RESUMO
Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells, and the loss of TERRA m6A/METTL3 results in telomere damage. We observed that m6A modification is abundant in R-loop enriched TERRA, and the m6A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m6A drives telomere targeting of TERRA via R-loops, and this m6A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+ NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+ NB.
Assuntos
Metiltransferases , Neuroblastoma , RNA Longo não Codificante , Humanos , Adenina/análogos & derivados , Metiltransferases/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Estruturas R-Loop , RNA Longo não Codificante/metabolismo , Telômero/genética , Homeostase do TelômeroRESUMO
BACKGROUND: Neuroblastoma (NB) is a heterogeneous embryonal malignancy and the deadliest tumor of infancy. It is a complex disease that can result in diverse clinical outcomes. In some children, tumors regress spontaneously. Others respond well to existing treatments. But for the high-risk group, which constitutes approximately 40% of all patients, the prognosis remains dire despite collaborative efforts in basic and clinical research. While its exact cellular origin is still under debate, NB is assumed to arise from the neural crest cell lineage including multipotent Schwann cell precursors (SCPs), which differentiate into sympatho-adrenal cell states eventually producing chromaffin cells and sympathoblasts. METHODS: To investigate clonal development of neuroblastoma cell states, we performed haplotype-specific analysis of human tumor samples using single-cell multi-omics, including joint DNA/RNA sequencing of sorted single cells (DNTR-seq). Samples were also assessed using immunofluorescence stainings and fluorescence in-situ hybridization (FISH). RESULTS: Beyond adrenergic tumor cells, we identify subpopulations of aneuploid SCP-like cells, characterized by clonal expansion, whole-chromosome 17 gains, as well as expression programs of proliferation, apoptosis, and a non-immunomodulatory phenotype. CONCLUSION: Aneuploid pre-malignant SCP-like cells represent a novel feature of NB. Genetic evidence and tumor phylogeny suggest that these clones and malignant adrenergic populations originate from aneuploidy-prone cells of migrating neural crest or SCP origin, before lineage commitment to sympatho-adrenal cell states. Our findings expand the phenotypic spectrum of NB cell states. Considering the multipotency of SCPs in development, we suggest that the transformation of fetal SCPs may represent one possible mechanism of tumor initiation in NB with chromosome 17 aberrations as a characteristic element.
Assuntos
Perfilação da Expressão Gênica , Neuroblastoma , Células de Schwann , Análise de Célula Única , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Neuroblastoma/metabolismo , Células de Schwann/metabolismo , Células de Schwann/patologia , Transcriptoma , Regulação Neoplásica da Expressão Gênica , Hibridização in Situ FluorescenteRESUMO
Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.
Assuntos
Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Células-Tronco Neurais/fisiologia , Anilidas/farmacologia , Animais , Síndrome do Nevo Basocelular/genética , Síndrome do Nevo Basocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Galectina 1/genética , Galectina 1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Humanos , Camundongos , Neoplasias Experimentais , Receptor Patched-1/genética , Piridinas/farmacologiaRESUMO
BACKGROUND: Transgenic animal models are crucial for the study of gene function and disease, and are widely utilized in basic biological research, agriculture and pharma industries. Since the current methods for generating transgenic animals result in the random integration of the transgene under study, the phenotype may be compromised due to disruption of known genes or regulatory regions. Unfortunately, most of the tools that predict transgene insertion sites from high-throughput data are not publicly available or not properly maintained. RESULTS: We implemented TC-hunter, Transgene-Construct hunter, an open tool that identifies transgene insertion sites and provides simple reports and visualization aids. It relies on common tools used in the analysis of high-throughput data and makes use of chimeric reads and discordant read pairs to identify and support the transgenic insertion site. To demonstrate its applicability, we applied TC-hunter to four transgenic mice samples harboring the human PPM1D gene, a model used in the study of malignant tumor development. We identified the transgenic insertion site in each sample and experimentally validated them with Touchdown-polymerase chain reaction followed by Sanger sequencing. CONCLUSIONS: TC-hunter is an accessible bioinformatics tool that can automatically identify transgene insertion sites from DNA sequencing data with high sensitivity (98%) and precision (92.45%). TC-hunter is a valuable tool that can aid in evaluating any potential phenotypic complications due to the random integration of the transgene and can be accessed at https://github.com/bcfgothenburg/SSF .
Assuntos
Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Camundongos Transgênicos , TransgenesRESUMO
BACKGROUND: p53 mutants contribute to the chronic inflammatory tumour microenvironment (TME). In this study, we address the mechanism of how p53 mutants lead to chronic inflammation in tumours and how to transform it to restore cancer immune surveillance. METHODS: Our analysis of RNA-seq data from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) project revealed that mutant p53 (mtp53) cancers correlated with chronic inflammation. We used cell-based assays and a mouse model to discover a novel gain of function of mtp53 and the effect of the mtp53 reactivating compound APR-246 on the anti-tumour immune response. RESULTS: We found that tumour samples from patients with breast carcinoma carrying mtp53 showed elevated Interferon (IFN) signalling, Tumour Inflammation Signature (TIS) score and infiltration of CD8+ T cells compared to wild type p53 (wtp53) tumours. We showed that the expression of IFN and immune checkpoints were elevated in tumour cells in a mtp53-dependent manner, suggesting a novel gain of function. Restoration of wt function to mtp53 by APR-246 induced the expression of endogenous retroviruses, IFN signalling and repressed immune checkpoints. Moreover, APR-246 promoted CD4+ T cells infiltration and IFN signalling and prevented CD8+ T cells exhaustion within the TME in vivo. CONCLUSIONS: Breast carcinomas with mtp53 displayed enhanced inflammation. APR-246 boosted the interferon response or represses immune checkpoints in p53 mutant tumour cells, and restores cancer immune surveillance in vivo.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Camundongos , Animais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Mutação com Ganho de Função , Neoplasias/genética , Interferons/genética , Interferons/metabolismo , Inflamação/genética , Microambiente Tumoral/genéticaRESUMO
PURPOSE OF REVIEW: The evolving information of the initiation, tumor cell heterogeneity, and plasticity of childhood neuroblastoma has opened up new perspectives for developing therapies based on detailed knowledge of the disease. RECENT FINDINGS: The cellular origin of neuroblastoma has begun to unravel and there have been several reports on tumor cell heterogeneity based on transcriptional core regulatory circuitries that have given us important information on the biology of neuroblastoma as a developmental disease. This together with new insight of the tumor microenvironment which acts as a support for neuroblastoma growth has given us the prospect for designing better treatment approaches for patients with high-risk neuroblastoma. Here, we discuss these new discoveries and highlight some emerging therapeutic options. Neuroblastoma is a disease with multiple facets. Detailed biological and molecular knowledge on neuroblastoma initiation, heterogeneity, and the communications between cells in the tumor microenvironment holds promise for better therapies.
Assuntos
Neuroblastoma , Humanos , Neuroblastoma/genética , Neuroblastoma/terapia , Microambiente TumoralRESUMO
Merkel cell polyomavirus (MCPyV) is a causal factor in Merkel cell carcinoma (MCC). The oncogenic potential is mediated through its viral oncoproteins large T-antigen (LT) and small T-antigen (sT). Cytokines produced by tumor cells play an important role in cancer pathogenesis, and viruses affect their expression. Therefore, we compared human cytokine and receptor transcript levels in virus positive (V+) and virus negative (V-) MCC cell lines. Increased expression of IL-33, a potent modulator of tumor microenvironment, was observed in V+ MCC cell lines when compared to V- MCC-13 cells. Transient transfection studies with luciferase reporter plasmids demonstrated that LT and sT stimulated IL-33, ST2/IL1RL1 and IL1RAcP promoter activity. The induction of IL-33 expression was confirmed by transfecting MCC-13 cells with MCPyV LT. Furthermore, recombinant human cytokine domain IL-33 induced activation of MAP kinase and NF-κB pathways, which could be blocked by a ST2 receptor antibody. Immunohistochemical analysis demonstrated a significantly stronger IL-33, ST2, and IL1RAcP expression in MCC tissues compared to normal skin. Of interest, significantly higher IL-33 and IL1RAcP protein levels were observed in MCC patient plasma compared to plasma from healthy controls. Previous studies have demonstrated the implication of the IL-33/STL2 pathway in cancer. Because our results revealed a T-antigens-dependent induction of the IL-33/ST2 axis, IL-33/ST2 may play a role in the tumorigenesis of MCPyV-positive MCC. Therefore, neutralizing the IL-33/ST2 axis may present a novel therapeutic approach for MCC patients.
Assuntos
Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Neoplasias Cutâneas , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Carcinogênese , Carcinoma de Célula de Merkel/patologia , Citocinas/metabolismo , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Poliomavírus das Células de Merkel/fisiologia , Neoplasias Cutâneas/patologia , Microambiente TumoralRESUMO
The majority of anti-cancer therapies target the proliferating tumor cells, while the tumor stroma, principally unaffected, survives, and provide a niche for surviving tumor cells. Combining tumor cell and stroma-targeting therapies thus have a potential to improve patient outcome. The neuroblastoma stroma contains cancer-associated fibroblasts expressing microsomal prostaglandin E synthase-1 (mPGES-1). mPGES-1-derived prostaglandin E2 (PGE2 ) is known to promote tumor growth through increased proliferation and survival of tumor cells, immune suppression, angiogenesis, and therapy resistance, and we, therefore, hypothesize that mPGES-1 constitutes an interesting stromal target. Here, we aimed to develop a relevant in vitro model to study combination therapies. Co-culturing of neuroblastoma and fibroblast cells in 3D tumor spheroids mimic neuroblastoma tumors with regard to the cyclooxygenase/mPGES-1/PGE2 pathway. Using the spheroid model, we show that the inhibition of fibroblast-derived mPGES-1 enhanced the cytotoxic effect of doxorubicin and vincristine and significantly reduced tumor cell viability and spheroid growth. Cyclic treatment with vincristine in combination with an mPGES-1 inhibitor abrogated cell repopulation. Moreover, inhibition of mPGES-1 potentiated the cytotoxic effect of vincristine on established neuroblastoma allografts in mice. In conclusion, we established a 3D neuroblastoma model, highlighting the potential of combining stromal targeting of mPGES-1 with tumor cell targeting drugs like vincristine.
Assuntos
Antineoplásicos/farmacologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neuroblastoma/metabolismo , Prostaglandina-E Sintases/metabolismoRESUMO
Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3ß-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.
Assuntos
Neuroblastoma , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Camundongos , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Inibidores de Proteínas Quinases , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Associadas a rho/metabolismoRESUMO
Neuroblastoma is the most common extracranical tumor of childhood and the most deadly tumor of infancy. It is characterized by early age onset and high frequencies of metastatic disease but also the capacity to spontaneously regress. Despite intensive therapy, the survival for patients with high-risk neuroblastoma and those with recurrent or relapsed disease is low. Hence, there is an urgent need to develop new therapies for these patient groups. The molecular pathogenesis based on high-throughput omics technologies of neuroblastoma is beginning to be resolved which have given the opportunity to develop personalized therapies for high-risk patients. Here we discuss the potential of developing targeted therapies against aberrantly expressed molecules detected in sub-populations of neuroblastoma patients and how these selected targets can be drugged in order to overcome treatment resistance, improve survival and quality of life for these patients and also the possibilities to transfer preclinical research into clinical testing.
Assuntos
Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular/métodos , Neuroblastoma/tratamento farmacológico , Neoplasias do Sistema Nervoso Periférico/tratamento farmacológico , Antineoplásicos/farmacologia , Criança , Descoberta de Drogas/métodos , Resistencia a Medicamentos Antineoplásicos , Drogas em Investigação/farmacologia , Drogas em Investigação/uso terapêutico , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neoplasias do Sistema Nervoso Periférico/metabolismo , Neoplasias do Sistema Nervoso Periférico/patologia , Qualidade de Vida , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/patologiaRESUMO
BACKGROUND: Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor mainly expressed by the cells of myeloid origin, where it mediates the innate immune response to bacterial formylated peptides. High expression of FPR1 has been detected in various cancers but the function of FPR1 in tumorigenesis is poorly understood. METHODS: Expression of FPR1 in neuroblastoma cell lines and primary tumors was studied using RT-PCR, western blotting, immunofluorescence and immunohistochemistry. Calcium mobilization assays and western blots with phospho-specific antibodies were used to assess the functional activity of FPR1 in neuroblastoma. The tumorigenic capacity of FPR1 was assessed by xenografting of neuroblastoma cells expressing inducible FPR1 shRNA, FPR1 cDNA or control shRNA in nude mice. RESULTS: FPR1 is expressed in neuroblastoma primary tumors and cell lines. High expression of FPR1 corresponds with high-risk disease and poor patient survival. Stimulation of FPR1 in neuroblastoma cells using fMLP, a selective FPR1 agonist, induced intracellular calcium mobilization and activation of MAPK/Erk, PI3K/Akt and P38-MAPK signal transduction pathways that were inhibited by using Cyclosporin H, a selective receptor antagonist for FPR1. shRNA knock-down of FPR1 in neuroblastoma cells conferred a delayed xenograft tumor development in nude mice, whereas an ectopic overexpression of FPR1 promoted augmented tumorigenesis in nude mice. CONCLUSION: Our data demonstrate that FPR1 is involved in neuroblastoma development and could represent a therapy option for the treatment of neuroblastoma.
Assuntos
Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Ciclosporina/farmacologia , Neuroblastoma/patologia , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/metabolismo , Animais , Cálcio/metabolismo , Criança , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Formil Peptídeo/genética , Transplante Heterólogo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: The non-canonical Wnt/Planar cell polarity (PCP) signaling pathway is a major player in cell migration during embryonal development and has recently been implicated in tumorigenesis. METHODS: Transfections with cDNA plasmids or siRNA were used to increase and suppress Prickle1 and Vangl2 expression in neuroblastoma cells and in non-tumorigenic cells. Cell viability was measured by trypan blue exclusion and protein expression was determined with western blotting. Transcriptional activity was studied with luciferase reporter assay and mRNA expression with real-time RT-PCR. Immunofluorescence stainings were used to study the effects of Vangl2 overexpression in non-tumorigenic embryonic cells. Statistical significance was tested with t-test or one-way ANOVA. RESULTS: Here we show that high expression of the PCP core genes Prickle1 and Vangl2 is associated with low-risk neuroblastoma, suppression of neuroblastoma cell growth and decreased Wnt/ß-catenin signaling. Inhibition of Rho-associated kinases (ROCKs) that are important in mediating non-canonical Wnt signaling resulted in increased expression of Prickle1 and inhibition of ß-catenin activity in neuroblastoma cells. In contrast, overexpression of Vangl2 in MYC immortalized neural stem cells induced accumulation of active ß-catenin and decreased the neural differentiation marker Tuj1. Similarly, genetically modified mice with forced overexpression of Vangl2 in nestin-positive cells showed decreased Tuj1 differentiation marker during embryonal development. CONCLUSIONS: Our experimental data demonstrate that high expression of Prickle1 and Vangl2 reduce the growth of neuroblastoma cells and indicate different roles of PCP proteins in tumorigenic cells compared to normal cells. These results suggest that the activity of the non-canonical Wnt/PCP signaling pathway is important for neuroblastoma development and that manipulation of the Wnt/PCP pathway provides a possible therapy for neuroblastoma.
Assuntos
Carcinogênese/genética , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas com Domínio LIM/biossíntese , Proteínas de Membrana/biossíntese , Neuroblastoma/genética , Proteínas Supressoras de Tumor/biossíntese , Animais , Movimento Celular/genética , Polaridade Celular/genética , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Proteínas de Membrana/genética , Camundongos , Neuroblastoma/patologia , Proteínas Supressoras de Tumor/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Quinases Associadas a rho/genéticaRESUMO
Epigenetic alterations and aberrant expression of genes controlling epigenetic mechanisms have been identified in several cancers, including medulloblastoma, the most common brain tumor in children. Here we show that combining drugs that inhibit two of the most important epigenetic factors, gene methylation and post-translational modifications of protein histone-associated DNA, with small molecule inhibitors of receptor tyrosine kinases induces apoptosis. The histone deacetylation inhibitor, 4-phenylbutyrate (4-PB) and the demethylation agent, 5-Aza-2'deoxycytidine (5-Aza-dC) had minor effects on medulloblastoma cell cytotoxity in single agent treatment whereas a significant enhancement in cell cytotoxity was seen when these drugs were combined with Gleevec. Triple treatment of medulloblastoma cells with 4-PB, 5-Aza and Gleevec were associated with reduced DNA methyltransferase activity, reduced global methylation and induction of apoptosis. Taken together these results suggest that a combination of these drugs may be beneficial in the treatment of medulloblastoma.
Assuntos
Epigênese Genética , Meduloblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Linhagem Celular Tumoral , Metilação de DNA , Humanos , Meduloblastoma/genéticaRESUMO
Neuroblastoma is a neural crest-derived tumor of the peripheral nervous system that is a leading cause of cancer-related deaths in children [...].
RESUMO
Neuroblastoma is a peripheral nervous system tumor that almost exclusively occurs in young children. Although intensified treatment modalities have led to increased patient survival, the prognosis for patients with high-risk disease is still around 50%, signifying neuroblastoma as a leading cause of cancer-related deaths in children. Neuroblastoma is an embryonal tumor and is shaped by its origin from cells within the neural crest. Hence, neuroblastoma usually presents with a low mutational burden and is, in the majority of cases, driven by epigenetically deregulated transcription networks. The recent development of Omic techniques has given us detailed knowledge of neuroblastoma evolution, heterogeneity, and plasticity, as well as intra- and intercellular molecular communication networks within the neuroblastoma microenvironment. Here, we discuss the potential of these recent discoveries with emphasis on new treatment modalities, including immunotherapies which hold promise for better future treatment regimens.
RESUMO
BACKGROUND: Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current preclinical in vivo model systems for MB have increased our understanding of molecular mechanisms regulating MB development. However, they may not be suitable for large-scale studies. The aim of this study was to investigate if a zebrafish-based xenograft model can recapitulate MB growth and enable rapid drug testing. METHODS: Nine different MB cell lines or patient-derived cells were transplanted into blastula-stage zebrafish embryos. Tumor development and migration were then monitored using live imaging. RNA sequencing was performed to investigate transcriptome changes after conditioning cells in neural stem cell-like medium. Furthermore, drug treatments were tested in a 96-well format. RESULTS: We demonstrate here that transplantation of MB cells into the blastula stage of zebrafish embryos leads to orthotopic tumor growth that can be observed within 24 hours after transplantation. Importantly, the homing of transplanted cells to the hindbrain region and the aggressiveness of tumor growth are enhanced by pre-culturing cells in a neural stem cell-like medium. The change in culture conditions rewires the transcriptome towards a more migratory and neuronal phenotype, including the expression of guidance molecules SEMA3A and EFNB1, both of which correlate with lower overall survival in MB patients. Furthermore, we highlight that the orthotopic zebrafish MB model has the potential to be used for rapid drug testing. CONCLUSION: Blastula-stage zebrafish MB xenografts present an alternative to current MB mouse xenograft models, enabling quick evaluation of tumor cell growth, neurotropism, and drug efficacy.
RESUMO
High-risk neuroblastoma has a poor prognosis despite intensive treatment, highlighting the need for new therapeutic strategies. Genetic alterations in activators and inactivators of Rho GTPase have been identified in neuroblastoma suggested to activate Rho/Rho-kinase (ROCK) signaling. ROCK has also been implicated in therapy resistance. Therefore, we have explored the efficacy of the dual ROCK inhibitor RKI-1447 in neuroblastoma, emphasizing combination strategies. Treatment with RKI-1447 resulted in decreased growth, increased cell death, and inhibition of N-MYC in vitro and in vivo. A combination screen revealed enhanced effects between RKI-1447 and BET inhibitors. Synergistic effects from RKI-1447 and the BET inhibitor, ABBV-075, were confirmed in various neuroblastoma models, including zebrafish. Interestingly, ABBV-075 increased phosphorylation of both myosin light chain 2 and cofilin, downstream effectors of ROCK, increases that were blocked by adding RKI-1447. The combination treatment also augmented an inhibitory effect on C-MYC and, less pronounced, N-MYC protein expression. BET inhibitors have shown preclinical efficacy against neuroblastoma, but acquired resistance has limited their therapeutic benefit. We reveal that the combination of ROCK and BET inhibitors offers a promising treatment approach that can potentially mitigate resistance to BET inhibitors and reduce toxicity.
RESUMO
Neuroblastoma is an aggressive pediatric cancer with a high rate of metastasis to the BM. Despite intensive treatments including high-dose chemotherapy, the overall survival rate for children with metastatic neuroblastoma remains dismal. Understanding the cellular and molecular mechanisms of the metastatic tumor microenvironment is crucial for developing new therapies and improving clinical outcomes. Here, we used single-cell RNA-Seq to characterize immune and tumor cell alterations in neuroblastoma BM metastases by comparative analysis with patients without metastases. Our results reveal remodeling of the immune cell populations and reprogramming of gene expression profiles in the metastatic niche. In particular, within the BM metastatic niche, we observed the enrichment of immune cells, including tumor-associated neutrophils, macrophages, and exhausted T cells, as well as an increased number of Tregs and a decreased number of B cells. Furthermore, we highlighted cell communication between tumor cells and immune cell populations, and we identified prognostic markers in malignant cells that are associated with worse clinical outcomes in 3 independent neuroblastoma cohorts. Our results provide insight into the cellular, compositional, and transcriptional shifts underlying neuroblastoma BM metastases that contribute to the development of new therapeutic strategies.
Assuntos
Medula Óssea , Neuroblastoma , Humanos , Criança , Medula Óssea/patologia , Neuroblastoma/genética , Análise de Célula Única , Microambiente TumoralRESUMO
BACKGROUND: Despite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets. METHODS: We collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis. RESULTS: The bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. CONCLUSIONS: These results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.
Assuntos
Carcinoma de Células Renais , Humanos , Carcinoma de Células Renais/genética , Células Estromais/patologia , Transdução de Sinais , Perfilação da Expressão Gênica , Análise de Célula Única , Microambiente TumoralRESUMO
Neuroblastoma is the most common and deadly tumor of childhood, where new therapy options for patients with high-risk disease are highly warranted. Human cytomegalovirus (HCMV) is prevalent in the human population and has recently been implicated in different cancer forms where it may provide mechanisms for oncogenic transformation, oncomodulation and tumor cell immune evasion. Here we show that the majority of primary neuroblastomas and neuroblastoma cell lines are infected with HCMV. Our analysis show that HCMV immediate-early protein was expressed in 100% of 36 primary neuroblastoma samples, and HCMV late protein was expressed in 92%. However, no infectious virus was detected in primary neuroblastoma tissue extracts. Remarkably, all six human neuroblastoma cell lines investigated contained CMV DNA and expressed HCMV proteins. HCMV proteins were expressed in neuroblastoma cells expressing the proposed stem cell markers CD133 and CD44. When engrafted into NMRI nu/nu mice, human neuroblastoma cells expressed HCMV DNA, RNA and proteins but did not produce infectious virus. The HCMV-specific antiviral drug valganciclovir significantly reduced viral protein expression and cell growth both in vitro and in vivo. These findings indicate that HCMV is important for the pathogenesis of neuroblastoma and that anti-viral therapy may be a novel adjuvant treatment option for children with neuroblastoma.