Medulloblastoma (MB) is the most common malignant brain tumor in children and among the subtypes, Group 3 MB has the worst outcome. Here, we perform an in vivo, patient-specific screen leading to the ...identification of Otx2 and c-MYC as strong Group 3 MB inducers. We validated our findings in human cerebellar organoids where Otx2/c-MYC give rise to MB-like organoids harboring a DNA methylation signature that clusters with human Group 3 tumors. Furthermore, we show that SMARCA4 is able to reduce Otx2/c-MYC tumorigenic activity in vivo and in human cerebellar organoids while SMARCA4 T910M, a mutant form found in human MB patients, inhibits the wild-type protein function. Finally, treatment with Tazemetostat, a EZH2-specific inhibitor, reduces Otx2/c-MYC tumorigenesis in ex vivo culture and human cerebellar organoids. In conclusion, human cerebellar organoids can be efficiently used to understand the role of genes found altered in cancer patients and represent a reliable tool for developing personalized therapies.
Medulloblastoma (MB) is a childhood malignant brain tumour comprising four main subgroups characterized by different genetic alterations and rate of mortality. Among MB subgroups, patients with ...enhanced levels of the c-MYC oncogene (MB
Group3
) have the poorest prognosis. Here we identify a previously unrecognized role of the pro-autophagy factor AMBRA1 in regulating MB. We demonstrate that AMBRA1 expression depends on c-MYC levels and correlates with Group 3 patient poor prognosis; also, knockdown of AMBRA1 reduces MB stem potential, growth and migration of MB
Group3
stem cells. At a molecular level, AMBRA1 mediates these effects by suppressing SOCS3, an inhibitor of STAT3 activation. Importantly, pharmacological inhibition of autophagy profoundly affects both stem and invasion potential of MB
Group3
stem cells
,
and a combined anti-autophagy and anti-STAT3 approach impacts the MB
Group3
outcome. Taken together, our data support the c-MYC/AMBRA1/STAT3 axis as a strong oncogenic signalling pathway with significance for both patient stratification strategies and targeted treatments of MB
Group3
.
Brain tumors are the leading cause of cancer‐related death in children. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of pediatric brain cancers are ...limited and hard to establish. We present a protocol that enables efficient generation, expansion, and biobanking of pediatric brain cancer organoids. Utilizing our protocol, we have established patient‐derived organoids (PDOs) from ependymomas, medulloblastomas, low‐grade glial tumors, and patient‐derived xenograft organoids (PDXOs) from medulloblastoma xenografts. PDOs and PDXOs recapitulate histological features, DNA methylation profiles, and intratumor heterogeneity of the tumors from which they were derived. We also showed that PDOs can be xenografted. Most interestingly, when subjected to the same routinely applied therapeutic regimens, PDOs respond similarly to the patients. Taken together, our study highlights the potential of PDOs and PDXOs for research and translational applications for personalized medicine.
Synopsis
Patient‐derived organoids (PDOs) and patient‐derived xenograft organoids (PDXOs) have been established through direct in vitro culture of primary tumors and patient‐derived xenograft (PDX)‐derived tumors with the goal to better model pediatric tumors of the central nervous system.
PDOs recapitulate cellular heterogeneity, histological features, mutational profiles (number of variants, microsatellite instability, tumor mutational burden) of the corresponding parental tumor even after 1 year of in vitro culture
ZFTA‐RELA EPN‐ and G3 MB‐PDOs recapitulate patients' response to routinely applied therapeutic regimens
Several PDOs and PDXOs were derived from human tumors for which there are currently very limited in vivo and in vitro models available
PDOs and PDXOs are available to the research community, and can be subjected to genetic manipulations, to drug screening, and translational applications for personalized medicine
Patient‐derived organoids (PDOs) and patient‐derived xenograft organoids (PDXOs) have been established through direct in vitro culture of primary tumors and patient‐derived xenograft (PDX)‐derived tumors with the goal to better model pediatric tumors of the central nervous system.
The transition of neural progenitors to differentiated postmitotic neurons is mainly considered irreversible in physiological conditions. In the present work, we show that Shh pathway activation ...through SmoM2 expression promotes postmitotic neurons dedifferentiation, re-entering in the cell cycle and originating medulloblastoma in vivo. Notably, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here, we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Indeed, postmitotic neurons re-entered the cell cycle and proliferated as a result of chromatin remodeling of neurons by BRPF1. Our model of brain cancer explains the onset of a subset of human medulloblastoma in adult individuals where granule neuron progenitors are no longer present.
Display omitted
•SmoM2 overexpression promotes cerebellar granule neurons dedifferentiation in vivo•SmoM2 and mutant BRPF1 cooperation in vivo mimics human adult SHH MBs•Granule neurons are putative cells of origin of adult SHH MBs•Truncated BRPF1 increases the accessibility of a subset of super-enhancers
Medulloblastoma is a brain tumor affecting the cerebellum of infants and adults. Aiello et al. establish a mouse model for adult onset, which allows investigation of the pathogenesis of the disease and identifies neurons as putative cells of origin.
Medulloblastoma and high-grade glioma represent the most aggressive and frequent lethal solid tumors affecting individuals during pediatric age. During the past years, several models have been ...established for studying these types of cancers. Human organoids have recently been shown to be a valid alternative model to study several aspects of brain cancer biology, genetics and test therapies. Notably, brain cancer organoids can be generated using genetically modified cerebral organoids differentiated from human induced pluripotent stem cells (hiPSCs). However, the protocols to generate them and their downstream applications are very rare. Here, we describe the protocols to generate cerebellum and forebrain organoids from hiPSCs, and the workflow to genetically modify them by overexpressing genes found altered in patients to finally produce cancer organoids. We also show detailed protocols to use medulloblastoma and high-grade glioma organoids for orthotopic transplantation and co-culture experiments aimed to study cell biology in vivo and in vitro, for lineage tracing to investigate the cell of origin and for drug screening. The protocol takes 60-65 d for cancer organoids generation and from 1-4 weeks for downstream applications. The protocol requires at least 3-6 months to become proficient in culturing hiPSCs, generating organoids and performing procedures on immunodeficient mice.
Abstract
Central nervous system (CNS) tumors are the most common solid tumors in the pediatric population and the main cause of death among childhood cancers. They present common features related to ...the development and produce symptoms based on the age of the child, their location and rate of tumor growth. Tumors of embryonal origin, such as medulloblastoma or primitive neuroectodermal tumor, have a higher incidence in younger patients, whereas older ones tend to present tumors of glial origin. Most of CNS-embryonal tumors are highly malignant and consequently leading to poor prognoses. Despite the understanding of the underlying molecular landscape of these tumors that has helped in clinical advances to improve life expectancies of the patients, there is still an emerging need to minimize morbidity and manage the long-term therapy effects. Developing new models to better understand the heterogeneity of these tumors and to test personalized treatment strategies remains a clinical challenge. We are willing to answer to this medical need with Patient-Derived Organoids (PDOs) established through the direct in vitro culture of primary tumors and patient derived xenograft (PDX)-derived tumors. We developed a method for generating PDOs from a variety of pediatric brain cancers that recapitulates the cellular heterogeneity, histological features and mutational profiles of the corresponding parental tumors, also confirmed by the DNA methylation profile analysis, nowadays used for an accurate routine CNS tumors diagnosis. Through this new model, we confirmed the action of some clinically used drugs, obtaining a proof of concept of their possible application as a reliable tool for drug screening. Further applications derive from the enrichment of PDOs culture media for possible tumor-specific antigens that could be useful to define tumor markers identifiable with diagnostic tests or as candidates for new therapies.
Abstract
Central nervous system (CNS) tumors are the most common solid tumors in the pediatric population and the main cause of death among childhood cancers. They present common features related to ...the development and produce symptoms based on their location, rate of tumor growth and age of the child. Tumors of embryonal origin, such as medulloblastoma or primitive neuroectodermal tumor, have a higher incidence in younger patients, whereas older ones tend to present tumors of glial origin. Most of CNS-embryonal tumors are highly malignant and consequently leading to poor prognoses. Despite the understanding of the underlying molecular landscape of these tumors that has helped in clinical advances to improve life expectancies of the patients, there is still an emerging need to minimize morbidity and manage the long-term therapy effects. Developing new models to better understand the heterogeneity of these tumors and to test personalized treatment strategies remains a clinical challenge. We are trying to answer to this medical need with Patient-Derived Organoids (PDOs) established through the direct in vitro culture of primary tumors and patient derived xenograft (PDX)-derived tumors. We have developed a method for generating PDOs from a variety of pediatric brain cancers that recapitulates the cellular heterogeneity, histological features and mutational profiles of the corresponding parental tumors. We have obtained promising results from the DNA methylation profile analysis, nowadays used for an accurate routine CNS tumors diagnosis. Through this new model, we confirmed the action of some clinically used drugs, obtaining a proof of concept of their possible application as a reliable tool for drug screening. Another interesting application derives from the enrichment of PDOs culture media for possible tumor-specific antigens: their identification could be of pivotal importance for the definition of tumor markers identifiable with diagnostic tests or as candidates for new therapies.
Lifespan is determined by complex and tangled mechanisms that are largely unknown. The early postnatal stage has been proposed to play a role in lifespan, but its contribution is still controversial. ...Here, we show that a short rapamycin treatment during early life can prolong lifespan in Mus musculus and Drosophila melanogaster. Notably, the same treatment at later time points has no effect on lifespan, suggesting that a specific time window is involved in lifespan regulation. We also find that sulfotransferases are upregulated during early rapamycin treatment both in newborn mice and in Drosophila larvae, and transient dST1 overexpression in Drosophila larvae extends lifespan. Our findings unveil a novel link between early‐life treatments and long‐term effects on lifespan.
Synopsis
This study reveals a link between early‐life treatments and long‐term effects on lifespan in Drosophila and mice.
A short rapamycin treatment during early life prolongs lifespan in Mus musculus and Drosophila melanogaster while the same treatment at later time points has no effect on lifespan.
Sulfotransferases are upregulated during early rapamycin treatment both in newborn mice and Drosophila larvae.
Transient dST1 sulfotransferase overexpression in Drosophila larvae extends lifespan.
This study reveals a link between early‐life treatments and long‐term effects on lifespan in Drosophila and mice.
Abstract
Brain cancer is now the deadliest form of childhood cancer in the United States. In particular, Group3 Medulloblastoma (MB) is the pediatric brain tumor with the highest morbidity and ...mortality. Patients with Group3 MB currently have the worst outcome and nearly 50% are metastatic at the time of diagnosis. However, the cellular and molecular mechanisms underlying Group3 MB are still unknown. What is still lacking in the field is the possibility to obtain tumors by direct genetic modification of mice and to be able to recapitulate the growth and metastasis formation of Group3 MB. Exploiting in-vivo transfection of mouse cerebellar cells with CRISPR-Cas9 and PiggyBac transposase systems, we tested different combinations of putative oncosuppressors and putative oncogenes, derived from human Medulloblastoma NGS data, for their ability to induce Group3 MB in mice. Surprisingly, concomitant overexpression of c-Myc and other transcription factors in mouse cerebellum is able to induce MB in a few months. The newly generated mouse model is able to fully recapitulate human Group 3 MB. Using this proposed patient-specific model, we were able to unravel the molecular aspects of Group 3 medulloblastoma tumorigenesis and identify molecules inhibiting tumor growth in a targeted manner.
Abstract
In the last years significant progress has been made to identify the cells of origin of many cancers and the majority of data points towards stem cells and progenitors. Notably, it is common ...opinion that these cells should possess a proliferative capacity, therefore postmitotic neurons have not been considered as suitable cell of origin of human and mouse cancer. Indeed, the transition of neural progenitor to differentiated postmitotic neurons is thought to be irreversible in physiological and pathological conditions. Here we show that neurons reprograming may occur upon oncogenes activation and it leads to brain cancer formation in vivo. Using a conditional Cre-recombination system we manage to recapitulate human cancer model in mice ad to demonstrate that the postmigratory mature granule neurons (MGNs) can be reprogrammed in-vivo. Interestingly, MGNs seem to possess defined characteristic that allow the reprogramming and the cancer formation, since activation of oncogenic pathways leads to cancer formation only in specific area of brain. For the first time we demonstrate the chance to reprogram postmitotic neurons into cancer cells shedding new light on the mechanisms of cancer formation. Postmitotic cells of other tissues may also possess tumor initianting cell features and our findings can be considered as a starting point to a new field in cancer biology.
PDF
