Metabolic Heterogeneity of Brain Tumor Cells of Proneural and Mesenchymal Origin.

Brain-tumor-initiating cells (BTICs) of proneural and mesenchymal origin contribute to the highly malignant phenotype of glioblastoma (GB) and resistance to current therapies. BTICs of different subtypes were challenged with oxidative phosphorylation (OXPHOS) inhibition with metformin to assess the differential effects of metabolic intervention on key resistance features. Whereas mesenchymal BTICs varied according to their invasiveness, they were in general more glycolytic and less responsive to metformin. Proneural BTICs were less invasive, catabolized glucose more via the pentose phosphate pathway, and responded better to metformin. Targeting glycolysis may be a promising approach to inhibit tumor cells of mesenchymal origin, whereas proneural cells are more responsive to OXPHOS inhibition. Future clinical trials exploring metabolic interventions should account for metabolic heterogeneity of brain tumors.

Stattic and metformin inhibit brain tumor initiating cells by reducing STAT3-phosphorylation.

Glioblastoma (GBM) is the most common and malignant type of primary brain tumor and associated with a devastating prognosis. Signal transducer and activator of transcription number 3 (STAT3) is an important pathogenic factor in GBM and can be specifically inhibited with Stattic. Metformin inhibits GBM cell proliferation and migration. Evidence from other tumor models suggests that metformin inhibits STAT3, but there is no specific data on brain tumor initiating cells (BTICs).We explored proliferation and migration of 7 BTICs and their differentiated counterparts (TCs) after treatment with Stattic, metformin or the combination thereof. Invasion was measured in situ on organotypic brain slice cultures. Protein expression of phosphorylated and total STAT3, as well as AMPK and mTOR signaling were explored using Western blot. To determine functional relevance of STAT3 inhibition by Stattic and metformin, we performed a stable knock-in of STAT3 in selected BTICs.Inhibition of STAT3 with Stattic reduced proliferation in all BTICs, but only in 4 out of 7 TCs. Migration and invasion were equally inhibited in BTICs and TCs. Treatment with metformin reduced STAT3-phosphorylation in all investigated BTICs and TCs. Combined treatment with Stattic and metformin led to significant additive effects on BTIC proliferation, but not migration or invasion. No additive effects on TCs could be detected. Stable STAT3 knock-in partly attenuated the effects of Stattic and metformin on BTICs.In conclusion, metformin was found to inhibit STAT3-phosphorylation in BTICs and TCs. Combined specific and unspecific inhibition of STAT3 might represent a promising new strategy in the treatment of glioblastoma.

Adaptive Immune Response to and Survival Effect of Temozolomide- and Valproic Acid-induced Autophagy in Glioblastoma.

BACKGROUND/AIM: The combination of radiotherapy, temozolomide and valproic acid (VPA) has shown some promise in retrospective analyses of patients with glioblastoma, although their mechanisms of action remain unknown. MATERIALS AND METHODS: We investigated the in vitro and in vivo effects of pretreating glioma cells with temozolomide and VPA as an immunization strategy to boost an adaptive immune response in a syngeneic mouse model. RESULTS: Temozolomide and VPA induced autophagy in GL261 glioma cells, and caused tumor antigen-specific T-cells to become activated effector T-cells. Mice with a pre-existing glioma showed no improvement in clinical outcome when immunized with temozolomide- and VPA-treated glioma cells. CONCLUSION: Although temozolomide and VPA treatment of glioma cells can boost the adaptive immune response, in the context of a vaccine therapy, additional factors are necessary to eradicate the tumor and improve survival.

Immune humanization of immunodeficient mice using diagnostic bone marrow aspirates from carcinoma patients.

Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses.

Classic bladder exstrophy: Frequent 22q11.21 duplications and definition of a 414 kb phenocritical region.

BACKGROUND: Classic bladder exstrophy (CBE) is the most common form of the bladder exstrophy and epispadias complex. Previously, we and others have identified four patients with a duplication of 22q11.21 among a total of 96 unrelated CBE patients. METHODS: Here, we investigated whether this chromosomal aberration was commonly associated with CBE/bladder exstrophy and epispadias complex in an extended case-control sample. Multiplex ligation-dependent probe amplification and microarray-based analysis were used to identify 22q11.21 duplications in 244 unrelated bladder exstrophy and epispadias complex patients (including 217 CBE patients) and 665 healthy controls. RESULTS: New duplications of variable size were identified in four CBE patients and one control. Pooling of our previous and present data (eight duplications in 313 CBE patients) yielded a combined odds ratio of 31.86 (95% confidence interval, 4.24-1407.97). Array-based sequence capture and high-throughput targeted re-sequencing established that all breakpoints resided within the low-copy repeats 22A to 22D. Comparison of the eight duplications revealed a 414 kb phenocritical region harboring 12 validated RefSeq genes. Characterization of these 12 candidate genes through whole-mount in situ hybridization of mouse embryos at embryonic day 9.5 suggested that CRKL, THAP7, and LZTR1 are CBE candidate genes. CONCLUSION: Our data suggest that duplication of 22q11.21 increases CBE risk and implicate a phenocritical region in disease formation.

In vivo knockdown of Brachyury results in skeletal defects and urorectal malformations resembling caudal regression syndrome.

The T-box transcription factor BRACHYURY (T) is a key regulator of mesoderm formation during early development. Complete loss of T has been shown to lead to embryonic lethality around E10.0. Here we characterize an inducible miRNA-based in vivo knockdown mouse model of T, termed KD3-T, which exhibits a hypomorphic phenotype. KD3-T embryos display axial skeletal defects caused by apoptosis of paraxial mesoderm, which is accompanied by urorectal malformations resembling the murine uro-recto-caudal syndrome and human caudal regression syndrome phenotypes. We show that there is a reduction of T in the notochord of KD3-T embryos which results in impaired notochord differentiation and its subsequent loss, whereas levels of T in the tailbud are sufficient for axis extension and patterning. Furthermore, the notochord in KD3-T embryos adopts a neural character and loses its ability to act as a signaling center. Since KD3-T animals survive until birth, they are useful for examining later roles for T in the development of urorectal tissues.

Murine expression and mutation analyses of the prostate androgen-regulated mucin-like protein 1 (Parm1) gene, a candidate for human epispadias.

BACKGROUND: Epispadias is the mildest phenotype of the human bladder exstrophy-epispadias complex (BEEC), and presents with varying degrees of severity. This urogenital birth defect results from a disturbance in the septation process, during which separate urogenital and anorectal components are formed through division of the cloaca. This process is reported to be influenced by androgen signaling. The human PARM1 gene encodes the prostate androgen-regulated mucin-like protein 1, which is expressed in heart, kidney, and placenta. METHODS: We performed whole mount in situ hybridization analysis of Parm1 expression in mouse embryos between gestational days (GD) 9.5 and 12.5, which are equivalent to human gestational weeks 4-6. Since the spatio-temporal localization of Parm1 corresponded to tissues which are affected in human epispadias, we sequenced PARM1 in 24 affected patients. RESULTS: We found Parm1 specifically expressed in the region of the developing cloaca, the umbilical cord, bladder anlage, and the urethral component of the genital tubercle. Additionally, Parm1 expression was detected in the muscle progenitor cells of the somites and head mesenchyme. PARM1 gene analysis revealed no alterations in the coding region of any of the investigated patients. CONCLUSIONS: These findings suggest that PARM1 does not play a major role in the development of human epispadias. However, we cannot rule out the possibility that a larger sample size would enable detection of rare mutations in this gene.

p63 (TP73L) a key player in embryonic urogenital development with significant dysregulation in human bladder exstrophy tissue.

Human bladder exstrophy-epispadias complex (BEEC) comprises a spectrum of urogenital anomalies in which part or all of the distal urinary tract fails to close. Several lines of evidence implicate genetic factors in the formation of BEEC. Among them a murine p63+/+ knockout model showed the full picture of classic exstrophy of the bladder and other urogenital defects within the BEEC spectrum. This led us to study in depth the role of p63 in urogenital development in mice and investigate the implication of p63 in human BEEC. Whole mount in situ analysis in mice was carried out to investigate the ventro-caudal expression of the p63 transcript at gestational days (GD) 9.5-12.5, the equivalent of human gestational weeks 4-6 (postulated time of BEEC organogenesis in humans). In addition, p63 expression analysis was performed in human blood and bladder derived samples of 15 BEEC newborns accompanied by sequencing analysis of their genomic DNA. We also conducted sequencing analysis of genomic DNA in additional 22 BEEC patients. In mouse embryos, p63 expression was detected at days 9.5-12.5 in the cloacal membrane and urethral epithelium, supporting its role in the morphogenesis of the external genitalia and the bladder. Tissue-specific expression of a novel and already-known mRNA isoforms were established and a reproducible dysregulation of variable p63 isoforms was observed in 11 of 15 patients indicating altered gene expression. However, no obvious p63 gene mutations were identified in any of the patients. Our findings strongly suggest that p63 is not only involved in embryonic formation of the urogenital and ventrocaudal anatomy but is also highly dysregulated in human BEEC bladder tissue. Since p63 has been shown to self-regulate its expression through a balance of its isoforms, the dysregulation observed may contribute to the formation of BEEC.

Embryonic expression of the cysteine rich protein 61 (CYR61) gene: A candidate for the development of human epispadias.

BACKGROUND: Isolated epispadias is the mildest phenotype of the exstrophy-epispadias complex, a urogenital birth defect of variable severity. The androgen receptor antagonist flutamide (FLU) is known to cause malformations in the rat genital and reproductive tract, and single-dose prenatal FLU exposure can induce epispadias in rat offspring. The Cyr61 gene exhibited the highest response to FLU in rat fetal testis, and we suggested it a promising candidate gene for epispadias in humans, because its protein product promotes proliferation, migration, and adhesion of endothelial cells and fibroblasts. METHODS: We used whole mount in situ analysis in mice to investigate ventrocaudal expression of the Cyr61 transcript at gestational days 9.5 to 11.5, which is the equivalent of human gestational weeks 4 to 6 (postulated time of epispadias organogenesis in humans). We also performed mutational analysis of the CYR61 gene in 11 patients with isolated epispadias and in additional eight patients with the related classic bladder exstrophy phenotype. RESULTS: Expression of Cyr61 was detected in endothelial cells of vessels surrounding the cloaca and the umbilical cord on gestational days 10 and 11.5. The mutation screening, however, revealed no alterations in the coding region of human CYR61. CONCLUSIONS: The spatiotemporal expression pattern observed suggests a role for Cyr61 in the development of the external genitalia. Our mutation screening study, however, could not confirm that mutations affecting the CYR61 gene are a frequent cause of epispadias or classic bladder exstrophy, although rare mutations might be detectable in larger patient samples.