immunoblotting and quantitative RT-PCR. Cells from the meningiomas were cultured and their CLIC2 expression was knockdown. Filter-based invasion assays and gelatin zymography were performed using the knocked-down meningioma cells. Rat C6 glioma cells stably expressing rat CLIC2 were established and transplanted into the right striatum of neonatal Wistar rats. Effects of CLIC2 on the survival periods of the animals were investigated. CLIC2 expression levels were high in the low-grade cases but low in the high-grade cases and highly invasive cases. Meningioma cells, of which CLIC2 expression was knocked-down, showed higher invasive activity than control cells. The CLIC2-knock down cells displayed increased activities of MMP-2 and MMP-9. Rat brain tumor models revealed that high expression of CLIC2 was correlated with smaller and less invasive brain tumors compared with those consisted of control cells. The rats transplanted with CLIC2-expressing cells survived longer periods than the rats with control C6 cells. These results suggest that CLIC2 plays a role in suppression of invasive activities of tumor cells.

The rise in population aging worldwide is causing an unparalleled increase in death from many cancers, including glioblastoma (GBM). Here, we have explored the impact of aging and rejuvenation on GBM tumorigenesis. Compared with old GBM, young GBM displayed elevated neuronal/synaptic signaling via brain-derived neurotrophic factor (BDNF) and SLIT and NTRK like-family member 6 (SLITRK6), promoting favorable survival rates. These effects were attributed to the rise in nicotinamide adenine dinucleotide (NAD+) levels, as brain rejuvenation by parabiosis or administration of nicotinamide mononucleotide (NMN) in mice elicited a younger phenotype with activated neuronal/synaptic signaling and improved outcomes. Our data indicate that age-associated NAD+ loss contributes to the highly aggressive GBM in the elderly. These findings have therapeutic implications in GBM and provide mechanistic insights into the exacerbation of GBM tumorigenesis with age.

Clinical outcomes for patients with glioblastoma (GBM) are extremely poor due to inevitable tumor recurrence even after extensive treatments. These recurrences are thought to manifest from cells located within the tumor edge. Despite this, the precise molecular mechanism governing GBM spatial phenotypic heterogeneity (e.g. edge vs. core) and subsequent tumor recurrence remains poorly elucidated. Here, using patient-derived GBM core and edge tissues, we analyzed transcriptional and metabolic signatures in an effort to determine how GBM facilitates the edge phenotype and its associated recurrence-initiating cells (RICs). In so doing, we unexpectedly identified CD38 as an essential protein in the formation of the edge phenotype and found a CD38-driven interaction between edge GBM cells and neighboring astrocytes that communally develops a GBM edge that is unresectable by surgery and retains RICs. These findings have profound implications for future clinical therapies and provide new mechanistic insights into both tumor progression and recurrence.

The poor prognosis of glioblastoma multiforme (GBM) may be due to the surviving glioma stem-like cells (GSCs) in the tumor periphery after tumor resection. We demonstrated that CD44-expressed GSCs existed much more in the tumor periphery of high invasive (HI) type GBM than low invasive (LI) type GBM. The HI type was significantly associated with worse outcome, but how GSCs with high CD44 expression relate to tumor progression remains unknown. In this study, we investigated effects of hypoxia on CD44-directed signal pathways, leading to tumor invasion and proliferation in GBM. We focused on the CD44 ligand osteopontin (OPN) because it is known hypoxia affects the interaction of CD44 and OPN which promotes stemness and proliferation of cancer stem cells. We examined mRNA expressions of hypoxia inducible factor (HIF)-1a, HIF-2a, CD44 and OPN in tumor tissues of GBM and investigated effects of hypoxia (1%O2:severe or 5%O2:moderate) on the expression of these molecules using cultured GSCs that were established from tumor tissues showing high CD44 expression in the periphery of GBMs. In addition, we analyzed the effects of OPN on invasive, migratory and proliferative activities of GSCs under the hypoxic conditions. OPN was much higher expressed in the tumor periphery of LI type GBM than HI type GBM. Severe hypoxia significantly increased the expressions of HIF-1a and CD44 but did not OPN. On the other hands, moderate hypoxia promoted the expressions of HIF-2a and OPN. Knockdown of HIF-2a significantly inhibited OPN expression. In addition, the more OPN was expressed in the cultured GSCs under moderate hypoxia, the more the GSCs proliferated and decreased their invasive and migratory activities. In conclusion, GSCs existing in the tumor periphery of GBM can migrate or proliferate by changing CD44-directed signal pathways. Moderate hypoxia promoted HIF-2a/OPN/CD44 pathway, resulting in phenotypic transition to high proliferative tumors.