First Transcriptome Analysis of Hepatoblastoma in Brazil: Unraveling the Pivotal Role of Noncoding RNAs and Metabolic Pathways.

Hepatoblastoma stands as the most prevalent liver cancer in the pediatric population. Characterized by a low mutational burden, chromosomal and epigenetic alterations are key drivers of its tumorigenesis. Transcriptome analysis is a powerful tool for unraveling the molecular intricacies of hepatoblastoma, shedding light on the effects of genetic and epigenetic changes on gene expression. In this study conducted in Brazilian patients, an in-depth whole transcriptome analysis was performed on 14 primary hepatoblastomas, compared to control liver tissues. The analysis unveiled 1,492 differentially expressed genes (1,031 upregulated and 461 downregulated), including 920 protein-coding genes (62%). Upregulated biological processes were linked to cell differentiation, signaling, morphogenesis, and development, involving known hepatoblastoma-associated genes (DLK1, MEG3, HDAC2, TET1, HMGA2, DKK1, DKK4), alongside with novel findings (GYNG4, CDH3, and TNFRSF19). Downregulated processes predominantly centered around oxidation and metabolism, affecting amines, nicotinamides, and lipids, featuring novel discoveries like the repression of SYT7, TTC36, THRSP, CCND1, GCK and CAMK2B. Two genes, which displayed a concordant pattern of DNA methylation alteration in their promoter regions and dysregulation in the transcriptome, were further validated by RT-qPCR: the upregulated TNFRSF19, a key gene in the embryonic development, and the repressed THRSP, connected to lipid metabolism. Furthermore, based on protein-protein interaction analysis, we identified genes holding central positions in the network, such as HDAC2, CCND1, GCK, and CAMK2B, among others, that emerged as prime candidates warranting functional validation in future studies. Notably, a significant dysregulation of non-coding RNAs (ncRNAs), predominantly upregulated transcripts, was observed, with 42% of the top 50 highly expressed genes being ncRNAs. An integrative miRNA-mRNA analysis revealed crucial biological processes associated with metabolism, oxidation reactions of lipids and carbohydrates, and methylation-dependent chromatin silencing. In particular, four upregulated miRNAs (miR-186, miR-214, miR-377, and miR-494) played a pivotal role in the network, potentially targeting multiple protein-coding transcripts, including CCND1 and CAMK2B. In summary, our transcriptome analysis highlighted disrupted embryonic development as well as metabolic pathways, particularly those involving lipids, emphasizing the emerging role of ncRNAs as epigenetic regulators in hepatoblastomas. These findings provide insights into the complexity of the hepatoblastoma transcriptome and identify potential targets for future therapeutic interventions.

Pharmacogenetics of the Primary and Metastatic Osteosarcoma: Gene Expression Profile Associated with Outcome.

Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. In recent decades, OS treatment has reached a plateau and drug resistance is still a major challenge. Therefore, the present study aimed to analyze the expression of the genes related to pharmacogenetics in OS. The expression of 32 target genes in 80 paired specimens (pre-chemotherapeutic primary tumor, post-chemotherapeutic primary tumor and pulmonary metastasis) obtained from 33 patients diagnosed with OS were analyzed by the real-time PCR methodology. As the calibrators (control), five normal bone specimens were used. The present study identified associations between the OS outcome and the expression of the genes TOP2A, DHFR, MTHFR, BCL2L1, CASP3, FASLG, GSTM3, SOD1, ABCC1, ABCC2, ABCC3, ABCC5, ABCC6, ABCC10, ABCC11, ABCG2, RALBP1, SLC19A1, SLC22A1, ERCC1 and MSH2. In addition, the expression of the ABCC10, GGH, GSTM3 and SLC22A1 genes were associated with the disease event, and the metastasis specimens showed a high expression profile of ABCC1, ABCC3 and ABCC4 genes and a low expression of SLC22A1 and ABCC10 genes, which is possibly an important factor for resistance in OS metastasis. Therefore, our findings may, in the future, contribute to clinical management as prognostic factors as well as possible therapeutic targets.

Quantitative expression evaluation of PRAME gene in osteosarcoma.

BACKGROUND: In a previous study, our group observed that 68% of the osteosarcoma (OS) samples presented PRAME (Preferentially Expressed Antigen in Melanoma) gene expression. In this work, we propose to investigate quantitatively gene expression of PRAME in distinct patients groups. METHODS AND RESULTS: 61 osteosarcoma samples, from 3 distinct patients groups were selected for this study: (1) Patients younger than 10 years old at diagnosis, (2) Patients that had poor evolution of disease and (3) Patients that were in remission of disease and had treatment with no intercurrences) PRAME gene expression levels were obtained using quantitative Real-Time Polymerase Chain Reaction method (qRT-PCR). Clinical parameters were collected from patient's medical charts. Results demonstrated an increase in PRAME gene expression in all samples, with high variation in expression levels, when considering all samples and when analyzed in each group. In addition, no statistical difference was found when considering clinical data collected or patients groups. CONCLUSION: PRAME gene expression quantitative investigation did not bring any complementary information beyond of what had already been observed in other qualitative investigations published by our group, there is no relation between PRAME gene expression levels and disease evolution. However, the findings in this work contribute for validation PRAME gene expression as a good biomarker to OS, which, in the future, may allow identification circulating tumor cell or molecules to contribute with early diagnostic of metastasis, a genuine problem in OS that determinate flattening in survival curves.

Copy Number Alterations in Hepatoblastoma: Literature Review and a Brazilian Cohort Analysis Highlight New Biological Pathways.

Hepatoblastoma (HB) is a rare embryonal tumor, although it is the most common pediatric liver cancer. The aim of this study was to provide an accurate cytogenomic profile of this type of cancer, for which information in cancer databases is lacking. We performed an extensive literature review of cytogenetic studies on HBs disclosing that the most frequent copy number alterations (CNAs) are gains of 1q, 2/2q, 8/8q, and 20; and losses at 1p and 4q. Furthermore, the CNA profile of a Brazilian cohort of 26 HBs was obtained by array-CGH; the most recurrent CNAs were the same as shown in the literature review. Importantly, HBs from female patients, high-risk stratification tumors, tumors who developed in older patients (> 3 years at diagnosis) or from patients with metastasis and/or deceased carried a higher diversity of chromosomal alterations, specifically chromosomal losses at 1p, 4, 11q and 18q. In addition, we distinguished three major CNA profiles: no detectable CNA, few CNAs and tumors with complex genomes. Tumors with simpler genomes exhibited a significant association with the epithelial fetal subtype of HBs; in contrast, the complex genome group included three cases with epithelial embryonal histology, as well as the only HB with HCC features. A significant association of complex HB genomes was observed with older patients who developed high-risk tumors, metastasis, and deceased. Moreover, two patients with HBs exhibiting complex genomes were born with congenital anomalies. Together, these findings suggest that a high load of CNAs, mainly chromosomal losses, particularly losses at 1p and 18, increases the tendency to HB aggressiveness. Additionally, we identified six hot-spot chromosome regions most frequently affected in the entire group: 1q31.3q42.3, 2q23.3q37.3, and 20p13p11.1 gains, besides a 5,3 Mb amplification at 2q24.2q24.3, and losses at 1p36.33p35.1, 4p14 and 4q21.22q25. An in-silico analysis using the genes mapped to these six regions revealed several enriched biological pathways such as ERK Signaling, MicroRNAs in Cancer, and the PI3K-Akt Signaling, in addition to the WNT Signaling pathway; further investigation is required to evaluate if disturbances of these pathways can contribute to HB tumorigenesis. The analyzed gene set was found to be associated with neoplasms, abnormalities of metabolism/homeostasis and liver morphology, as well as abnormal embryonic development and cytokine secretion. In conclusion, we have provided a comprehensive characterization of the spectrum of chromosomal alterations reported in HBs and identified specific genomic regions recurrently altered in a Brazilian HB group, pointing to new biological pathways, and relevant clinical associations.

Hepatoblastomas exhibit marked NNMT downregulation driven by promoter DNA hypermethylation.

Hepatoblastomas exhibit the lowest mutational burden among pediatric tumors. We previously showed that epigenetic disruption is crucial for hepatoblastoma carcinogenesis. Our data revealed hypermethylation of nicotinamide N-methyltransferase, a highly expressed gene in adipocytes and hepatocytes. The expression pattern and the role of nicotinamide N-methyltransferase in pediatric liver tumors have not yet been explored, and this study aimed to evaluate the effect of nicotinamide N-methyltransferase hypermethylation in hepatoblastomas. We evaluated 45 hepatoblastomas and 26 non-tumoral liver samples. We examined in hepatoblastomas if the observed nicotinamide N-methyltransferase promoter hypermethylation could lead to dysregulation of expression by measuring mRNA and protein levels by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays. The potential impact of nicotinamide N-methyltransferase changes was evaluated on the metabolic profile by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Significant nicotinamide N-methyltransferase downregulation was revealed in hepatoblastomas, with two orders of magnitude lower nicotinamide N-methyltransferase expression in tumor samples and hepatoblastoma cell lines than in hepatocellular carcinoma cell lines. A specific TSS1500 CpG site (cg02094283) of nicotinamide N-methyltransferase was hypermethylated in tumors, with an inverse correlation between its methylation level and nicotinamide N-methyltransferase expression. A marked global reduction of the nicotinamide N-methyltransferase protein was validated in tumors, with strong correlation between gene and protein expression. Of note, higher nicotinamide N-methyltransferase expression was statistically associated with late hepatoblastoma diagnosis, a known clinical variable of worse prognosis. In addition, untargeted metabolomics analysis detected aberrant lipid metabolism in hepatoblastomas. Data presented here showed the first evidence that nicotinamide N-methyltransferase reduction occurs in hepatoblastomas, providing further support that the nicotinamide N-methyltransferase downregulation is a wide phenomenon in liver cancer. Furthermore, this study unraveled the role of DNA methylation in the regulation of nicotinamide N-methyltransferase expression in hepatoblastomas, in addition to evaluate the potential effect of nicotinamide N-methyltransferase reduction in the metabolism of these tumors. These preliminary findings also suggested that nicotinamide N-methyltransferase level may be a potential prognostic biomarker for hepatoblastoma.

Frequency of Pathogenic Germline Variants in Cancer-Susceptibility Genes in Patients With Osteosarcoma.

Importance: Osteosarcoma, the most common malignant bone tumor in children and adolescents, occurs in a high number of cancer predisposition syndromes that are defined by highly penetrant germline mutations. The germline genetic susceptibility to osteosarcoma outside of familial cancer syndromes remains unclear. Objective: To investigate the germline genetic architecture of 1244 patients with osteosarcoma. Design, Setting, and Participants: Whole-exome sequencing (n = 1104) or targeted sequencing (n = 140) of the DNA of 1244 patients with osteosarcoma from 10 participating international centers or studies was conducted from April 21, 2014, to September 1, 2017. The results were compared with the DNA of 1062 individuals without cancer assembled internally from 4 participating studies who underwent comparable whole-exome sequencing and 27 173 individuals of non-Finnish European ancestry who were identified through the Exome Aggregation Consortium (ExAC) database. In the analysis, 238 high-interest cancer-susceptibility genes were assessed followed by testing of the mutational burden across 736 additional candidate genes. Principal component analyses were used to identify 732 European patients with osteosarcoma and 994 European individuals without cancer, with outliers removed for patient-control group comparisons. Patients were subsequently compared with individuals in the ExAC group. All data were analyzed from June 1, 2017, to July 1, 2019. Main Outcomes and Measures: The frequency of rare pathogenic or likely pathogenic genetic variants. Results: Among 1244 patients with osteosarcoma (mean [SD] age at diagnosis, 16 [8.9] years [range, 2-80 years]; 684 patients [55.0%] were male), an analysis restricted to individuals with European ancestry indicated a significantly higher pathogenic or likely pathogenic variant burden in 238 high-interest cancer-susceptibility genes among patients with osteosarcoma compared with the control group (732 vs 994, respectively; P = 1.3 × 10-18). A pathogenic or likely pathogenic cancer-susceptibility gene variant was identified in 281 of 1004 patients with osteosarcoma (28.0%), of which nearly three-quarters had a variant that mapped to an autosomal-dominant gene or a known osteosarcoma-associated cancer predisposition syndrome gene. The frequency of a pathogenic or likely pathogenic cancer-susceptibility gene variant was 128 of 1062 individuals (12.1%) in the control group and 2527 of 27 173 individuals (9.3%) in the ExAC group. A higher than expected frequency of pathogenic or likely pathogenic variants was observed in genes not previously linked to osteosarcoma (eg, CDKN2A, MEN1, VHL, POT1, APC, MSH2, and ATRX) and in the Li-Fraumeni syndrome-associated gene, TP53. Conclusions and Relevance: In this study, approximately one-fourth of patients with osteosarcoma unselected for family history had a highly penetrant germline mutation requiring additional follow-up analysis and possible genetic counseling with cascade testing.

MAPK7 variants related to prognosis and chemotherapy response in osteosarcoma.

Osteosarcoma (OS) is a class of cancer originating from the bone, affecting mainly children and young adults. Our previous study showed that MAPK7 gene overexpression was significantly associated with tumor progression, poor treatment response, and worse overall survival, suggesting that MAPK7 could play an important role in OS tumorigenesis. We have investigated if MAPK7 overexpression was a result of any genomic changes in OS tumor specimens. We identified five SNPs (Single Nucleotide Polymorphism) previously described in databases, dbSNP and COSMIC, and identified two single nucleotide substitution not yet described. We found, in prechemotherapy specimens, a significant association of MAPK7 rs2233072G allele variant with metastasis at diagnosis and relapse (0.0909 and 0.0455, respectively). In post-chemotherapy, rs1054206GG specimen's genotype was associated with osteoblastic histological type (P= 0.0249) and presented decreased MAPK7 gene expression when compared with pre-chemotherapy specimens of same patients (P = 0.0095). Interestingly, it was observed some SNPs genotype exchange after chemotherapy. Our data indicated that MAPK7 gene expression associated with genotype exchange after chemotherapy, and these SNPs associated with important clinical parameters might be a valuable indicator for predicting in OS.

New therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X.

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

TET Upregulation Leads to 5-Hydroxymethylation Enrichment in Hepatoblastoma.

Hepatoblastoma is an embryonal liver tumor carrying few genetic alterations. We previously disclosed in hepatoblastomas a genome-wide methylation dysfunction, characterized by hypermethylation at specific CpG islands, in addition to a low-level hypomethylation pattern in non-repetitive intergenic sequences, in comparison to non-tumoral liver tissues, shedding light into a crucial role for epigenetic dysregulation in this type of cancer. To explore the underlying mechanisms possibly related to aberrant epigenetic modifications, we evaluated the expression profile of a set of genes engaged in the epigenetic machinery related to DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L, UHRF1, TET1, TET2, and TET3), as well as the 5-hydroxymethylcytosine (5hmC) global level. We observed in hepatoblastomas a general disrupted expression of these genes from the epigenetic machinery, mainly UHRF1, TET1, and TET2 upregulation, in association with an enrichment of 5hmC content. Our findings support a model of active demethylation by TETs in hepatoblastoma, probably during early stages of liver development, which in combination with UHRF1 overexpression would lead to DNA hypomethylation and an increase in overall 5hmC content. Furthermore, our data suggest that decreased 5hmC content might be associated with poor survival rate, highlighting a pivotal role of epigenetics in hepatoblastoma development and progression.

Intravenous Grafts of Human Amniotic Fluid-Derived Stem Cells Reduce Behavioral Deficits in Experimental Ischemic Stroke.

Amniotic fluid has been investigated as new cell source for stem cells in the development of future cell-based transplantation. This study reports isolation of viable human amniotic fluid-derived stem cells, labeled with multimodal iron oxide nanoparticles, and its effect on focal cerebral ischemia-reperfusion injury in Wistar rats. Middle cerebral artery occlusion of 60 min followed by reperfusion for 1 h, 6 h, and 24 h was employed in the present study to produce ischemia and reperfusion-induced cerebral injury in rats. Tests were employed to assess the functional outcome of the sensorimotor center activity in the brain, through a set of modified neurological severity scores used to assess motor and exploratory capacity 24 h, 14, and 28 days after receiving cellular therapy via tail vein. In our animal model of stroke, transplanted cells migrated to the ischemic focus, infarct volume decreased, and motor deficits improved. Therefore, we concluded that these cells appear to have beneficial effects on the ischemic brain, possibly based on their ability to enhance endogenous repair mechanisms.

Intravenous grafts of human amniotic fluid-derived stem cells reduce behavioral deficits in experimental ischemic stroke

Amniotic fluid has been investigated as new cell source for stem cells in the development of future cell-based transplantation. This study reports isolation of viable human amniotic fluid-derived stem cells, labeled with multimodal iron oxide nanoparticles, and its effect on focal cerebral ischemia–reperfusion injury in Wistar rats. Middle cerebral artery occlusion of 60 min followed by reperfusion for 1 h, 6 h, and 24 h was employed in the present study to produce ischemia and reperfusion-induced cerebral injury in rats. Tests were employed to assess the functional outcome of the sensorimotor center activity in the brain, through a set of modified neurological severity scores used to assess motor and exploratory capacity 24 h, 14, and 28 days after receiving cellular therapy via tail vein. In our animal model of stroke, transplanted cells migrated to the ischemic focus, infarct volume decreased, and motor deficits improved. Therefore, we concluded that these cells appear to have beneficial effects on the ischemic brain, possibly based on their ability to enhance endogenous repair mechanisms.

New therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

Intravenous grafts of human amniotic fluid-derived stem cells reduce behavioral deficits in experimental ischemic stroke

Amniotic fluid has been investigated as new cell source for stem cells in the development of future cell-based transplantation. This study reports isolation of viable human amniotic fluid-derived stem cells, labeled with multimodal iron oxide nanoparticles, and its effect on focal cerebral ischemia–reperfusion injury in Wistar rats. Middle cerebral artery occlusion of 60 min followed by reperfusion for 1 h, 6 h, and 24 h was employed in the present study to produce ischemia and reperfusion-induced cerebral injury in rats. Tests were employed to assess the functional outcome of the sensorimotor center activity in the brain, through a set of modified neurological severity scores used to assess motor and exploratory capacity 24 h, 14, and 28 days after receiving cellular therapy via tail vein. In our animal model of stroke, transplanted cells migrated to the ischemic focus, infarct volume decreased, and motor deficits improved. Therefore, we concluded that these cells appear to have beneficial effects on the ischemic brain, possibly based on their ability to enhance endogenous repair mechanisms.

New therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo.

BACKGROUND: Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. METHODS/RESULTS: We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. CONCLUSIONS: Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.

Valproic acid treatment response in vitro is determined by TP53 status in medulloblastoma.

PURPOSE: Histone deacetylate inhibitors (HDACi), as valproic acid (VA), have been reported to enhance efficacy and to prevent drug resistance in some tumors, including medulloblastoma (MB). In the present study, we investigated VA role, combined to cisplatin (CDDP) in cell viability and gene expression of MB cell lines. METHODS: Dose-response curve determined IC50 values for each treatment: (1) VA single, (2) CDDP single, and (3) VA and CDDP combined. Cytotoxicity and flow cytometry evaluated cell viability after exposure to treatments. Quantitative PCR evaluated gene expression levels of AKT, CTNNB1, GLI1, KDM6A, KDM6B, NOTCH2, PTCH1, and TERT, before and after treatment. Besides, we performed next-generation sequencing (NGS) for PTCH1, TERT, and TP53 genes. RESULTS: The most effective treatment to reduce viability was combined for D283MED and ONS-76; and CDDP single for DAOY cells (p < 0.0001). TERT, GLI1, and AKT genes were overexpressed after treatments with VA. D283MED and ONS-76 cells presented variants in TERT and PTCH1, respectively and DAOY cell line presented a TP53 mutation. CONCLUSIONS: MB tumors belonging to SHH molecular subgroup, with TP53MUT, would be the ones that present high risk in relation to VA use during the treatment, while TP53WT MBs can benefit from VA therapy, both SHH and groups 3 and 4. Our study shows a new perspective about VA action in medulloblastoma cells, raising the possibility that VA may act in different patterns. According to the genetic background of MB cell, VA can stimulate cell cycle arrest and apoptosis or induce resistance to treatment via signaling pathways activation.

Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo

Background Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. Methods/results We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. Conclusions Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology

Background: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

Tropism of mesenchymal stem cell toward CD133+ stem cell of glioblastoma in vitro and promote tumor proliferation in vivo

Background Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. Methods/results We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. Conclusions Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology

Background: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.